WO2000071565A2 - Fluorescent protein indicators - Google Patents

Fluorescent protein indicators Download PDF

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Publication number
WO2000071565A2
WO2000071565A2 PCT/US2000/013684 US0013684W WO0071565A2 WO 2000071565 A2 WO2000071565 A2 WO 2000071565A2 US 0013684 W US0013684 W US 0013684W WO 0071565 A2 WO0071565 A2 WO 0071565A2
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WO
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Prior art keywords
fluorescent protein
polypeptide
moiety
receptor
nucleic acid
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PCT/US2000/013684
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French (fr)
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WO2000071565A3 (en
WO2000071565A9 (en
Inventor
Roger Y. Tsien
Geoffrey A. Baird
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The Regents Of The University Of California
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Priority claimed from US09/316,920 external-priority patent/US6699687B1/en
Priority claimed from US09/316,919 external-priority patent/US6469154B1/en
Application filed by The Regents Of The University Of California filed Critical The Regents Of The University Of California
Priority to AU52751/00A priority Critical patent/AU5275100A/en
Publication of WO2000071565A2 publication Critical patent/WO2000071565A2/en
Publication of WO2000071565A3 publication Critical patent/WO2000071565A3/en
Priority to US09/999,745 priority patent/US7060793B2/en
Publication of WO2000071565A9 publication Critical patent/WO2000071565A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae

Definitions

  • the invention relates generally to fluorescent proteins and more particularly to compositions and methods for measuring the response of a sensor polypeptide to an environmental (e g , biological, chemical, electrical or physiological) parameter
  • Fluorescent Ca' + indicators such as fura-2, ⁇ ndo-1 , fluo-3, and Calcium-Green have been the mainstay of intracellular Ca "+ measurement and imaging (see, for example, U S Pat No 4,603,209 and U S Pat No 5,049,673)
  • These relatively low molecular weight indicators can suffer from many technical problems relating to ester loading, leakage of the dyes from the cell, compartmentation in organelles, and perturbation of the indicators by cellular constituents
  • photoprotein aequo ⁇ n is targetable, the photoresponse to Ca ⁇ + is low since it is chemiluminescent Moreover, aequo ⁇ ns need to incorporate exogenous coelenterazine
  • CaM calmoduhn
  • cyclic AMP can be detected by fluorescence resonance energy transfer between separately labeled proteins that associate with each other but are not covalently attached to each other See, U S Pat No 5,439,797
  • GFP The Aequorea Green Fluorescent Protein
  • FRET fluorescence resonance energy transfer
  • a sensor polypeptide when a sensor polypeptide is inserted into an Aequorea-r ated fluorescent protein (e g , Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP) or Cyan Fluorescent Protein (CFP)) to form a construct, interaction of the sensor polypeptide with a biological, chemical, electrical or physiological parameter, for example, results in a change in fluorescence of the fluorescent protein.
  • GFP Green Fluorescent Protein
  • YFP Yellow Fluorescent Protein
  • CFP Cyan Fluorescent Protein
  • Such constructs are useful in measuring interactions of a sensor polypeptides with environmental stimuli in vitro or in vivo or in measuring particular characteristics of a cell (e g , redox potential, intracellular ion concentration). These constructs rely on the responsiveness of a sensor polypeptide inserted within a GFP- sensor-related protein itself to influence the actual fluorescence of the fluorophore and not the interaction of tandem fluorescent molecules
  • the present invention provides an isolated nucleic acid sequence which encodes a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide.
  • the fluorescent protein moiety can be any fluorescent protein, for example, an Aequorea-r ated fluorescent protein moiety.
  • the Aeqi ⁇ re ⁇ -related fluorescent protein can be, for example, a GFP, CFP or YFP moiety
  • the sensor polypeptide may be any polypeptide moiety, for example, a moiety that undergoes a conformational change upon interaction with a molecule, oxidation-reduction, or changes in electrical or chemical potential.
  • the indicator may further include a linker moiety, linking the N- and C-terminal amino acids of the sensor polypeptide to the fluorescent protein
  • the linker may be any moiety that provides for linking of the sensor polypeptide to the fluorescent protein moiety such as for example, a nucleic acid that encodes GGTGEL (SEQ ID NOJ) or FKTRHN (SEQ ID NO.2).
  • Two or more linker moieties may be attached to two separate polypeptides, that together form a sensor polypeptide.
  • the indicator may have a localization sequence, for localizing the indicator, for example, to a particular organelle of a cell.
  • the sensor polypeptide or linker moiety may be inserted at numerous sites including, for example, one or more amino acids between residues 128 - 148, residues 155-160, residues 168-176 or residues 227-229 of the fluorescent protein moiety (e g , GFP) More particularly Y145 is used for insertion of the linker or sensor polypeptide.
  • the present invention provides a transgenic non- human animal having a nucleic acid sequence which encodes a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
  • the present invention provides an expression vector having expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator
  • the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
  • the present invention provides a host cell transfected with an expression vector having an expression control sequence operatively linked to a sequence coding for the expression of a fluorescent indicator
  • the host cell can be any host cell capable of transfection and expression of the indicator such as, for example, a prokaryote (e g , E Coli), a eukaryotic cell (e g , a yeast cell) or a mammalian cell
  • the present invention provides a fluorescent indicator polypeptide, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
  • the present invention provides a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive WO 00/71565 PCT/USOO/l 3684 to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide the responsiveness resulting in protonation or deprotonation of the chromophore of the fluorescent protein moiety
  • the present invention provides a method for detecting the presence of a environmental parameter in a sample, by contacting the sample with a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical, or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide, and detecting a change in fluorescence wherein a change is indicative of the presence of a parameter which affects the sensor polypeptide.
  • the invention provides an isolated nucleic acid sequence encoding a circularly pe ⁇ nuted fluorescent protein and the polypeptide encoded thereby, having a linker moiety linking the amino-terminal and carboxy- terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-termmal and carboxy-terminal amino acids of the fluorescent protein.
  • the invention provides an expression vector comprising expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator, the indicator having a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein WO 00/71565 PCT/USOO/l 3684 the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy- terminal amino acids of the fluorescent protein.
  • the invention provides a host cell containing the expression vector.
  • the invention provides a method of producing a nucleic acid sequence encoding a fluorescent indicator, by linking a nucleic acid sequence encoding a linker moiety to the 5' nucleotide of a polynucleotide encoding a fluorescent protein, circularizing the polynucleotide with the nucleic acid sequence encoding the linker sequence, and cleaving the circularized polynucleotide with a nuclease, wherein cleavage linearizes the circularized polynucleotide.
  • the invention provides a method of producing a circularly permuted fluorescent protein by expressing a nucleic acid sequence encoding a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.
  • FIG. 1 shows the designs of a calmodulin or a Zif268 insertion into a fluorescent indicator of the present invention.
  • FIG. 2A shows pH effects on a calmodulin insertion indicator.
  • FIG 2B shows titration curves for a calmodulin insertion indicator
  • FIG 3 further shows the effects of calcium concentration on fluorescence of a fluorescent indicator of the invention containing a sensor polypeptide of calmodulin
  • FIG 4 show the effect of Zn concentration on fluorescence of a fluorescent indicator of the invention containing a sensor polypeptide of inserted Zif polypeptide
  • FIG 5 shows the overall design of a circularly permuted polypeptide
  • FIG 6 shows the result of random digestion of a circularly permuted nucleic acid sequences
  • FIG 7 shows insertions into GFP
  • FIG 8 shows a schematic diagram of potential insertional sites and sites for circular permutations in GFP
  • the present invention relates to polynucleotides encoding fluorescent indicators, fluorescent indicators having a sensor polypeptide, and methods of producing and using the same
  • the inventors have discovered a number of sites in Aequot e ⁇ -related fluorescent protein moieties that aie tolerant to insertions and rearrangements
  • the inventors have additionally discovered that the insertion of sensory polypeptides into such sites results in polypeptides that are useful in detection of chemical, biological, electrical or physiological parameters, for example
  • Such insertion sites include, for example, one or more amino acids between residues 128 - 148, residues 155-160, residues 168-176 or residues 227-229 of the fluorescent protein moiety
  • Other positions which may tolerate insertions include, but are not limited to, residues 49-50, 78-79, 1 16-1 17, 134-135, 140-141 , 157-158, 172-173, 194-195, 189-190 and 213-214 (see Abedi et al , Nucleic Acids Research, 26(2) 623-630 (1998), the disclosure of which is incorporated herein) More specifically, the insertion is a Y145
  • a sensor polypeptide is inserted into an Aequoi e ⁇ -related fluorescent protein (e g , GFP, YFP or CFP) that provides a response related to an interaction with a biological, chemical, electrical or physiological parameter, the responsiveness results in a change in fluorescence of the fluorescent protein
  • an Aequoi e ⁇ -related fluorescent protein e g , GFP, YFP or CFP
  • Such constructs are useful in measuring interactions of a sensor polypeptides with environmental stimuli in vitro or in v ⁇ o
  • These new constructs rely on, for example, detectable changes within a GFP-sensor-related protein itself to influence the actual fluorescence of the fluorophoie and not the interaction of tandem fluorescent molecules
  • calmodulin is inserted into YFP at position Y145
  • interaction of calmodulin with its ligand e g , calcium
  • the indicators of the present invention are advantageous due to their reduced size as compared to the FRET-based sensors desc ⁇ bed above
  • the reduced size has importance in allowing the indicator to measure chemical, biological, electrical or physiological interactions with the sensor polypeptide in, for example, subcellular compartments previously inaccessible to the larger, FRET-based sensors
  • the maximal change in fluorescence intensity observed in the present indicators e g , up to 8 fold increase
  • those in the cameleons e g , FRET-based sensors
  • the invention provides polynucleotides and nucleic acid sequences encoding fluorescent indicators having a fluorescent protein moiety and a sensor polypeptide, or fragments thereof, inserted in operable association into the fluorescent protein moiety, in which the sensor polypeptide is responsive to an environmental parameter (e g , a chemical, a biological, a electrical, or a physiological parameter) Accordingly, the responsiveness of the sensor polypeptide causes a change in fluorescence of the fluorescent indicator The degree of change in the fluorescence of the indicator is sensitive to pH
  • "operatively inserted” or “operably inserted” is meant between two amino acids of a polypeptide or two nucleotides of a nucleic acid sequence Accordingly, insertion excludes ligating or attaching a polypeptide to the last terminal amino acid or nucleotide in a sequence
  • a "detectable change” or “responsiveness” means any response of a polypeptide to a chemical, biological, electrical, or physiological parameter or stimuli
  • a response includes small changes, for example, a shift in the orientation of an amino acid or peptide fragment of the sensor polypeptide as well as, for example, a change in the primary, secondary, or tertiary structure of a polypeptide, including for example, changes in protonation, electrical and chemical potential and or coniormation
  • Conformation is the three-dimensional arrangement of the primary, secondary and tertiary structure of a molecule including side groups in the molecule, a change in conformation occurs when the three-dimensional structure of a molecule changes Examples of conformational changes include a shift from -helix to a -sheet or a shift from -sheet to a -helix It is understood that detectable changes need not be a conformational change, so long as the fluorescence of the fluorescent protein moiety is altered
  • Fragments are a portion of a naturally occurring sensor protein which can exist in at least two different states or conformations Fragments can have the same or substantially the same amino acid sequence as the naturally occurring protein "Substantially the same” means that an amino acid sequence is largely, but not entirely, the same, but retains a functional activity of the sequence to which it is related In general two amino acid sequences are substantially the same” or “substantially homologous” if they are at least 85% identical Fragments which have different three dimensional structures as the naturally occurring protein are also included
  • responsive as used herein is intended to encompass any response of a polypeptide which is related to an interaction of a chemical, biological, electrical, or physiological parameter with a sensor polypeptide (e g , conformational change in a voltage-gated ion channel (e g , Shaker) in detection of membrane voltage across a biological membrane, phosphorylation of a hormone receptor resulting in a conformational change in the receptor upon hormone stimulation) WO
  • Example of sensor polypeptide useful in the present invention include calmodulin, a calmoduhn-related protein moiety, recove ⁇ n, a nucleoside diphosphate or t ⁇ phosphate binding protein, an ⁇ nos ⁇ tol-l,4,5-t ⁇ phosphate receptor, a cyclic nucleotide receptor, a nitric oxide receptor, a growth factor receptor, a hormone receptor, a ligand-binding domain of a hormone receptor, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, a cytokine receptor, a growth factor receptor, a neurotransmitter receptor, a hgand-gated channel, a voltage-gated channel, a protein kinase C, a domain of protein kinase C.
  • a cGMP-dependent protein kinase an inositol polyphosphate receptor, a phosphate receptor, a carbohydrate receptor, an SH2 domain, an SH3 domain, a PTB domain, an antibody, an antigen- binding site from an antibody, a single-chain antibody, a zinc-finger domain, a protein kinase substrate, a protease substrate, a phosphorylation domain, a redox sensitive loop, a loop containing at least two cysteines that can form a cyclic disulfide, and a fluorescent protein moiety
  • the fluorescent protein contains a second fluorescent protein and a sensor moiety within the insert
  • utilization of FRET based techniques to analyze or detect changes in chemical, biological or electrical parameters may be performed
  • binding of an analyte such as calcium to a sensor polypeptide such as calmodulin would change the distance or angular orientation of the two fluorescent protein chromophores relative to each other and thereby modulate FRET.
  • a circularly permuted fluorescent protein may be tandemly or msertionally fused via a sensor moiety to a second fluorescent protein (which itself may optionally be a circular permutation) so that the FRET between the two fluorescent proteins changes in response to chemical, biological, or electrical parameters
  • Classes of sensor polypeptides which may be used in the compositions and methods of the invention include, but are not limited to, channel proteins, receptors, enzymes, and G-proteins
  • Channel polypeptides useful with the invention include, but are not limited to voltage-gated ion channels including the potassium, sodium, chloride, G-protem- responsive, and calcium channels
  • a "channel polypeptide” is typically a WO 00/71565 PCT/USOO/l 3684 polypeptide embedded in the cell membrane which is part of a structure that determines what particle sizes and or charges are allowed to diffuse into the cell
  • Channel polypeptides include the "voltage-gated ion channels", which are proteins imbedded in a cell membrane that serve as a crossing point for the regulated transfer of a specific ion or group of ions across the membrane
  • Shaker potassium channels or dihydropu ⁇ dine receptors from skeletal muscle may be advantageously used in the present invention
  • Table 1 Several ion channel polypeptides of use with the invention are listed in Table 1
  • Channels also include those activated by intracellular signals such as those where the signal is by binding of ligand such as calcium, cyclic nucleotides, G- proteins, phosphoinositols, arachidonic acid, for example, and those where the signal is by a covalent modification such as phosphorylation, enzymatic cleavage, oxidation/reduction, and acetylation, for example
  • ligand such as calcium, cyclic nucleotides, G- proteins, phosphoinositols, arachidonic acid, for example
  • Channel proteins also include those activated by extracellular ligands (e g , lonotropic receptors) These can be activated by acetylcholine, biogenic amines, amino acids, and ATP, for example
  • a "receptor polypeptide” is a polypeptide found on a cell, often on a membrane, that can combine with a specific type of molecule, e g , a ligand, which alters a function of the cell
  • Receptor polypeptides of use with the invention include, but are not limited to, the growth factor receptors, hormone receptors, cytokine WO 00/71565 PCT/USOO/l 3684 receptors, chemokine receptors, neurotransmitter receptors, hgand-gated channels, and steroid receptors. Specifically polypeptides encoding insulin-like growth factor, insulin, somatostatin, glucagon.
  • interleukins e g , IL-2, transforming growth factors (TGF- ⁇ , TGF- ⁇ ), platelet-dern ed growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), fibroblast growth factor (FGF), interferon- ⁇ (IFN- ⁇ ), and GM-CSF receptors
  • TGF- ⁇ , TGF- ⁇ ), platelet-dern ed growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), fibroblast growth factor (FGF), interferon- ⁇ (IFN- ⁇ ), and GM-CSF receptors are of use with the invention
  • Receptors such as those where binding of ligand is transmitted to a G-protein (e g , for 7-transmembrane receptors) or kinase domains (for single transmembrane receptors) can be used with the invention
  • G-protein e g , for 7-transmembrane receptor
  • Enzyme is a polypeptide that acts as a catalyst, which speeds the rate at which biochemical reactions proceed do not alter the direction or nature of the reaction.
  • Enzyme polypeptides useful in the invention include, but are not limited to, protein kinases, catalyses, amidase, phosphatases, guanylyl and adenylyl cyclases, and poxygenases
  • Polypeptides encoding the se ⁇ ne/threonine protein kinases are of use with the invention.
  • Several genes encoding human enzymes of use with the invention are listed in Table 3. TABLE 3 ENZYMES
  • the responsiveness of the sensor polypeptide (e g , a change in conformation or state) that occurs in response to interaction of a sensor polypeptide with a chemical, biological, electrical or physiological parameter will, as discovered by the inventors, cause a change in fluorescence of the fluorescence indicator
  • the change can be the result of an alteration in the environment, structure, protonation or ohgome ⁇ zation status of the fluorescent indicator or chromophore.
  • the molecular component responsible for a conformational change is known for many enzymes (e g , Blostien, R., et al (1997) J. Biol.
  • optical properties (e g , fluorescence) of the indicator which can be altered in response to the conformational change in the sensor polypeptide include, but are not limited to, changes in the excitation or emission spectrum, quantum yield, extinction coefficient, excited life-time and degree of self-quenching for example
  • the cause of the changes in these parameters may include but are not limited to changes in the environment, changes in the rotational or vibrational freedom of the sensor, changes in the angle of the sensor with respect to the exciting light or the optical detector apparatus, changes in the protonation or deprotonation of amino acids or side groups associated with a chromophore or changes in distance or dipole orientation between sensors on associated responsive polypeptides
  • insertion of a peptide or protein in place of tyrosine- 145 (Y145) in mutants of GFP increases the sensitivity of fluorescence to quenching by acidic pH WO 00/71565 PCT/USOO/l 3684
  • the sensor polypeptide is operably inserted into an optically active polypeptide (e g , a fluorescent protein moiety)
  • an optically active polypeptide e g , a fluorescent protein moiety
  • a protein-based "optically active polypeptide” is a polypeptide which contains a means for emitting light Fluorescence is one optical property of an optically active polypeptide which can be used as the means of detecting the responsiveness of the sensor or responsive polypeptide of the fluorescent indicator or circularly permuted fluorescent proteins of the invention
  • fluorescent property refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum and emission wavelength maximum, the ratio of excitation amplitudes at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy
  • a measurable difference in any one of these properties between the active and inactive states suffices for the utility of the fluorescent protein
  • excitation radiation from an excitation source having a first wavelength, passes through excitation optics The excitation optics cause the excitation radiation to excite the sample
  • fluorescent proteins in the sample emit radiation which has a wavelength that is different from the excitation wavelength Collection optics then collect the emission from the sample.
  • the device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned
  • a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed
  • the multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer
  • the computer also can transform the data collected during the assay into another format for presentation
  • Other means of measuring fluorescence can also be used with the invention
  • any fluorescent protein can be used in the invention, including proteins that fluoresce due to intramolecular rearrangements or the addition of cofactors that promote fluorescence
  • green fluorescent proteins of cnida ⁇ ans which act as their energy-transfer acceptors in bioluminescence
  • a green fluorescent protein is a protein that emits green light
  • BFP blue fluorescent protein
  • YFP yellow fluorescent protein
  • CFP cyan fluorescent protein
  • GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, the sea pansy, Renilla remformis, and Phiahdium gregarium See, Ward, W W., et al ,
  • WlB l included the following mutations" F64L;S65T; Y66W; F99S; and V163A.
  • fluorescent proteins can be used in the fluorescent indicators, such as, for example, yellow fluorescent protein from Vibrio fischeri strain Y-l , Pe ⁇ dinin- chlorophyll a binding protein from the dmofiagellate Symbwdinium sp phycobihproteins from marine cyanobacte ⁇ a such as Synechococcus, e.g., phycoeryth ⁇ n and phycocyamn, or oat phytochromes from oat reconstructed with phycoerythrobilm.
  • yellow fluorescent protein from Vibrio fischeri strain Y-l Pe ⁇ dinin- chlorophyll a binding protein from the dmofiagellate Symbwdinium sp phycobihproteins from marine cyanobacte ⁇ a such as Synechococcus, e.g., phycoeryth ⁇ n and phycocyamn, or oat phytochromes from oat reconstructed with phycoerythrobil
  • the fluorescent indicators can be produced as chimeric proteins by recombinant DNA technology.
  • Recombinant production of fluorescent proteins involves expressing nucleic acids having sequences that encode the proteins.
  • Nucleic acids encoding fluorescent proteins can be obtained by methods known in the art.
  • a nucleic acid encoding the protein can be isolated by polymerase chain reaction of cDNA from A victoria using p ⁇ mers based on the DNA sequence of J victoria green fluorescent protein PCR methods are described in, for example, U.S. Pat. No. 4,683,195; Mul s, et al Cold Spring Harbor Symp. Quant. Biol. 51 :263 (1987), and Erhch, ed., PCR Technology, (Stockton Press, NY, 1989).
  • Mutant versions of fluorescent proteins can be made by site-specific mutagenesis of other nucleic acids encoding fluorescent proteins, or by random mutagenesis caused by increasing the error rate of PCR of the original polynucleotide with 0 1 mM MnCl 2 and unbalanced nucleotide concentrations.
  • the sensor polypeptide is operably inserted to an optically active polypeptide, which responds (e.g , a conformation change)to, for example, a cell signaling event.
  • a cell signaling event e.g , a conformation change
  • Cell signaling events that occur in vivo can be of very short duration.
  • the optically active polypeptides of the invention allow measurement of the optical parameter, such as fluorescence, which is altered in response to the cell signal, over the same time period that the event actually occurs.
  • the response can be measured after the event occurs (over a longer time period) as the response that occurs in an optically active polypeptide can be of a longer duration than the cell signaling event itself.
  • the invention provides isolated nucleic acid sequences which encode fluorescent indicator polypeptides having operatively inserted therein a sensor polypeptide, or fragment thereof, which normally exists in one state e g , conformational shape or charge, prior to an interaction with a chemical, biological, electrical or physiological parameter at which time it undergoes a response during or after the interaction of the chemical, biological, electrical or physiological parameter with the sensor polypeptide
  • Polynucleotide or “nucleic acid sequence” refers to a polymeric form of nucleotides at least 10 bases in length
  • isolated nucleic acid sequence is meant a polynucleotide that is no longer immediately contiguous with both of the coding sequences with which it was immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic cell or organism, or which exists as a separate molecule (e g a cDNA) independent of other sequences
  • the nucleotides of the invention can be ⁇ bonucleotides, deoxy ⁇ bonucleotides, or modified forms of either nucleotide
  • the term includes single and double stranded forms of DNA.
  • Nucleic acid sequences which encode a fluorescent indicator of the invention, wherein the indicator includes a sensor polypeptide, or fragment thereof, which normally has two or more states or conformational arrangements, and which undergoes a response during interaction with a chemical, biological, electrical or physiological parameter can be operatively linked to expression control sequences "Operatively linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • expression control sequences refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked
  • Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (.
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and chimeric partner sequences
  • Expression control sequences can include a promoter
  • promoter minimal sequence sufficient to direct transcription
  • promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents, such elements may be located in the 5' or 3' regions of the gene
  • constitutive and inducible promoters are included in the invention (see e g , Bitter et al , 1987, Methods in Enzymology 153 516-544)
  • inducible promoters such as pL of bacteriophage , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used
  • promoters derived from the genome of mammalian cells e g , metallothionein promoter
  • fiom mammalian viruses e g , the retrovirus long terminal repeat, the adenovirus late
  • fluorescent protein and “fluorescent protein moiety” are used interchangeably and refer to any protein capable of emitting light when excited with appropriate electromagnetic radiation, and which has an amino acid sequence that is either natural or engineered and is derived from the amino acid sequence of an
  • fluorescent indicator refers to a fluorescent protein having a sensor polypeptide whose emitted light varies with the response state or conformation of the sensor polypeptide upon interaction with a chemical, biological, electrical or physiological parameter
  • the term also refers to a fluorescent protein whose ammo acid sequence has been circularly permuted
  • the fluorescent indicators of the invention are also sensitive to pH in the range of about 5 to about 10
  • the invention provides, for example, a functional engineered fluorescent protein indicator whose amino acid sequence is substantially identical to the 238 amino acid Aequoi ea victona green fluorescence protein (SEQ ID NO 3)
  • the invention also includes functional polypeptide fragments of a fluorescent indicator
  • the term "functional polypeptide fragment” refers to a polypeptide which possesses biological function or activity which is identified through a defined functional assay
  • the term "functional fragments of a functional engineered fluorescent protein” refers to fragments of a functional engineered protein that retain a function of the engineered fluorescent protein, e g , the ability to fluoresce in a manner which is dependent upon interactions of a chemical, biological, electrical or physiological parameter with a sensor polypeptide over the pH range 5 to 10
  • substantially identical or “substantially homologous” is meant a protein or polypeptide that retains the activity of a functional engineered fluorescent indicator, or nucleic acid sequence or polynucleotide encoding the same, and which exhibits at least 80%, preferably 85%, more preferably 90%, and most preferably 95%) homology to a reference amino acid or nucleic acid sequence
  • the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids
  • the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 1 10 nucleotides
  • the reference amino acid sequence or nucleic acid sequence is considered homologous if the reference ammo acid sequence is 80-95% homologous to any portion of the amino acid or nucleic acid sequence in question For example, where a circular
  • substantially identical is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one ammo acid for another of the same class (e g , valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein (e.g., assayed as described herein).
  • such a sequence is at least 85%, more preferably 90%, more preferably 95%, more preferably 98%, and most preferably 99%) identical at the amino acid sequence to one of the sequences of EGFP (SEQ ID NOJ), EYFP (SEQ ID NO:5), ECFP (SEQ ID NO 6), EYFP-V68L/Q69K (SEQ ID NO-7), YFP H148G (SEQ ID NO:8), or YFP H 148Q (SEQ ID NO"9).
  • EGFP EYFP
  • EYFP SEQ ID NO:5
  • EYFP EYFP-V68L/Q69K
  • SEQ ID NO-7 EYFP H148G
  • SEQ ID NO:8 EYFP H 148Q
  • sequence analysis software e.g.,
  • the amino acid sequence of the protein includes one of the following sets of substitutions in the ammo acid sequence of the Aequorea green fluorescent protein (SEQ ID NO 3) F64L/S65T/H231 L, referred to herein as EGFP (SEQ ID NO 4), S65G/S72A/T203Y/H231L, referred to herein as EYFP (SEQ ID NO 5), S65G/V68L/Q69K/S72A/T203Y/H231L, referred to herein as EYFP- V68L/Q69K (SEQ ID NO 7),
  • ECFP K26R/F64L/S65T/Y66W/N146I/M153T/V163A/NJ64H/H231L
  • the amino acid sequences of EGFP, EYFP, ECFP, and EYFP-V68L/Q69K are shown in Tables 5-8, respectively
  • the numbering of the amino acids conforms to that in native Aequorea GFP
  • the first serine is amino acid number 2 even if a valine (ammo acid no l a) has been inserted to optimize ribosome initiation
  • F64L corresponds to a substitution of leucine for phenylalanine in the 64th ammo acid following the initiating methionine
  • the amino acid sequence of the protein is based on the sequence of the wild-type A equora green fluorescent protein, but includes the substitution H148G (SEQ ID NO:8) or H148Q (SEQ ID NO:9).
  • these substitutions can be present along with other substitutions, e.g., the proteins can include the substitutions S65G/V68L/S72A/H 148G/Q80R/T203 Y (SEQ ID NO:8), which is referred to herein as the "YFP H148G mutant," S65G/V68L/S72A/H148Q/Q80R/T203Y, which is referred to herein as the "YFP H148Q mutant" (SEQ ID NO:9), as well as EYFP-H148G (SEQ ID NO: 10) and EFP-H148Q (SEQ ID NO: 1 1).
  • the amino acid sequences of these mutants are shown in Tables 9-12, respectively. Table 9. Amino Acid Sequence of YFP H148
  • EYFP-H148Q (SEQ ID NOJ 1) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG KLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYIMA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSA LSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
  • the protein or polypeptide is substantially purified.
  • substantially pure protein or polypeptide is meant an functional engineered fluorescent polypeptide which has been separated from components which naturally accompany it.
  • the protein or polypeptide is substantially pure when it is at least 60%), by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%o, more preferably at least 90%, and most preferably at least 99%, by weight, of the protein.
  • a substantially pure protein may be obtained, for example, by extraction from a natural source (e.g., a plant cell); by expression of a recombinant nucleic acid WO 00/71565 PCT/USOO/l 3684 encoding a functional engineered fluorescent protein; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., those described in column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
  • a protein or polypeptide is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state.
  • a protein or polypeptide which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components.
  • substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. Coli or other prokaryotes.
  • the invention also provides polynucleotides encoding the functional engineered fluorescent protein described herein.
  • These polynucleotides include DNA, cDNA, and RNA sequences which encode functional engineered fluorescent proteins. It is understood that all polynucleotides encoding functional engineered fluorescent proteins are also included herein, as long as they encode a protein or polypeptide whose fluorescent emission intensity changes as pH varies between 5 and 10.
  • Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For example, the polynucleotide may be subjected to site-directed mutagenesis.
  • the polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the functional engineered fluorescent protein or derivative is functionally unchanged.
  • a polynucleotide sequence encoding a functional engineered fluorescent protein that includes one of the following sets of substitutions in the amino acid sequence of the Aequorea green fluorescent protein (S ⁇ Q ID NOJ): S65G/S72A/T203Y /H231L, S65G/V68L/Q69K/S72 A/T203 Y/H231 L, or K26R/F64L/S65T/Y66W/N 1461/ M153T/V163A/N 164H/H231L.
  • S ⁇ Q ID NOJ Aequorea green fluorescent protein
  • the DNA sequences encoding ⁇ GFP, ⁇ YFP, ⁇ CFP, ⁇ YFP-V68L/Q69K, YFP H 148G, and YFP H148Q are those shown in Table 13-20 (SEQ ID NOs: 12 to 19), respectively.
  • the nucleic acid encoding functional engineered fluorescent proteins may be chosen to reflect the codon choice in the native A victoria coding sequence, or, alternatively, may be chosen to reflect the optimal codon frequencies used in the organism in which the proteins will be expressed
  • nucleic acids encoding a target functional engineered protein to be expressed in a human cell may have use a codon choice that is optimized for mammals, or especially humans.
  • the nucleic acid sequences encoding the fluorescent indicator or circularly permuted fluorescent protein of the invention may be inserted into a recombinant expression vector
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the nucleic acid sequences encoding the chimeric peptides of the invention
  • the expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells
  • Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al , Gene, 56 125, 1987), the pMSXND expression vector, or adeno or vaccinia viral vectors for expression in mammalian cells (Lee and Nathans, J Biol Chem., 263 3521 , 1988), baculovirus-de ⁇ ved vectors for expression in insect cells, cauliflower
  • the nucleic acid sequences encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention can also include a localization sequence to direct the indicator to particular cellular sites by fusion to appropriate organellar targeting signals or localized host proteins
  • a polynucleotide encoding a localization sequence, or signal sequence can be ligated or fused at the 5' terminus of a polynucleotide encoding the fluorescence indicator such that the signal peptide is located at the amino terminal end of the resulting chimeric polynucleotide/polypeptide.
  • the signal peptide is believed to function to transport the chimeric polypeptide across the endoplasmic reticulum
  • the secretory protein is then transported through the Golgi apparatus, into secretory vesicles and into the extracellular space or, preferably, the external environment.
  • Signal peptides which can be utilized according to the invention include pre-pro peptides which contain a proteolytic enzyme recognition site
  • Other signal peptides with similar properties to those described herein are known to those skilled in the art, or can be readily ascertained without undue experimentation
  • the localization sequence can be a nuclear localization sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, or a localized protein Localization sequences can be targeting sequences which are described, for example, in "Protein Targeting' " .
  • the localization sequence can also be a localized protein
  • Some important localization sequences include those targeting the nucleus (KKKRK) (SE Q ID NO:20), mitochondrion (amino terminal MLRTSSLFTRRVQPSLFRNILRLQST (SEQ ID NO"21)), endoplasmic reticulum (KDEL (SEQ ID NO.22) at C-terminus, assuming a signal sequence present at N-terminus), peroxisome (SKF at C-termmus), synapses (S/TDV or fusion to GAP 43, kinesin and tau) prenylation or insertion into plasma membrane (CaaX (SEQ ID NO.23), CC, CXC, or CCXX (SEQ ID NO:24) at C- terminus), cytoplasmic side of plasma membrane (chime ⁇ c to SNAP-25), or the Golgi apparatus (chimeric to fu ⁇ n).
  • agents which induce a sensor polypeptide include agents that contain any of the amino acid sequences in Table 21 , or a portion thereof with the proviso that the parameter must bind to a calmodulin sensor polypeptide.
  • the parameter can be a subsequence of a calmoduhn-binding domain
  • the moieties listed in Table 21 are recognized by the sensor polypeptide CaM. See, for example, C ⁇ vici, A. & Ikura, M. Annu. Rev. Biophys Biomol. Struct. 24 84-1 16 (1995).
  • the parameter can be modified to enhance the response of the fluorescent indicator to the parameter.
  • Other parameter are known in the art for other sensor polypeptides.
  • AC adenylyl cyclase
  • BBMHCI brush-border myosin heavy chain-I
  • CaMKII calmodulin kinase II
  • CBP2 calmodulin binding pept ⁇ de-2
  • GIP gastrin inhibitory peptide
  • HIV-1 gpl60 human immunodeficiency virus envelope glycoprotein 160
  • HSP heat-shock protein
  • MARCKS myristoylated alaminte- ⁇ ch C kinase substrate
  • MHC myosin heavy chain
  • NOS nitric oxide synthase
  • PDE phosphodiesterase
  • PFK phosphofructokinase
  • PhK phosphorylase kinase
  • sk- smMLCK
  • VIP vasoactive intestinal peptide
  • the preferred linker moiety is a peptide between about one and 30 amino acid residues in length, preferably between about two and 15 amino acid residues
  • One preferred linker moiety is a -Gly-Gly- linker.
  • the linker moiety can include flexible spacer amino acid sequences, such as those known in single-chain antibody research
  • the linker moiety can be GGGGS (SEQ ID NO"56)(GGGGS)n, GKSSGSGSESKS (SEQ ID NO"57), GSTSGSGKSSEGKG (SEQ ID NO”58), GSTSGSGKSSEGSGSTKG (SEQ ID NO 59) GSTSGSGKSSEGKG (SEQ ID NO 60), GSTSGSGKPGSGEGSTKG (SEQ ID NO 61), EGKSSGSGSESKEF (SEQ ID NO:62), GGTGEL (SEQ ID NO" 1), FKTRHN (SEQ ID NO 2), or GGTGGS
  • any of a number of suitable transcription and translation elements including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc may be used in the expression vector (see, e g , Bitter, et al , Methods in Enzymology 153:516-544, 1987). These elements are well known to one of skill in the art
  • a number of expression vectors may be advantageously selected depending upon the intended use. For example, when large quantities of a protein of the invention is desired, vectors which direct the expression of high levels of chimeric protein products that are readily purified may be desirable Those which are engineered to contain a cleavage site to aid in protein recovery are preferred
  • yeast a number of vectors containing constitutive or inducible promoters may be used.
  • Current Protocols in Molecular Biology Vol 2, Ed Ausubel, et al , Greene Publish Assoc & Wiley Interscience, Ch 13, 1988, Grant, et al , Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds Wu & Grossman, 31987. Acad Press, N Y , Vol 153, pp 516-544, 1987.
  • An alternative expression system which could be used to express the proteins of the invention is an insect system
  • Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes
  • the virus grows in Spodoptera frugiperda cells
  • the sequence encoding a protein of the invention may be cloned into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter)
  • Successful insertion of the sequences coding for a protein of the invention will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus (i e , virus lacking the proteinaceous coat coded for by the polyhedrin gene)
  • These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, see Smith, et al , J Viol 46-584, 1983, Smith, U S Patent
  • transformation is meant a permanent or transient genetic change induced in a cell following incorporation of new DNA (. e DNA exogenous to the cell) Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
  • transformed cell is meant a cell into which (or into an ancestor of which has been introduced), by means of recombinant DNA techniques, a DNA molecule encoding a fluorescent indicator or circularly permuted fluorescent protein having an optically active polypeptide having operatively inserted therein a sensor polypeptide, or fragment thereof, which normally has two or more states, and which is affected by a chemical, biological, electrical or physiological parameter.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. Coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl? method by procedures well known in the art.
  • MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
  • Eukaryotic cells can also be cotransfected with DNA sequences encoding the chimeric polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) adenovirus, vaccinia virus, or bovine papilloma vims, to transiently infect or transform eukaryotic cells and express the protein.
  • a eukaryotic viral vector such as simian virus 40 (SV40) adenovirus, vaccinia virus, or bovine papilloma vims
  • SV40 simian virus 40
  • vaccinia virus vaccinia virus
  • bovine papilloma vims bovine papilloma vims
  • Eukaryotic systems and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur.
  • Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and, secretion of the gene product should be used as host cells for the expression of fluorescent indicator
  • host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38
  • Mammalian cell systems hich utilize recombinant viruses or viral elements to direct expression may be engineered
  • the nucleic acid sequences encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention may be ligated to an adenovirus transcription/translation control complex, e g , the late promoter and tripartite leader sequence
  • This nucleic acid sequence may then be inserted in the adenovirus genome by in vitro or in vivo recombination Insertion in a non-essential region of the viral genome (e g , region El or E3) will result in a recombinant virus that is viable and capable of expressing the fluorescent indicator in infected hosts (e g , see Logan & Shenk, Proc Natl Acad Sci USA, 81 3655-3659, 1984)
  • the vaccinia virus 7 5K promoter may be used (e g , see, Mackett, et al
  • hypoxanthine- guamne phosphoribosyltransferase Szybalska & Szybalski, Proc Natl Acad Sci USA, 48 2026, 1962
  • adenine phosphoribosyltransferase Lowy, et al , Cell, 22 817, 1980
  • antimetabohte resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al , Proc Natl Acad Sci USA, 77 3567, 1980, O'Hare, et al , Proc Natl Acad Sci USA, 8 1527, 1981), gpt, which confers resistance to mycophenohc acid (Mulligan & Berg, Proc Natl Acad Sci USA, 78 2072, 1981 , ne
  • a fluorescent indicator or circularly permuted fluorescent protein of the invention can be produced by expression of nucleic acid encoding the protein in prokaryotes These include but are not limited to microorganisms such as bacteria transformed with recombinant bacte ⁇ ophage DNA, plasmid DNA or cosmid DNA expression vectors encoding a chimeric protein of the invention
  • a primary advantage of the optically active polypeptides of the invention is that they are prepared by normal protein biosynthesis, thus avoiding organic synthesis and the requirement for customized unnatural amino acid analogs
  • the constructs can be expressed in E Coli in large scale for in vitro assays Purification from bacte ⁇ a is simplified when the sequences include tags for one-step purification by nickel-chelate chromatography
  • the construct can also contain a tag to simplify isolation of the fluorescent indicator For example, a polyhistidine tag of, e g , six histidine residues, can be inco ⁇ orated at the ammo terminal end of the fluorescent protein The polyhist
  • Techniques for the isolation and purification of either microbially or eukaryotically expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromato graphic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies or antigen
  • nucleic acid sequence can also function as the concentration or available parameter in another embodiment of the invention
  • a response may result from the interaction of a nucleic acid sequence with a sensor polypeptide comprising a DNA binding protein motif
  • the invention features a method for determining the presence of a chemical, biological, electrical or physiological parameter, by contacting the sample with a fluorescent indicator or circularly permuted fluorescent protein of the invention, exciting the indicator or protein, and measuring the amount of an optical property of the indicator or protein in the presence and absence of a parameter, such that a change in the optical property is indicative of an affect of the parameter on the indicator or protein
  • a series of standards, with known levels of activity can be used to generate a standard curve
  • the optical event such as intensity of fluorescence, that occurs following exposure of the sample to the fluorescent indicator or protein is measured, and the amount of the optical property is then compared to the standard curve.
  • a standard with a known level of activity, can be used to generate a standard curve, or to provide reference standards
  • the optical event, such as fluorescence, that occurs following exposure of the sample to the fluorescent indicator or protein is measured, and the amount of the optical property (e g , fluorescence) is then compared to the standard in order to generate a relative measure of the affect of the sample on the fluorescent indicator.
  • the invention features a method for determining if a cell exhibits an activity, which includes transfecting the cell with a nucleic acid encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention; exciting the fluorescent indicator or circularly permuted fluorescent protein; and measuring the amount of an optical property in the presence of the activity and in the absence of the activity, such that a change in the optical property is indicative of activity.
  • the optical property is calibrated against standard measurements to yield an absolute amount of protein activity.
  • the invention additionally, features methods for determining transient changes in a chemical, biological, electrical or physiological parameter, by contacting the sample with a fluorescent indicator or circularly permuted fluorescent protein of the invention and measuring a change in the optical property of the indicator over time.
  • the cell containing a nucleic acid sequence encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention can be used to co-transfect other genes of interest in order to determine the effect of the gene product of that gene on the cell or the sensor polypeptide of the fluorescent indicator or circularly permuted fluorescent protein. Therefore, a cell containing such a nucleic acid sequence is a composition provided by the present invention
  • the invention can be used in screening assays to determine whether a compound (e.g., a drug, a chemical or a biologic) alters the activity of a particular protein, i.e., the sensor polypeptide (e.g., ligand binding to a receptor).
  • the assay is performed on a sample containing the chimeric protein in vitro.
  • a sample containing a known amount of activity such as an enzymatic activity, is mixed with a fluorescent indicator substrate of the invention, with the co- factors required for activity, and with a test compound.
  • the amount of the enzyme activity in the sample is then determined by measuring the amount of an optical property, such as a fluorescent property, at least a first and second time after contact between the sample, the chimeric protein substrate of the invention, and any co- factors or components required to conduct the reaction, and the test compound.
  • the amount of activity per mole of enzyme for example, in the presence of the test compound is compared with the activity per mole of enzyme in the absence of the test compound.
  • a difference indicates that the test compound alters the activity of the enzyme.
  • a change in the optical parameter by any measurable amount between activity in the presence of the test compound as compared with the activity in the absence of the test compound, is indicative of activity.
  • the ability of a compound to alter the activity of a particular protein (i.e., a sensor polypeptide) in vivo is determined.
  • a particular protein i.e., a sensor polypeptide
  • in vivo assay cells transfected with a expression vector encoding a substrate of the invention are exposed to different amounts of the test compound, and the effect on the optical parameter, such as fluorescence, in each cell can be determined.
  • the difference is calibrated against standard measurements to yield an absolute amount of protein activity.
  • This provides a method for screening for compounds which affect cellular events (e.g., receptor-ligand binding, protein-protein interactions or protein kinase activation). In a given cell type, any measurable change between activity in the presence of the test compound as compared with the activity in the absence of the test compound, is indicative of activity.
  • kits for determining the presence of an activity in a sample may contain a container containing a chimeric protein comprising an optically active polypeptide having operatively inserted therein a sensor polypeptide, or fragment thereof, which is affected by a change in a parameter or the env ironment, wherein optical properties of the sensor are altered in response to the change
  • a kit of the invention contains an isolated nucleic acid sequence which encodes a chimeric protein comprising an optically active polypeptide having operatively insereted therein a sensor polypeptide, or fragment thereof, which is affected by a change in a parameter or the environment, wherein optical properties of the sensor are altered in response to the change
  • the nucleic acid sequence of the later kit may be contained in a host cell, preferably stably transfected The cell could optionally be transiently transfected Thus, the cell acts as an indicator kit in itself
  • the present invention relates to transgenic animals that have cells that express an optically active polypeptide having operatively inserted therein a sensor polypeptide, or fragment thereof, which normally is capable of existing in two or more states, and which causes a change in the optical properties of the optically active polypeptide upon environmental conditions or parameters
  • Transgenic animals expressing high levels of the tagged transgene may be obtained, for example, by over-expression of the transgene with an enhanced promoter and/or with high copy numbers of the transgene
  • the transgenic animal may be heterozygous or homozygous for an ablated or disrupted endogenous indicator gene
  • non-human animals comprise any non-human animal having nucleic acid sequence which encodes a fluorescent indicator or circularly permuted fluorescent protein of the invention
  • non-human animals include vertebrates such as rodents, non-human primates, sheep, dog, cow, pig, amphibians, reptiles and fish
  • Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse
  • the "transgenic non-human animals” of the invention are produced by introducing "transgenes" into the germhne of the non-human animal Embryonal target cells at various developmental stages can be used to introduce transgenes Different methods are used depending on the stage of development of the embryonal target cell
  • the zygote is the best target for micro- mjection In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 pi of DNA solution
  • the use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will
  • transgenic is used to describe an animal which includes exogenous genetic material within all of its cells
  • a "transgenic” animal can be produced by cross-breeding two chimeric animals which include exogenous genetic material within cells used in reproduction Twenty-five percent of the resulting offspring will be transgenic / e , animals which include the exogenous genetic material within all of their cells in both alleles 50% of the resulting animals will include the exogenous genetic material within one allele and 25% will include no exogenous genetic mate ⁇ al
  • Viral infection can also be used to introduce transgene into a non-human animal (e g , retroviral, adenoviral or any other RNA or DNA viral vectors)
  • a non-human animal e g , retroviral, adenoviral or any other RNA or DNA viral vectors
  • the developing non-human embryo can be cultured in vitro to the blastocyst stage Dunng this time, the blastomeres can be targets for retro viral infection (Jaenich, R , Proc Natl Acad Sci USA 73 1260-1264, 1976) Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan, et al (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Sp ⁇ ng Harbor, N Y )
  • the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner, et al ,
  • ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J Evans et al Nature 292" 154- 156, 1981 , M O Bradley et al.,
  • Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. (For review see Jaenisch, R., Science 240 1468-1474, 1988).
  • Transformed means a cell into which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a heterologous nucleic acid molecule.
  • Heterologous refers to a nucleic acid sequence that either originates from another species or is modified from either its original form or the form primarily expressed in the cell
  • Transgene means any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism (i e , either stably integrated or as a stable extrachromosomal element) which develops from that cell
  • a transgene may include a gene which is partly or entirely heterologous (i e , foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism Included within this definition is a transgene created by the providing of an RNA sequence which is transcribed into DNA and then inco ⁇ orated into the genome.
  • transgenes of the invention include DNA sequences which encode the fluorescent indicator or circularly permuted fluorescent protein of the invention which may be expressed in a transgenic non-human animal
  • transgenic as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout
  • gene knockout refers to the targeted disruption of a gene in vivo with complete loss of function that has been achieved by any transgenic technology familiar to those in the art.
  • transgenic animals having gene knockouts are those in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination
  • transgenic includes any transgenic technology familiar to those in the art which can produce an organism carrying an introduced transgene or one in which an endogenous gene has been rendered non-functional or "knocked out"
  • Sites in, for example, GFP mutants which can tolerate insertions of sensor polypeptides can be identified by generating mutant proteins by manipulating the DNA sequence such that a variety of different insertions are produced and screening the mutants by flow cytometry for mutants which retain fluorescence.
  • Such insertions may include replacement of certain amino acids, as well as the addition of a new sequence without a corresponding deletion or replacement in the sequence of the fluorescent protein. Variants identified in this fashion reveal sites which can tolerate insertions while retaining fluorescence.
  • circularly permutation techniques are also useful in identifying sites in fluorescent proteins which are capable of tolerating insertions while retain the ability to fluoresce
  • Such techniques include are exemplified herein as well as known to those of skill in the art (see, for example, Graf et al , Proc Natl. Acad. Sci USA, 93: 1 1591-1 1596 (October 1996), the disclosure of which is inco ⁇ orated herein)
  • the original N- and C-terminal amino acids of a fluorescent protein are engineered to be linked by a linker moiety
  • linker moieties include those described above, as well as other easily ascertain by one skilled in the art
  • This is typically performed at the nucleic acid level resulting in a polynucleotide sequence wherein the 5' codon encoding the N-terminal ammo acid is linked to the 3' codon encoding the C-terminal amino acid, resulting in a circularized fluorescent protein nucleic acid sequence
  • the circularized sequence is then cleaved with a nuclease to create a linear polynucleotide sequence, the cleavage site corresponding to an ammo acid in of the fluorescent protein
  • the cleavage of the circularized sequence is either random or specific depending on the desired product, nuclease, and desired sequence
  • the linearized polynucleotide which contains sequence homologous to the starting fluorescent protein sequence, is cloned into an expression vector and
  • polypeptide sequence is then screened, for example by flow cytometry, for polypeptides retaining the ability to fluoresce. Accordingly, polypeptides which retain the ability to fluorescence correspondingly, via identification of the cleavage site, identify amino acids which can tolerate insertions without destroying the ability of the fluorescent protein to fluoresce
  • Bacteria used in this study were BL21(DE3) Gold cells from Stratagene Transformation was performed by electroporating cells suspended in 10%) glycerol directly with a ligation mixture (0 1cm cuvette, 12 5 kV/cm, 200 ⁇ , 25uFd).
  • Yellow GFP mutants with peptide insertions replacing Y145 were made by performing two separate polymerase chain reactions (PCRs)
  • the first PCR amplified the N terminal piece of YFP to include a 5' BamHl site and 3' replacement of Y 145 with the hexapeptide linker GGTGEL (coded for by DNA containing Kpnl and Sacl restriction sites for subsequent cloning).
  • the second PCR amplified the C terminal piece of YFP to include the 5' linker (GGTGEL) replacing Y145 and a 3' EcoRI site.
  • Protein pH titrations were carried out in 125mM KCl, 20mM NaCl, 0.5mM CaCl 2 , 0.5mM MgCl 2 , and 50mM buffer. Buffers were chosen to span a wide pH range, and included citrate (pH4-5), MES (pH 5.5-6.5), HEPES (pH 7-8..5), glycine (pH 8.8-10.7), and phosphate (pHl 1.3-13.2).
  • Zinc titrations of YFP-zinc finger insertions were done in 50mM MOPS, pH 7.0. A fluorescence emission spectrum was taken of the protein in buffer containing 50uM EDTA, and then small aliquots of ZnCl were added, and subsequent spectra were recorded. ( Figure 4).
  • Titration curves were generated by averaging the three intensity values for each pH (for microplate data) or be integrating the total emission intensity (for full spectra), plotting these data versus analyte concentration, and fitting a sigmoidal curve to the data.
  • YFP containing calmodulin replacing Y145 show an pH-sensitive increase in fluorescence intensity on calcium binding, with an apparent Kd of fluorescence of 7 ⁇ M.
  • the increase in fluorescence observed at constant pH reflects a shift of 1 pKa unit between the calcium-free and calcium-bound states of the protein, as observed in pH titrations done in the presence of or absence of free calcium ( Figure 2).
  • the absorbance of this protein changes from a predominantly ultraviolet, non-fluorescent band to a predominantly blue, fluorescent band on calcium binding at constant pH.
  • YFPs containing a zinc-finger motif dem ed from z ⁇ f268 also increase in fluorescence on binding zinc
  • the change in fluorescence for Zn-sensmg YFPs is substantially less than that of Calmoduhn-YFPs described above
  • Zn finger motifs contain cysteine residues in close proximity, they can be prone to oxidation, which would prevent zinc binding
  • the change of inserted proteins on substrate binding is responsible for the change in pKa and therefore the change in fluorescence
  • zinc-finger motifs adopt a non-optimal conformation on binding zinc, leaving the chromophore still vulnerable to protonation
  • each of the three reasons listed above concerning the performance of YFP-Calmoduhn Insertions could in principle also apply analogously to the zinc finger insertions
  • the zmc-finger inserted YFP reported here is just a first generation prototype of what will likely become a powerful new class of indicators
  • Circular Permutations of GFP mutants with a new N terminus at Y 145 were made by performing two separate PCRs
  • the first PCR amplified the C terminal piece of a GFP mutant (N terminal in the final permuted gene) to have a 5' BamHl site, the mutation Y145M, and a 3' hexapeptide linker (GGTGGS) containing a Kpnl site
  • the second PCR amplified the N terminal piece of the GFP mutant (C terminal in the final permuted gene) to have a 5' hexapeptide linker (GGTGGS) containing a Kpnl site, and a 3' EcoRI site
  • the first PCR product was digested with sequentially with BamHl and Kpnl
  • the second PCR product was digested sequentially with EcoRI and Kpnl
  • the fragments were purified by agarose gel electrophoresis
  • the N and C terminal PCR fragments were then clon
  • a plasmid consisting of the YC3J cDNA cloned into the BamHl and EcoRI sites of pRSETB was digested with BamHl and Sacl, and the 4.2 kb DNA fragment (YC3J X CFP) was agarose gel purified.
  • the circularly permuted CFP cDNA was then ligated to the YC3J ⁇ CFP fragment, and the DNA was transformed into BL21(DE3) Gold cells as desc ⁇ bed above.
  • Random Circular Permutations were based on the method of Graf, et al with major modifications because their original conditions were found to be unsuccessful when applied to GFP cDNAs. Through extensive testing, it was found that circular permutation required 1) reducing the concentration of DNA used when circularizing the gene from 300 ⁇ g/mL to 5 ⁇ g/mL, 2) increasing the amount of DNAse used to relinearize the fragment from 1 U/mg DNA to 100 U/mg DNA, 3) changing the temperature of DNAse incubation from 16 degrees to 22 degrees Celsius, and switching the DNA repair enzyme used from T4 to T7 DNA polymerase. Accordingly, the method of Graf et al , was substantially modified as follows.
  • An expression vector for the random circular permutations was made by ligating an oligonucleotide containing a 5' EcoRV site and three downstream stop codons in each reading frame between the BamHl and EcoRI sites of pRSET B.
  • This vector (“pRSET triple stop”) was digested with EcoRV, treated with Alkaline Phosphatase, and purified by agarose gel electrophoresis
  • the circularly permuted GFP gene described above was amplified by PCR with primers that created a final PCR amp con starting and ending at an Xhol site (ctcgag) coding for residues L141 and E142.
  • the PCR product was digested with Xhol and cloned into the Xhol site of pBluesc ⁇ pt.
  • This plasmid was amplified in bacteria, purified with a Qiagen maxi- prep, digested with Xhol , and the ⁇ 730bp fragment was agarose gel-purified to yield a linearized GFP gene.
  • the linear fragment was circularized at a concentration of 5 ⁇ g/mL with 8000 U/mL T4 DNA gase (New England Biolabs) overnight at 16°C.
  • T4 DNA gase New England Biolabs
  • the DNA was digested with DNAse (100 U/mg DNA, Pharmacia) for 15 minutes at room temperature in 50mM T ⁇ s HC1, pH7 5 and ImM MnC12 Digestion was stopped by phenol extraction, then subsequent phenol/chloroform/Isoamyl Alcohol and Chloroform/Isoamyl alcohol extractions
  • the DNA was ethanol precipitated, resuspended in lx synthesis buffer (Stratagene) and incubated with T7 DNA polymerase (Stratagene) and T4 DNA ligase (Stratagene) at room temperature for 1 hour to repair DNA nicks and fill sticky ends ( Figure 6)
  • LB/agar plates containing ampicillin usually displayed a few thousand colonies per plate and were screened by digital imaging of fluorescence
  • the plates were illuminated with a 150 W xenon arc lamp through a 450-490 nm bandpass filter and a pair of fiber optic light guides (Oriel Instruments) positioned to illuminate the top surface of the agar as evenly as possible
  • the emitted fluorescence was selected by a 510-550 nm bandpass filter and focused by a f/1 2 camera lens (Nikon) onto a cooled charge-coupled-device camera (Photometries)
  • Digital images from the camera were analyzed with Metafluor software (Universal Imaging Co ⁇ ) Out of approximately 25,000 bacterial colonies screened, about 200 became fluorescent after 24 hours at 4°C, and 144 of these were picked for plasmid minipreps and restriction analysis All plasmid minipreps were digested with Hmdlll and Kpnl to analyze the site of permuted termini (Hi
  • GFP forms a fluorescent circularly permuted protein when its native N and C termini are connected with the hexapeptide linker GGTGGS and new N termini are formed at E142, Y143, Y145, H148, D155, H169, E172, D173, A227, or 1229 (See Table 22).
  • the permuted protein with the N terminus at Y 145 was made and studied for the Cyan, Green, and Yellow mutants of GFP (cpCFP, cpGFP, cpYFP). In each case, the protein had a higher pKa of fluorescence than its native counte ⁇ art, although the fluorescence spectra were similar. This suggests that interrupting GFP and its mutants at Y 145 generally increases the chromophore's sensitivity to protonation, which is in agreement with the results obtained from the GFP insertions described above
  • *Start ⁇ ng Amino Acid is the first amino acid for which is not coded for by the expression vector (which may also have been mutated), e g E142M means that the GFP starts at Position 142, but the glutamate residue has been changed to methionine by the cloning process.
  • cpGFP mutants Another possible use of cpGFP mutants is in making novel insertions of GFP into other proteins for use as biosensors GFP, because its termini are close in space, can be inserted into other proteins, but only rarely to date has it been shown to sense a conformational change in such a construct
  • cpGFP mutants When cpGFP mutants are inserted into a protein, they are topologically similar to the GFP insertion constructs described above, and they might reasonably be expected to have similar sensing properties as GFP insertions (Figure 7)
  • GFP mutants with peptide insertions replacing Y145 were made by performing two separate polymerase chain reactions (PCRs)
  • the first PCR amplified the N-terminal piece of GFP to include a 5' BAMH1 site and 3' replacement of Y145 wit the hexapeptide linker GGTGEL (coded for by DNA containing Kpnl and Sacl restriction sites for subsequent cloning)
  • the second PCR amplified the C-terminal piece of GFP to include the 5' linker (GGTGEL) replacing Y145 and a 3' EcoRI site
  • GGTGEL 5' linker replacing Y145 and a 3' EcoRI site
  • Any sensor polypeptide can be inserted into a fluorescence protein (e g , GFP, YFP, or CFP) by analogy to the method described above and put in a cell by introducing the cDNA coding for the protein into the cell in a vector that directs protein production The indicator is then visualized using a fluorescence detector

Abstract

The present invention provides polypeptide and polynucleotides encoding fluorescent indicators having inserted within a fluorescent moiety a sensor polypeptide. Also provided are methods of using the fluorescent indicator. Circularly permuted fluorescent polypeptides and polynucleotides are also provided.

Description

FLUORESCENT PROTEIN INDICATORS
This invention was made with Government support under Grant No NS27177, awarded by the National Institutes of Health The Government has certain rights in this invention
TECHNICAL FIELD OF THE INVENTION
The invention relates generally to fluorescent proteins and more particularly to compositions and methods for measuring the response of a sensor polypeptide to an environmental (e g , biological, chemical, electrical or physiological) parameter
BACKGROUND
Fluorescent Ca'+ indicators such as fura-2, ιndo-1 , fluo-3, and Calcium-Green have been the mainstay of intracellular Ca"+ measurement and imaging (see, for example, U S Pat No 4,603,209 and U S Pat No 5,049,673) These relatively low molecular weight indicators can suffer from many technical problems relating to ester loading, leakage of the dyes from the cell, compartmentation in organelles, and perturbation of the indicators by cellular constituents Although
Figure imgf000002_0001
photoprotein aequoπn is targetable, the photoresponse to Ca~+ is low since it is chemiluminescent Moreover, aequoπns need to incorporate exogenous coelenterazine
Many effects of Ca2+ in cells are mediated by Ca2+ binding to calmoduhn (CaM), which causes CaM to bind and activate target proteins or peptide sequences Based on the NMR structure of CaM bound to the 26-residue M 13 Ca2+-bιndιng peptide of myosin light-chain kinase, Porumb et al fused the C-terminus of CaM via a Gly-Gly spacer to M13 Ca2^ binding switches the resulting hybrid protein (CaM- M13) from a dumbbell-like extended form to a compact globular form similar to the CaM-M13 lntermolecular complex (see, Porumb, T , et al , Prot Engineering 7 109- 115 (1994))
Measurement of a binding member concentration in vitro or in vivo by non- mvasive techniques can help elucidate the physiological function of the binding WO 00/71565 PCT/USOO/l 3684 member This can also aid in identifying changes that occur in a cell or organism in response to physiological stimuli For example, cyclic AMP can be detected by fluorescence resonance energy transfer between separately labeled proteins that associate with each other but are not covalently attached to each other See, U S Pat No 5,439,797
The Aequorea
Figure imgf000003_0001
Green Fluorescent Protein (GFP) is useful as a marker for gene expression, as a fluorescent tag to aid in visualizing protein trafficking, and as a component of indicator systems that allow fluorescent sensing of small molecules and pH Currently, GFPs use as a biosensor is limited to those systems that use GFP fusion proteins as partners for fluorescence resonance energy transfer (FRET) or those that use the subcellular redistribution of GFP fusion proteins as indicators of substrate concentration or the measurement of pH
Currently, fluorescent molecules designed to measure interactions of proteins rely on cameleon molecules of tandem GFP constructs In these constructs, conformational changes occur and alter the FRET between the GFPs and a ratiometπc color change is noted Such cameleon or FRET-sensitive constructs are large molecules in which protein conformation influences FRET efficiency of two GFPs of different colors Although insertions into Green Fluorescent Protein have been attempted (see Abedi et al , Nucleic Acids Research, 26(2) 623-630 (1998)) such insertions have been made to optimize the presentation of short peptide libraries and not to present binding molecules or sensor polypeptides Additionally, such insertions have been only short insertions of about six ammo acids in length Until now, however, it has not been possible to make a single GFP molecules' fluorescence sensitive to a substrate other than hydrogen ions There currently is a desire for smaller constructs useful in measuring interactions of molecules in vitro and in vivo
SUMMARY OF THE INVENTION The inventors have discovered that when a sensor polypeptide is inserted into an Aequorea-r ated fluorescent protein (e g , Green Fluorescent Protein (GFP), Yellow Fluorescent Protein (YFP) or Cyan Fluorescent Protein (CFP)) to form a construct, interaction of the sensor polypeptide with a biological, chemical, electrical or physiological parameter, for example, results in a change in fluorescence of the fluorescent protein. Such constructs are useful in measuring interactions of a sensor polypeptides with environmental stimuli in vitro or in vivo or in measuring particular characteristics of a cell (e g , redox potential, intracellular ion concentration). These constructs rely on the responsiveness of a sensor polypeptide inserted within a GFP- sensor-related protein itself to influence the actual fluorescence of the fluorophore and not the interaction of tandem fluorescent molecules
Accordingly, the present invention provides an isolated nucleic acid sequence which encodes a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide. The fluorescent protein moiety can be any fluorescent protein, for example, an Aequorea-r ated fluorescent protein moiety. More specifically, the Aeqiωreα-related fluorescent protein can be, for example, a GFP, CFP or YFP moiety The sensor polypeptide may be any polypeptide moiety, for example, a moiety that undergoes a conformational change upon interaction with a molecule, oxidation-reduction, or changes in electrical or chemical potential. The indicator may further include a linker moiety, linking the N- and C-terminal amino acids of the sensor polypeptide to the fluorescent protein The linker may be any moiety that provides for linking of the sensor polypeptide to the fluorescent protein moiety such as for example, a nucleic acid that encodes GGTGEL (SEQ ID NOJ) or FKTRHN (SEQ ID NO.2). Two or more linker moieties may be attached to two separate polypeptides, that together form a sensor polypeptide. Additionally, the indicator may have a localization sequence, for localizing the indicator, for example, to a particular organelle of a cell. The sensor polypeptide or linker moiety may be inserted at numerous sites including, for example, one or more amino acids between residues 128 - 148, residues 155-160, residues 168-176 or residues 227-229 of the fluorescent protein moiety (e g , GFP) More particularly Y145 is used for insertion of the linker or sensor polypeptide. In another embodiment, the present invention provides a transgenic non- human animal having a nucleic acid sequence which encodes a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
In yet another embodiment, the present invention provides an expression vector having expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator The indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
In another embodiment, the present invention provides a host cell transfected with an expression vector having an expression control sequence operatively linked to a sequence coding for the expression of a fluorescent indicator The host cell can be any host cell capable of transfection and expression of the indicator such as, for example, a prokaryote (e g , E Coli), a eukaryotic cell (e g , a yeast cell) or a mammalian cell
In yet a further embodiment, the present invention provides a fluorescent indicator polypeptide, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
In another embodiment, the present invention provides a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive WO 00/71565 PCT/USOO/l 3684 to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide the responsiveness resulting in protonation or deprotonation of the chromophore of the fluorescent protein moiety
In yet another embodiment, the present invention provides a method for detecting the presence of a environmental parameter in a sample, by contacting the sample with a fluorescent indicator or chimeric construct, the indicator having a sensor polypeptide which is responsive to a chemical, biological, electrical, or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide, and detecting a change in fluorescence wherein a change is indicative of the presence of a parameter which affects the sensor polypeptide.
In another embodiment, the invention provides an isolated nucleic acid sequence encoding a circularly peπnuted fluorescent protein and the polypeptide encoded thereby, having a linker moiety linking the amino-terminal and carboxy- terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-termmal and carboxy-terminal amino acids of the fluorescent protein.
In another embodiment, the invention provides an expression vector comprising expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator, the indicator having a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein WO 00/71565 PCT/USOO/l 3684 the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy- terminal amino acids of the fluorescent protein. In a further embodiment, the invention provides a host cell containing the expression vector.
In yet another embodiment, the invention provides a method of producing a nucleic acid sequence encoding a fluorescent indicator, by linking a nucleic acid sequence encoding a linker moiety to the 5' nucleotide of a polynucleotide encoding a fluorescent protein, circularizing the polynucleotide with the nucleic acid sequence encoding the linker sequence, and cleaving the circularized polynucleotide with a nuclease, wherein cleavage linearizes the circularized polynucleotide.
In yet another embodiment, the invention provides a method of producing a circularly permuted fluorescent protein by expressing a nucleic acid sequence encoding a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
DESCRIPTION OF THE DRAWINGS FIG. 1 shows the designs of a calmodulin or a Zif268 insertion into a fluorescent indicator of the present invention.
FIG. 2A shows pH effects on a calmodulin insertion indicator. FIG 2B shows titration curves for a calmodulin insertion indicator
FIG 3 further shows the effects of calcium concentration on fluorescence of a fluorescent indicator of the invention containing a sensor polypeptide of calmodulin
FIG 4 show the effect of Zn concentration on fluorescence of a fluorescent indicator of the invention containing a sensor polypeptide of inserted Zif polypeptide
FIG 5 shows the overall design of a circularly permuted polypeptide
FIG 6 shows the result of random digestion of a circularly permuted nucleic acid sequences
FIG 7 shows insertions into GFP
FIG 8 shows a schematic diagram of potential insertional sites and sites for circular permutations in GFP
DETAILED DESCRIPTION OF THE INVENTION The present invention relates to polynucleotides encoding fluorescent indicators, fluorescent indicators having a sensor polypeptide, and methods of producing and using the same
The inventors have discovered a number of sites in Aequot eα-related fluorescent protein moieties that aie tolerant to insertions and rearrangements The inventors have additionally discovered that the insertion of sensory polypeptides into such sites results in polypeptides that are useful in detection of chemical, biological, electrical or physiological parameters, for example Such insertion sites include, for example, one or more amino acids between residues 128 - 148, residues 155-160, residues 168-176 or residues 227-229 of the fluorescent protein moiety Other positions which may tolerate insertions include, but are not limited to, residues 49-50, 78-79, 1 16-1 17, 134-135, 140-141 , 157-158, 172-173, 194-195, 189-190 and 213-214 (see Abedi et al , Nucleic Acids Research, 26(2) 623-630 (1998), the disclosure of which is incorporated herein) More specifically, the insertion is a Y145
Additionally, the inventors have discovered that w hen a sensor polypeptide is inserted into an Aequoi eα-related fluorescent protein (e g , GFP, YFP or CFP) that provides a response related to an interaction with a biological, chemical, electrical or physiological parameter, the responsiveness results in a change in fluorescence of the fluorescent protein Such constructs are useful in measuring interactions of a sensor polypeptides with environmental stimuli in vitro or in vι\o These new constructs rely on, for example, detectable changes within a GFP-sensor-related protein itself to influence the actual fluorescence of the fluorophoie and not the interaction of tandem fluorescent molecules For example, when calmodulin is inserted into YFP at position Y145, interaction of calmodulin with its ligand (e g , calcium) results in a change in the brightness of the fluorescent protein of between 2-8 fold
The indicators of the present invention, are advantageous due to their reduced size as compared to the FRET-based sensors descπbed above The reduced size has importance in allowing the indicator to measure chemical, biological, electrical or physiological interactions with the sensor polypeptide in, for example, subcellular compartments previously inaccessible to the larger, FRET-based sensors In addition, the maximal change in fluorescence intensity observed in the present indicators (e g , up to 8 fold increase) are much larger than those in the cameleons (e g , FRET-based sensors), which show only a 2 fold change in yellow to cyan intensity ratio
Accordingly, the invention provides polynucleotides and nucleic acid sequences encoding fluorescent indicators having a fluorescent protein moiety and a sensor polypeptide, or fragments thereof, inserted in operable association into the fluorescent protein moiety, in which the sensor polypeptide is responsive to an environmental parameter (e g , a chemical, a biological, a electrical, or a physiological parameter) Accordingly, the responsiveness of the sensor polypeptide causes a change in fluorescence of the fluorescent indicator The degree of change in the fluorescence of the indicator is sensitive to pH As used herein, "operatively inserted" or "operably inserted" is meant between two amino acids of a polypeptide or two nucleotides of a nucleic acid sequence Accordingly, insertion excludes ligating or attaching a polypeptide to the last terminal amino acid or nucleotide in a sequence
As used herein, a "detectable change" or "responsiveness" means any response of a polypeptide to a chemical, biological, electrical, or physiological parameter or stimuli A response includes small changes, for example, a shift in the orientation of an amino acid or peptide fragment of the sensor polypeptide as well as, for example, a change in the primary, secondary, or tertiary structure of a polypeptide, including for example, changes in protonation, electrical and chemical potential and or coniormation "Conformation" is the three-dimensional arrangement of the primary, secondary and tertiary structure of a molecule including side groups in the molecule, a change in conformation occurs when the three-dimensional structure of a molecule changes Examples of conformational changes include a shift from -helix to a -sheet or a shift from -sheet to a -helix It is understood that detectable changes need not be a conformational change, so long as the fluorescence of the fluorescent protein moiety is altered
"Fragments" as used herein are a portion of a naturally occurring sensor protein which can exist in at least two different states or conformations Fragments can have the same or substantially the same amino acid sequence as the naturally occurring protein "Substantially the same" means that an amino acid sequence is largely, but not entirely, the same, but retains a functional activity of the sequence to which it is related In general two amino acid sequences are substantially the same" or "substantially homologous" if they are at least 85% identical Fragments which have different three dimensional structures as the naturally occurring protein are also included The term "responsive" as used herein is intended to encompass any response of a polypeptide which is related to an interaction of a chemical, biological, electrical, or physiological parameter with a sensor polypeptide (e g , conformational change in a voltage-gated ion channel (e g , Shaker) in detection of membrane voltage across a biological membrane, phosphorylation of a hormone receptor resulting in a conformational change in the receptor upon hormone stimulation) WO 00/71565 PCT/USOO/l 3684
Example of sensor polypeptide useful in the present invention include calmodulin, a calmoduhn-related protein moiety, recoveπn, a nucleoside diphosphate or tπphosphate binding protein, an ιnosιtol-l,4,5-tπphosphate receptor, a cyclic nucleotide receptor, a nitric oxide receptor, a growth factor receptor, a hormone receptor, a ligand-binding domain of a hormone receptor, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, a cytokine receptor, a growth factor receptor, a neurotransmitter receptor, a hgand-gated channel, a voltage-gated channel, a protein kinase C, a domain of protein kinase C. a cGMP-dependent protein kinase, an inositol polyphosphate receptor, a phosphate receptor, a carbohydrate receptor, an SH2 domain, an SH3 domain, a PTB domain, an antibody, an antigen- binding site from an antibody, a single-chain antibody, a zinc-finger domain, a protein kinase substrate, a protease substrate, a phosphorylation domain, a redox sensitive loop, a loop containing at least two cysteines that can form a cyclic disulfide, and a fluorescent protein moiety
Where the fluorescent protein contains a second fluorescent protein and a sensor moiety within the insert, utilization of FRET based techniques to analyze or detect changes in chemical, biological or electrical parameters may be performed For example, binding of an analyte such as calcium to a sensor polypeptide such as calmodulin would change the distance or angular orientation of the two fluorescent protein chromophores relative to each other and thereby modulate FRET. A circularly permuted fluorescent protein may be tandemly or msertionally fused via a sensor moiety to a second fluorescent protein (which itself may optionally be a circular permutation) so that the FRET between the two fluorescent proteins changes in response to chemical, biological, or electrical parameters
Classes of sensor polypeptides, which may be used in the compositions and methods of the invention include, but are not limited to, channel proteins, receptors, enzymes, and G-proteins
Channel polypeptides useful with the invention include, but are not limited to voltage-gated ion channels including the potassium, sodium, chloride, G-protem- responsive, and calcium channels A "channel polypeptide" is typically a WO 00/71565 PCT/USOO/l 3684 polypeptide embedded in the cell membrane which is part of a structure that determines what particle sizes and or charges are allowed to diffuse into the cell Channel polypeptides include the "voltage-gated ion channels", which are proteins imbedded in a cell membrane that serve as a crossing point for the regulated transfer of a specific ion or group of ions across the membrane Specifically, Shaker potassium channels or dihydropuπdine receptors from skeletal muscle may be advantageously used in the present invention Several ion channel polypeptides of use with the invention are listed in Table 1
TABLE 1 ION CHANNELS
Figure imgf000012_0001
Channels also include those activated by intracellular signals such as those where the signal is by binding of ligand such as calcium, cyclic nucleotides, G- proteins, phosphoinositols, arachidonic acid, for example, and those where the signal is by a covalent modification such as phosphorylation, enzymatic cleavage, oxidation/reduction, and acetylation, for example Channel proteins also include those activated by extracellular ligands (e g , lonotropic receptors) These can be activated by acetylcholine, biogenic amines, amino acids, and ATP, for example
A "receptor polypeptide" is a polypeptide found on a cell, often on a membrane, that can combine with a specific type of molecule, e g , a ligand, which alters a function of the cell Receptor polypeptides of use with the invention include, but are not limited to, the growth factor receptors, hormone receptors, cytokine WO 00/71565 PCT/USOO/l 3684 receptors, chemokine receptors, neurotransmitter receptors, hgand-gated channels, and steroid receptors. Specifically polypeptides encoding insulin-like growth factor, insulin, somatostatin, glucagon. interleukins, e g , IL-2, transforming growth factors (TGF-α , TGF-β ), platelet-dern ed growth factor (PDGF), epidermal growth factor (EGF), nerve growth factor (NGF), fibroblast growth factor (FGF), interferon-γ (IFN- γ), and GM-CSF receptors are of use with the invention Receptors such as those where binding of ligand is transmitted to a G-protein (e g , for 7-transmembrane receptors) or kinase domains (for single transmembrane receptors) can be used with the invention These can be activated by acetylcholine, biogenic amines, amino acids, ATP, and many peptides, such as opioids, hypothalamic-releasing hormones, neurohypophyseal hormones, pituitary hormones, tachykinins, secretms, insulins, somatostatms. and gastrointestinal peptides. Several receptor polypeptides of use with the invention are listed in Table 2
TABLE 2: RECEPTORS
Figure imgf000013_0001
An "enzyme" is a polypeptide that acts as a catalyst, which speeds the rate at which biochemical reactions proceed do not alter the direction or nature of the reaction. Enzyme polypeptides useful in the invention include, but are not limited to, protein kinases, catalyses, amidase, phosphatases, guanylyl and adenylyl cyclases, and poxygenases Polypeptides encoding the seπne/threonine protein kinases are of use with the invention. Several genes encoding human enzymes of use with the invention are listed in Table 3. TABLE 3 ENZYMES
Figure imgf000014_0001
The responsiveness of the sensor polypeptide (e g , a change in conformation or state) that occurs in response to interaction of a sensor polypeptide with a chemical, biological, electrical or physiological parameter will, as discovered by the inventors, cause a change in fluorescence of the fluorescence indicator The change can be the result of an alteration in the environment, structure, protonation or ohgomeπzation status of the fluorescent indicator or chromophore. The molecular component responsible for a conformational change is known for many enzymes (e g , Blostien, R., et al (1997) J. Biol. Chem., 272 24987-93; Shoelstein, Se E , et al , (1993) EMBO J., 12-795-802), receptors (e g Moyle, W R., et al , (1995) J Biol Chem., 270 20020- 20031 ; Baron, V., et al , (\992), J. Biol Chem. 267.23290-23294), and channels (e g, Bouzat. A., et al., (1994) Neuron, 13 1395-1402; Dulhanty, A.M., (1994) Biochemistry, 33 "4072-79) polypeptide The optical properties (e g , fluorescence) of the indicator which can be altered in response to the conformational change in the sensor polypeptide include, but are not limited to, changes in the excitation or emission spectrum, quantum yield, extinction coefficient, excited life-time and degree of self-quenching for example The cause of the changes in these parameters may include but are not limited to changes in the environment, changes in the rotational or vibrational freedom of the sensor, changes in the angle of the sensor with respect to the exciting light or the optical detector apparatus, changes in the protonation or deprotonation of amino acids or side groups associated with a chromophore or changes in distance or dipole orientation between sensors on associated responsive polypeptides
For example, insertion of a peptide or protein in place of tyrosine- 145 (Y145) in mutants of GFP increases the sensitivity of fluorescence to quenching by acidic pH WO 00/71565 PCT/USOO/l 3684
When the inserted sensor polypeptide responds to a chemical, biological, electrical, or physiological parameter and undergoes a detectable change (e g , a change in conformation), such interactions change the fluorescence via a shift in the acid sensitivity For example, when calmodulin, a calcium sensing protein, replaces residue Y145 in a yellow mutant of GFP, calcium binding increases the fluorescence by up to 8-fold, depending on the pH at which the measurement is made Other sites for insertion into GFP or GFP-mutants are allowable where circular permutation is tolerated, as discussed more fully below
In the fluorescent indicator proteins of the invention, the sensor polypeptide is operably inserted into an optically active polypeptide (e g , a fluorescent protein moiety) A protein-based "optically active polypeptide" is a polypeptide which contains a means for emitting light Fluorescence is one optical property of an optically active polypeptide which can be used as the means of detecting the responsiveness of the sensor or responsive polypeptide of the fluorescent indicator or circularly permuted fluorescent proteins of the invention As used herein, the term "fluorescent property" refers to the molar extinction coefficient at an appropriate excitation wavelength, the fluorescence quantum efficiency, the shape of the excitation spectrum or emission spectrum, the excitation wavelength maximum and emission wavelength maximum, the ratio of excitation amplitudes at two different wavelengths, the ratio of emission amplitudes at two different wavelengths, the excited state lifetime, or the fluorescence anisotropy A measurable difference in any one of these properties between the active and inactive states suffices for the utility of the fluorescent protein substrates of the invention in assays for activity A measurable difference can be determined by determining the amount of any quantitative fluorescent property, e g , the amount of fluorescence at a particular wavelength, or the integral of fluorescence over the emission spectrum Optimally, the protein substrates are selected to have fluorescent properties that are easily distinguishable in the un-activated and activated conformational states
One means of measuring fluorescence in a sample uses a fluoπmeter. In general, excitation radiation, from an excitation source having a first wavelength, passes through excitation optics The excitation optics cause the excitation radiation to excite the sample In response, fluorescent proteins in the sample emit radiation which has a wavelength that is different from the excitation wavelength Collection optics then collect the emission from the sample. The device can include a temperature controller to maintain the sample at a specific temperature while it is being scanned For example, a multi-axis translation stage moves a microtiter plate holding a plurality of samples in order to position different wells to be exposed The multi-axis translation stage, temperature controller, auto-focusing feature, and electronics associated with imaging and data collection can be managed by an appropriately programmed digital computer The computer also can transform the data collected during the assay into another format for presentation Other means of measuring fluorescence can also be used with the invention
Methods of performing assays on fluorescent materials are well known in the art and are described in, e g , Lakowicz, J R., Principles of Fluorescence Spectroscopy, New York'Plenum Press (1983): Herman, B., Resonance energy transfer microscopy, in" Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol 30, ed. Taylor, D L. & Wang, Y.-L , San Diego: Academic Press (1989), pp. 219-243, Turro, NJ , Modern Molecular Photochemistry, Menlo Park Benjamin/Cummings Publishing Col, Inc (1978), pp. 296-361.
Any fluorescent protein can be used in the invention, including proteins that fluoresce due to intramolecular rearrangements or the addition of cofactors that promote fluorescence For example, green fluorescent proteins of cnidaπans, which act as their energy-transfer acceptors in bioluminescence, are suitable fluorescent proteins for use in the fluorescent indicators. A green fluorescent protein ("GFP") is a protein that emits green light, a blue fluorescent protein ("BFP") is a protein that emits blue hght, a yellow fluorescent protein ("YFP") is one that emits yellow light, and a cyan fluorescent protein ("CFP") is one that emits a greenish-blue light. GFPs have been isolated from the Pacific Northwest jellyfish, Aequorea victoria, the sea pansy, Renilla remformis, and Phiahdium gregarium See, Ward, W W., et al ,
Photochem Photobiol., 35:803-808 (1982); and Levine, L D , et al , Comp. Biochem Physiol., 72B 77-85 (1982). A variety of Aequorea-related GFPs having useful excitation and emission spectra have been engineered b modifying the amino acid sequence of a naturally occurring GFP from Aequoi eα x ictoriα (See, Prasher, D C , et αl , Gene, 1 1 1 229- 233 (1992), Heim, R , et α/ , Proc Natl Acad Sci , USA, 91 12501-04 (1994), U S Patent No 5,491,084, 5,625,048, International application PCT/US95 14692, filed 1 1/10/95) The cDNA of GFP can be concatenated with those encoding many other proteins, the resulting chimeπcs often are fluorescent and retain the biochemical features of the partner proteins (See, Cubitt, A B , et αl , Trends Biochem Sci 20 448-455 (1995)) Mutagenesis studies have produced may GFP mutants, some having shifted wavelengths of excitation or emission (see, Heim, R & Tsien, R Y Current Biol 6 178-182 (1996)) Suitable pairs, for example a blue-shifted GFP mutant P4-3 (Y66H/Y145F) and an improved green mutant S65T can respectively serve as a donor and an acceptor for fluorescence resonance energy transfer (FRET) See, Tsien, R Y , et αl , Trends Cell Biol 3 242-245 (1993) Such proteins are included in the invention sensor A fluorescent piotein is an "Je -.oreα-related fluorescent protein" if any contiguous sequence of 150 amino acids of the fluorescent protein has at least 85% sequence identity with an amino acid sequence, either contiguous or non-contiguous, from the wild type Aequoreα green fluorescent protein More preferably, a fluorescent protein is an Aequoi eα-related fluorescent protein if any contiguous sequence of 200 ammo acids of the fluorescent protein has at least 95% sequence identity with an amino acid sequence, either contiguous or noncontiguous, from the wild type Aequoreα green fluorescent protein For example, where circularly permuted fluorescent proteins are concerned, any continuous sequence of the circularly permuted sequence which has identity to an Aequoreα- related fluorescent protein, as described above, whether further N- or C- terminal than the comparison sequence is considered related Similarly, the fluorescent protein can be related to Renilla or Phia dium wild-type fluorescent proteins using the same standards Some Aequoreα-r ated engineered versions descπbed in Table 4 Other variants or mutants are within the scope of the invention as descπbed, for example, in the Examples WO 00/71565 PCT USOO/l 3684
TABLE 4
Figure imgf000018_0001
An additional clone, WlB l included the following mutations" F64L;S65T; Y66W; F99S; and V163A.
Other fluorescent proteins can be used in the fluorescent indicators, such as, for example, yellow fluorescent protein from Vibrio fischeri strain Y-l , Peπdinin- chlorophyll a binding protein from the dmofiagellate Symbwdinium sp phycobihproteins from marine cyanobacteπa such as Synechococcus, e.g., phycoerythπn and phycocyamn, or oat phytochromes from oat reconstructed with phycoerythrobilm. These fluorescent proteins have been descπbed in Baldwin, T.O., et al, Biochemistry 29 5509-5515 (1990), Morris, BJ., et al , Plant Molecular Biology, 24-673-677 (1994), and Wilbanks, S.M., et al , j. Biol. Chem. 268: 1226- 1235 (1993), and Li et al , Biochemistry 34:7923-7930 (1995), Murphy, J.T., & Lagaπas, J.C., Current Biology 7: 870-876 (1997)
The fluorescent indicators can be produced as chimeric proteins by recombinant DNA technology. Recombinant production of fluorescent proteins involves expressing nucleic acids having sequences that encode the proteins. Nucleic acids encoding fluorescent proteins can be obtained by methods known in the art. For example, a nucleic acid encoding the protein can be isolated by polymerase chain reaction of cDNA from A victoria using pπmers based on the DNA sequence of J victoria green fluorescent protein PCR methods are described in, for example, U.S. Pat. No. 4,683,195; Mul s, et al Cold Spring Harbor Symp. Quant. Biol. 51 :263 (1987), and Erhch, ed., PCR Technology, (Stockton Press, NY, 1989). Mutant versions of fluorescent proteins can be made by site-specific mutagenesis of other nucleic acids encoding fluorescent proteins, or by random mutagenesis caused by increasing the error rate of PCR of the original polynucleotide with 0 1 mM MnCl2 and unbalanced nucleotide concentrations.
In the chimeric proteins of the invention, the sensor polypeptide is operably inserted to an optically active polypeptide, which responds (e.g , a conformation change)to, for example, a cell signaling event. Cell signaling events that occur in vivo can be of very short duration. The optically active polypeptides of the invention allow measurement of the optical parameter, such as fluorescence, which is altered in response to the cell signal, over the same time period that the event actually occurs. Alternatively, the response can be measured after the event occurs (over a longer time period) as the response that occurs in an optically active polypeptide can be of a longer duration than the cell signaling event itself. Nucleic Acid Constructs of the Invention
In another embodiment, the invention provides isolated nucleic acid sequences which encode fluorescent indicator polypeptides having operatively inserted therein a sensor polypeptide, or fragment thereof, which normally exists in one state e g , conformational shape or charge, prior to an interaction with a chemical, biological, electrical or physiological parameter at which time it undergoes a response during or after the interaction of the chemical, biological, electrical or physiological parameter with the sensor polypeptide
"Polynucleotide" or "nucleic acid sequence" refers to a polymeric form of nucleotides at least 10 bases in length By "isolated nucleic acid sequence" is meant a polynucleotide that is no longer immediately contiguous with both of the coding sequences with which it was immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryotic or eukaryotic cell or organism, or which exists as a separate molecule (e g a cDNA) independent of other sequences The nucleotides of the invention can be πbonucleotides, deoxyπbonucleotides, or modified forms of either nucleotide The term includes single and double stranded forms of DNA.
Nucleic acid sequences which encode a fluorescent indicator of the invention, wherein the indicator includes a sensor polypeptide, or fragment thereof, which normally has two or more states or conformational arrangements, and which undergoes a response during interaction with a chemical, biological, electrical or physiological parameter can be operatively linked to expression control sequences "Operatively linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. An expression control sequence operatively linked to a coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the expression control sequences As used herein, the term "expression control sequences" refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (. e , ATG) in front of a protein-encoding gene, splicing signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and stop codons The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and chimeric partner sequences Expression control sequences can include a promoter
By "promoter" is meant minimal sequence sufficient to direct transcription Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific, tissue- specific, or inducible by external signals or agents, such elements may be located in the 5' or 3' regions of the gene Both constitutive and inducible promoters, are included in the invention (see e g , Bitter et al , 1987, Methods in Enzymology 153 516-544) For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage , plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e g , metallothionein promoter) or fiom mammalian viruses (e g , the retrovirus long terminal repeat, the adenovirus late promoter, the vaccinia virus 7 5K promoter, CMV promoter) may be used Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention
The term "fluorescent protein," and "fluorescent protein moiety" are used interchangeably and refer to any protein capable of emitting light when excited with appropriate electromagnetic radiation, and which has an amino acid sequence that is either natural or engineered and is derived from the amino acid sequence of an
Aequoi eα-related fluorescent protein The term "fluorescent indicator" refers to a fluorescent protein having a sensor polypeptide whose emitted light varies with the response state or conformation of the sensor polypeptide upon interaction with a chemical, biological, electrical or physiological parameter The term also refers to a fluorescent protein whose ammo acid sequence has been circularly permuted The fluorescent indicators of the invention are also sensitive to pH in the range of about 5 to about 10 Thus, the invention provides, for example, a functional engineered fluorescent protein indicator whose amino acid sequence is substantially identical to the 238 amino acid Aequoi ea victona green fluorescence protein (SEQ ID NO 3)
The invention also includes functional polypeptide fragments of a fluorescent indicator As used herein, the term "functional polypeptide fragment" refers to a polypeptide which possesses biological function or activity which is identified through a defined functional assay The term "functional fragments of a functional engineered fluorescent protein" refers to fragments of a functional engineered protein that retain a function of the engineered fluorescent protein, e g , the ability to fluoresce in a manner which is dependent upon interactions of a chemical, biological, electrical or physiological parameter with a sensor polypeptide over the pH range 5 to 10
Minor modifications of the functional engineered fluorescent indicator may result in proteins which have substantially equivalent activity as compared to the unmodified counterpart polypeptide as described herein Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous All of the polypeptides produced by these modifications are included herein as long as fluorescence of the engineered protein still exists
By "substantially identical" or "substantially homologous" is meant a protein or polypeptide that retains the activity of a functional engineered fluorescent indicator, or nucleic acid sequence or polynucleotide encoding the same, and which exhibits at least 80%, preferably 85%, more preferably 90%, and most preferably 95%) homology to a reference amino acid or nucleic acid sequence For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 1 10 nucleotides The reference amino acid sequence or nucleic acid sequence is considered homologous if the reference ammo acid sequence is 80-95% homologous to any portion of the amino acid or nucleic acid sequence in question For example, where a circularly permuted polypeptide sequence has been generated, the sequence will typically have an amino acid sequence wherein a carboxy terminal sequence is now more amino terminal than the original fluorescent protein. In such instances, the circularly permuted sequence is considered homologous because the carboxy terminal sequence is still present in the circularly permuted fluorescent protein even though it is now more N-terminal.
By "substantially identical" is meant an amino acid sequence which differs only by conservative amino acid substitutions, for example, substitution of one ammo acid for another of the same class (e g , valine for glycine, arginine for lysine, etc.) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein ( e.g., assayed as described herein). Preferably, such a sequence is at least 85%, more preferably 90%, more preferably 95%, more preferably 98%, and most preferably 99%) identical at the amino acid sequence to one of the sequences of EGFP (SEQ ID NOJ), EYFP (SEQ ID NO:5), ECFP (SEQ ID NO 6), EYFP-V68L/Q69K (SEQ ID NO-7), YFP H148G (SEQ ID NO:8), or YFP H 148Q (SEQ ID NO"9). As discussed in the previous paragraph and more fully below, circularly permuted sequences fall within the definition of "substantially identical", for example if one or more amino acids of a circularly permuted polypeptide sequence is changed as described herein.
Homology is typically measured using sequence analysis software (e.g.,
Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, substitutions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; seπne, threonine; lysine, arginine; and phenylalanine, tyrosine. WO 00/71565 PCT/USOO/l 3684
In some embodiments, the amino acid sequence of the protein includes one of the following sets of substitutions in the ammo acid sequence of the Aequorea green fluorescent protein (SEQ ID NO 3) F64L/S65T/H231 L, referred to herein as EGFP (SEQ ID NO 4), S65G/S72A/T203Y/H231L, referred to herein as EYFP (SEQ ID NO 5), S65G/V68L/Q69K/S72A/T203Y/H231L, referred to herein as EYFP- V68L/Q69K (SEQ ID NO 7),
K26R/F64L/S65T/Y66W/N146I/M153T/V163A/NJ64H/H231L, referred to herein as ECFP (SEQ ID NO 6) The amino acid sequences of EGFP, EYFP, ECFP, and EYFP-V68L/Q69K are shown in Tables 5-8, respectively The numbering of the amino acids conforms to that in native Aequorea GFP Thus, the first serine is amino acid number 2 even if a valine (ammo acid no l a) has been inserted to optimize ribosome initiation Thus, F64L corresponds to a substitution of leucine for phenylalanine in the 64th ammo acid following the initiating methionine
Table 5. EGFP Amino Acid Sequence (SEQ ID NOJ)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG KLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFK-EDGNJLGHKLEYNYNSHNVYIMAD KQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALS KDPNEKRDHMVLLEFVTAAGITLGMDELYK
Table 6. EYFP Amino Acid Sequence (SEQ ID NO:5)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG KLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDF EDGNILGH LEYNY SHNVYIMA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSA LSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
Table 7. EYFPN68L/Q69K Amino Acid Sequence (SEQ ID ΝO:7)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG KLPVPWPTLVTTFGYGLKCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNY SHNVYIMAD KQKNGIKVNF IRH IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALS KDPNEKRDHMVLLEFVTAAGITLGMDELYK
Table 8. ECFP Amino Acid Sequence (SEQ ID NO:6) MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTLKFICTTG KLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITAD KQKNGIKAHFKIRH IEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALS KDPNEKRDHMVLLEFVTAAGITLGMDELYK
In other embodiments, the amino acid sequence of the protein is based on the sequence of the wild-type A equora green fluorescent protein, but includes the substitution H148G (SEQ ID NO:8) or H148Q (SEQ ID NO:9). In specific embodiments, these substitutions can be present along with other substitutions, e.g., the proteins can include the substitutions S65G/V68L/S72A/H 148G/Q80R/T203 Y (SEQ ID NO:8), which is referred to herein as the "YFP H148G mutant," S65G/V68L/S72A/H148Q/Q80R/T203Y, which is referred to herein as the "YFP H148Q mutant" (SEQ ID NO:9), as well as EYFP-H148G (SEQ ID NO: 10) and EFP-H148Q (SEQ ID NO: 1 1). The amino acid sequences of these mutants are shown in Tables 9-12, respectively. Table 9. Amino Acid Sequence of YFP H148G (SEQ ID NO:8)
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLV RIELKGIDFKEDGNILGHKLEYNYNSGNVYIMADK QKNGIKV FKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSK DPNEKRDHMVLLEFVTAAGITHGMDELYK
Table 10. Amino Acid Sequence of YFP H148Q (SEQ ID NO:9)
MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGK LPVPWPTLVTTFGYGLQCFARYPDHMKRHDFFKSAMPEGYVQERTIFFKDDG NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYIMADK QKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSK DPNEKRDHMVLLEFVTAAGITHGMDELYK
Table 1 1. Amino Acid Sequence of EYFP-H148G (SEQ ID NO: 10)
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG KLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSGNVYIMA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSA LSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
Table 12. Amino acid Sequence of EYFP-H148Q (SEQ ID NOJ 1) MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTG KLPVPWPTLVTTFGYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKD DGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSQNVYIMA DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSYQSA LSKDPNEKRDHMVLLEFVTAAGITLGMDELYK
In some embodiments, the protein or polypeptide is substantially purified. By "substantially pure protein or polypeptide" is meant an functional engineered fluorescent polypeptide which has been separated from components which naturally accompany it. Typically, the protein or polypeptide is substantially pure when it is at least 60%), by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%o, more preferably at least 90%, and most preferably at least 99%, by weight, of the protein. A substantially pure protein may be obtained, for example, by extraction from a natural source (e.g., a plant cell); by expression of a recombinant nucleic acid WO 00/71565 PCT/USOO/l 3684 encoding a functional engineered fluorescent protein; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., those described in column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.
A protein or polypeptide is substantially free of naturally associated components when it is separated from those contaminants which accompany it in its natural state. Thus, a protein or polypeptide which is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. Coli or other prokaryotes.
The invention also provides polynucleotides encoding the functional engineered fluorescent protein described herein. These polynucleotides include DNA, cDNA, and RNA sequences which encode functional engineered fluorescent proteins. It is understood that all polynucleotides encoding functional engineered fluorescent proteins are also included herein, as long as they encode a protein or polypeptide whose fluorescent emission intensity changes as pH varies between 5 and 10. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For example, the polynucleotide may be subjected to site-directed mutagenesis. The polynucleotides of the invention include sequences that are degenerate as a result of the genetic code. Therefore, all degenerate nucleotide sequences are included in the invention as long as the amino acid sequence of the functional engineered fluorescent protein or derivative is functionally unchanged.
Specifically disclosed herein is a polynucleotide sequence encoding a functional engineered fluorescent protein that includes one of the following sets of substitutions in the amino acid sequence of the Aequorea green fluorescent protein (SΕQ ID NOJ): S65G/S72A/T203Y /H231L, S65G/V68L/Q69K/S72 A/T203 Y/H231 L, or K26R/F64L/S65T/Y66W/N 1461/ M153T/V163A/N 164H/H231L. In specific embodiments, the DNA sequences encoding ΕGFP, ΕYFP, ΕCFP, ΕYFP-V68L/Q69K, YFP H 148G, and YFP H148Q are those shown in Table 13-20 (SEQ ID NOs: 12 to 19), respectively. The nucleic acid encoding functional engineered fluorescent proteins may be chosen to reflect the codon choice in the native A victoria coding sequence, or, alternatively, may be chosen to reflect the optimal codon frequencies used in the organism in which the proteins will be expressed Thus, nucleic acids encoding a target functional engineered protein to be expressed in a human cell may have use a codon choice that is optimized for mammals, or especially humans.
Table 13. EGFP Nucleic Acid Sequence (SEQ ID NOJ 2)
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCT ACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGAC TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACA ACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTC AAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTA CCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC ACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGG CATGGACGAGCTGTACAAGTAA
Table 14. EYFP Nucleic Acid Sequence (SEQ ID NO: 13) ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTA CGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACT TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTAC CAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGC ATGGACGAGCTGTACAAGTAA Table 15. ECFP Nucleic Acid Sequence (SEQ ID NO: 14)
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAGGTTCAGCGTGTCCGGCGAG GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCT GGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGAC TTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGTACCATCTTC TTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGG GCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAG GACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAA CGTCTATATCACCGCCGACAAGCAGAAGAACGGCATCAAGGCCCACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTAC CAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGC ATGGACGAGCTGTACAAGTAA
Table 16. EYFP-V68L/Q69K Nucleic Acid Sequence (SEQ ID NO: 15)
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT
CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG
GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTA CGGCCTGAAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACT TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAA CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTAC CAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGC ATGGACGAGCTGTACAAGTAA
Table 17. Nucleotide Sequence of the YFP H148G Coding Region (SEQ ID NOJ 6)
ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGA ATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTG AAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTG GAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTC TTCAATGCTTTGCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCA AGAGTGCCATGCCCGAAGGTTATGTTCAGGAAAGAACTATATTTTTCAAA GATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATAC CCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAA ACATTCTTGGACACAAATTGGAATACAACTATAACTCAGGCAATGTATAC ATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAG
ACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAA
ATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGT
CCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATG
GTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAA
CTATACAAA
Table 18. Nucleotide Sequence of the YFP H148Q Coding Region (SEQ ID NOJ7)
ATGAGTAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGA ATTAGATGGTGATGTTAATGGGCACAAATTTTCTGTCAGTGGAGAGGGTG AAGGTGATGCAACATACGGAAAACTTACCCTTAAATTTATTTGCACTACTG GAAAACTACCTGTTCCATGGCCAACACTTGTCACTACTTTCGGTTATGGTC TTCAATGCTTTGCAAGATACCCAGATCATATGAAACGGCATGACTTTTTCA AGAGTGCCATGCCCGAAGGTTATGTTCAGGAAAGAACTATATTTTTCAAA GATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATAC CCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAA ACATTCTTGGACACAAATTGGAATACAACTATAACTCAGGCAATGTATAC ATCATGGCAGACAAACAAAAGAATGGAATCAAAGTTAACTTCAAAATTAG ACACAACATTGAAGATGGAAGCGTTCAACTAGCAGACCATTATCAACAAA ATACTCCAATTGGCGATGGCCCTGTCCTTTTACCAGACAACCATTACCTGT CCTATCAATCTGCCCTTTCGAAAGATCCCAACGAAAAGAGAGACCACATG GTCCTTCTTGAGTTTGTAACAGCTGCTGGGATTACACATGGCATGGATGAA CTATACAAA
Table 19. Nucleotide Sequence of the EYFP-H148G Coding Region (SEQ ID
NOJ 8)
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTA CGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACT TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCGGCAA CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTAC CAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGC ATGGACGAGCTGTACAAGTAA Table 20 Nucleotide Sequence of the EYFP-H148Q Coding Region (SEQ ID
NO 19)
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAG GGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCAC CACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCTTCGGCTA CGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACT TCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCT TCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGG CGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCAGAA CGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTAC CAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA CTACCTGAGCTACCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCG ATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGC ATGGACGAGCTGTACAAGTAA The functional engineered fluorescent protein can also include a targeting sequence to direct the fluorescent protein to particular cellular sites by fusion to appropriate organellar targeting signals or localized host proteins A polynucleotide encoding a targeting sequence can be ligated to the 5' terminus of a polynucleotide encoding the fluorescence such that the targeting peptide is located at the amino terminal end of the resulting fusion polynucleotide/polypeptide The targeting sequence can be, e g , a signal peptide In the case of eukaryotes, the signal peptide is believed to function to transport the fusion polypeptide across the endoplasmic reticulum The secretory protein is then transported through the Golgi apparatus, into secretory vesicles and into the extracellular space or, preferably, the external environment Signal peptides which can be utilized according to the invention include pre-pro peptides which contain a proteolytic enzyme recognition site Other signal peptides with similar properties are known to those skilled in the art, or can be readily ascertained without undue experimentation
In the present invention, the nucleic acid sequences encoding the fluorescent indicator or circularly permuted fluorescent protein of the invention may be inserted into a recombinant expression vector The term "recombinant expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the nucleic acid sequences encoding the chimeric peptides of the invention The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al , Gene, 56 125, 1987), the pMSXND expression vector, or adeno or vaccinia viral vectors for expression in mammalian cells (Lee and Nathans, J Biol Chem., 263 3521 , 1988), baculovirus-deπved vectors for expression in insect cells, cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV
The nucleic acid sequences encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention can also include a localization sequence to direct the indicator to particular cellular sites by fusion to appropriate organellar targeting signals or localized host proteins A polynucleotide encoding a localization sequence, or signal sequence, can be ligated or fused at the 5' terminus of a polynucleotide encoding the fluorescence indicator such that the signal peptide is located at the amino terminal end of the resulting chimeric polynucleotide/polypeptide. In the case of eukaryotes, the signal peptide is believed to function to transport the chimeric polypeptide across the endoplasmic reticulum The secretory protein is then transported through the Golgi apparatus, into secretory vesicles and into the extracellular space or, preferably, the external environment. Signal peptides which can be utilized according to the invention include pre-pro peptides which contain a proteolytic enzyme recognition site Other signal peptides with similar properties to those described herein are known to those skilled in the art, or can be readily ascertained without undue experimentation
The localization sequence can be a nuclear localization sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, or a localized protein Localization sequences can be targeting sequences which are described, for example, in "Protein Targeting'". Chapter 35 of Stryer, L , Biochemistry (4th ed ), W H Freeman, 1995 The localization sequence can also be a localized protein Some important localization sequences include those targeting the nucleus (KKKRK) (SE Q ID NO:20), mitochondrion (amino terminal MLRTSSLFTRRVQPSLFRNILRLQST (SEQ ID NO"21)), endoplasmic reticulum (KDEL (SEQ ID NO.22) at C-terminus, assuming a signal sequence present at N-terminus), peroxisome (SKF at C-termmus), synapses (S/TDV or fusion to GAP 43, kinesin and tau) prenylation or insertion into plasma membrane (CaaX (SEQ ID NO.23), CC, CXC, or CCXX (SEQ ID NO:24) at C- terminus), cytoplasmic side of plasma membrane (chimeπc to SNAP-25), or the Golgi apparatus (chimeric to fuπn). The construction of expression vectors and the expression of genes in transfected cells involves the use of molecular cloning techniques also well known in the art. Sambrook et al , Molecular Cloning — A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, (1989) and Current Protocols in Molecular Biology, F M. Ausubel et al , eds., (Current
Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., most recent Supplement) These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. (See, for example, the techniques described in Mamatis, et al, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, NN., 1989).
Examples of agents which induce a sensor polypeptide, include agents that contain any of the amino acid sequences in Table 21 , or a portion thereof with the proviso that the parameter must bind to a calmodulin sensor polypeptide. The parameter can be a subsequence of a calmoduhn-binding domain The moieties listed in Table 21 are recognized by the sensor polypeptide CaM. See, for example, Cπvici, A. & Ikura, M. Annu. Rev. Biophys Biomol. Struct. 24 84-1 16 (1995). The parameter can be modified to enhance the response of the fluorescent indicator to the parameter. Other parameter are known in the art for other sensor polypeptides.
TABLE 21
Figure imgf000033_0001
WO 00/71565 PCT/USOO/l 3684
Figure imgf000034_0001
Abbreviations: AC, adenylyl cyclase; BBMHCI, brush-border myosin heavy chain-I; CaMKII, calmodulin kinase II; CBP2, calmodulin binding peptιde-2; GIP, gastrin inhibitory peptide; HIV-1 gpl60, human immunodeficiency virus envelope glycoprotein 160; HSP, heat-shock protein; MARCKS, myristoylated alaminte-πch C kinase substrate; MHC, myosin heavy chain; NOS, nitric oxide synthase; PDE, phosphodiesterase; PFK, phosphofructokinase; PhK, phosphorylase kinase; sk-, smMLCK, skeletal muscle- and smooth muscle-myosin light chain kinase; VIP, vasoactive intestinal peptide Where a linker moiety is present, the length of the linker moiety is chosen to optimize the kinetics and specificity of responsiveness of the sensor polypeptide induced by the interaction of the chemical, biological, electrical or physiological parameter with the sensor polypeptide The linker moiety should be long enough and flexible enough to allow the sensor polypeptide to freely interact and respond to a particular parameter The linker moiety is, preferably, a peptide moiety. The preferred linker moiety is a peptide between about one and 30 amino acid residues in length, preferably between about two and 15 amino acid residues One preferred linker moiety is a -Gly-Gly- linker. The linker moiety can include flexible spacer amino acid sequences, such as those known in single-chain antibody research For example, the linker moiety can be GGGGS (SEQ ID NO"56)(GGGGS)n, GKSSGSGSESKS (SEQ ID NO"57), GSTSGSGKSSEGKG (SEQ ID NO"58), GSTSGSGKSSEGSGSTKG (SEQ ID NO 59) GSTSGSGKSSEGKG (SEQ ID NO 60), GSTSGSGKPGSGEGSTKG (SEQ ID NO 61), EGKSSGSGSESKEF (SEQ ID NO:62), GGTGEL (SEQ ID NO" 1), FKTRHN (SEQ ID NO 2), or GGTGGS
(SEQ ID NO"63) Linking moieties are described, for example, in Huston, J.S., et al , PNAS 85:5879-5883 (1988), Whitlow, M., et al , Protein Engineering 6"989-995 (1993), and Newton, D.L., et al , Biochemistry 35 545-553 (1996)
Depending on the vector utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc may be used in the expression vector (see, e g , Bitter, et al , Methods in Enzymology 153:516-544, 1987). These elements are well known to one of skill in the art
In bacterial systems a number of expression vectors may be advantageously selected depending upon the intended use. For example, when large quantities of a protein of the invention is desired, vectors which direct the expression of high levels of chimeric protein products that are readily purified may be desirable Those which are engineered to contain a cleavage site to aid in protein recovery are preferred
In yeast, a number of vectors containing constitutive or inducible promoters may be used For a review see, Current Protocols in Molecular Biology, Vol 2, Ed Ausubel, et al , Greene Publish Assoc & Wiley Interscience, Ch 13, 1988, Grant, et al , Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds Wu & Grossman, 31987. Acad Press, N Y , Vol 153, pp 516-544, 1987. Glover, DNA Cloning, Vol II, IRL Press, Wash , D C , Ch 3, 1986, and Bitter, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds Berger & Kimmel, Acad Press, N Y , Vol 152, pp 673-684, 1987, and The Molecular Biology of the Yeast Saccharomyces, Eds Strathern et al , Cold Spring Harbor Press, Vols I and II, 1982 A constitutive yeast promoter such as ADH or LEU2 or an inducible promoter such as GAL may be used (Cloning in Yeast, Ch 3, R Rothstein In DNA Cloning Vol 1 1, A Practical Approach, Ed DM Glover, IRL Press, Wash , D C , 1986) Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome
An alternative expression system which could be used to express the proteins of the invention is an insect system In one such system, Autographa calif ornica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes The virus grows in Spodoptera frugiperda cells The sequence encoding a protein of the invention may be cloned into non-essential regions (for example, the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter) Successful insertion of the sequences coding for a protein of the invention will result in inactivation of the polyhedrin gene and production of non- occluded recombinant virus (i e , virus lacking the proteinaceous coat coded for by the polyhedrin gene) These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed, see Smith, et al , J Viol 46-584, 1983, Smith, U S Patent No 4,215,051 Another alternative expression system includes plant cell systems infected with recombinant virus expression vectors (e g , cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e g , Ti plasmid) containing a fluorescent indicator or circularly permuted fluorescent protein
By "transformation" is meant a permanent or transient genetic change induced in a cell following incorporation of new DNA (. e DNA exogenous to the cell) Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.
By "transformed cell" is meant a cell into which (or into an ancestor of which has been introduced), by means of recombinant DNA techniques, a DNA molecule encoding a fluorescent indicator or circularly permuted fluorescent protein having an optically active polypeptide having operatively inserted therein a sensor polypeptide, or fragment thereof, which normally has two or more states, and which is affected by a chemical, biological, electrical or physiological parameter.
Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art. Where the host is prokaryotic, such as E. Coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl? method by procedures well known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransfected with DNA sequences encoding the chimeric polypeptide of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) adenovirus, vaccinia virus, or bovine papilloma vims, to transiently infect or transform eukaryotic cells and express the protein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Preferably, a eukaryotic host is utilized as the host cell as described herein. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host, are known in the art.
Eukaryotic systems, and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur. Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and, secretion of the gene product should be used as host cells for the expression of fluorescent indicator Such host cell lines may include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and WI38
Mammalian cell systems hich utilize recombinant viruses or viral elements to direct expression may be engineered For example, when using adenovirus expression vectors, the nucleic acid sequences encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention may be ligated to an adenovirus transcription/translation control complex, e g , the late promoter and tripartite leader sequence This nucleic acid sequence may then be inserted in the adenovirus genome by in vitro or in vivo recombination Insertion in a non-essential region of the viral genome (e g , region El or E3) will result in a recombinant virus that is viable and capable of expressing the fluorescent indicator in infected hosts (e g , see Logan & Shenk, Proc Natl Acad Sci USA, 81 3655-3659, 1984) Alternatively, the vaccinia virus 7 5K promoter may be used (e g , see, Mackett, et al , Proc Natl Acad Sci USA ,79 7415-7419, 1982, Mackett, et al , J Virol 49 857-864, 1984, Panicah, et α , Proc Natl Acad Sci USA 79 4927-4931, 1982) Of particular interest are vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al , Mol Cell Biol 1 486, 1981) Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression These vectors can be used for stable expression by including a selectable marker in the plasmid, such as the neo gene Alternatively, the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the fluorescent indicator gene in host cells (Cone & Mulligan, Proc Natl Acad Sci USA, 81 6349-6353, 1984) High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters For long-term, high-yield production of recombinant proteins, stable expression is preferred Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the cDNA encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention controlled by appropriate expression control elements (e g , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc ), and a selectable marker The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines For example, following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media A number of selection systems may be used, including but not limited to the herpes simplex \ irus thymidine kinase (Wigler, et al , Cell. 1 1 223, 1977), hypoxanthine- guamne phosphoribosyltransferase (Szybalska & Szybalski, Proc Natl Acad Sci USA, 48 2026, 1962), and adenine phosphoribosyltransferase (Lowy, et al , Cell, 22 817, 1980) genes can be employed in tk-, hgprt- or aprt- cells respectively Also, antimetabohte resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al , Proc Natl Acad Sci USA, 77 3567, 1980, O'Hare, et al , Proc Natl Acad Sci USA, 8 1527, 1981), gpt, which confers resistance to mycophenohc acid (Mulligan & Berg, Proc Natl Acad Sci USA, 78 2072, 1981 , neo. which confers resistance to the aminoglycoside G-418 (Colberre- Garapin, et al , J Mol Biol , 150 1, 1981), and hygro, which confers resistance to hygromycm (Santerre, et al , Gene, 30 147, 1984) genes Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan, hisD, which allows cells to utilize histmol in place of histidine (Hartman & Mulligan, Proc Natl Acad Sci USA, 85 8047, 1988), and ODC (or thine decarboxylase) which confers resistance to the ornithme decarboxylase inhibitor, 2-(dιfluoromethyl)-DL-ornιthιne, DFMO (McConlogue L , In Current Communications in Molecular Biolog) , Cold Spring Harbor Laboratory, ed , 1987)
A fluorescent indicator or circularly permuted fluorescent protein of the invention can be produced by expression of nucleic acid encoding the protein in prokaryotes These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteπophage DNA, plasmid DNA or cosmid DNA expression vectors encoding a chimeric protein of the invention A primary advantage of the optically active polypeptides of the invention is that they are prepared by normal protein biosynthesis, thus avoiding organic synthesis and the requirement for customized unnatural amino acid analogs The constructs can be expressed in E Coli in large scale for in vitro assays Purification from bacteπa is simplified when the sequences include tags for one-step purification by nickel-chelate chromatography The construct can also contain a tag to simplify isolation of the fluorescent indicator For example, a polyhistidine tag of, e g , six histidine residues, can be incoφorated at the ammo terminal end of the fluorescent protein The polyhistidine tag allows convenient isolation of the protein in a single step by nickel- chelate chromatography Alternatively, the substrates can be expressed directly in a desired host cell for assays in situ
Techniques for the isolation and purification of either microbially or eukaryotically expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromato graphic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies or antigen
It should be understood that a nucleic acid sequence can also function as the concentration or available parameter in another embodiment of the invention For example, a response may result from the interaction of a nucleic acid sequence with a sensor polypeptide comprising a DNA binding protein motif
Screening Assays
The invention features a method for determining the presence of a chemical, biological, electrical or physiological parameter, by contacting the sample with a fluorescent indicator or circularly permuted fluorescent protein of the invention, exciting the indicator or protein, and measuring the amount of an optical property of the indicator or protein in the presence and absence of a parameter, such that a change in the optical property is indicative of an affect of the parameter on the indicator or protein A series of standards, with known levels of activity, can be used to generate a standard curve The optical event, such as intensity of fluorescence, that occurs following exposure of the sample to the fluorescent indicator or protein is measured, and the amount of the optical property is then compared to the standard curve. A standard, with a known level of activity, can be used to generate a standard curve, or to provide reference standards The optical event, such as fluorescence, that occurs following exposure of the sample to the fluorescent indicator or protein is measured, and the amount of the optical property (e g , fluorescence) is then compared to the standard in order to generate a relative measure of the affect of the sample on the fluorescent indicator.
In another embodiment, the invention features a method for determining if a cell exhibits an activity, which includes transfecting the cell with a nucleic acid encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention; exciting the fluorescent indicator or circularly permuted fluorescent protein; and measuring the amount of an optical property in the presence of the activity and in the absence of the activity, such that a change in the optical property is indicative of activity. Typically, the optical property is calibrated against standard measurements to yield an absolute amount of protein activity.
The invention additionally, features methods for determining transient changes in a chemical, biological, electrical or physiological parameter, by contacting the sample with a fluorescent indicator or circularly permuted fluorescent protein of the invention and measuring a change in the optical property of the indicator over time.
It is understood that the cell containing a nucleic acid sequence encoding a fluorescent indicator or circularly permuted fluorescent protein of the invention can be used to co-transfect other genes of interest in order to determine the effect of the gene product of that gene on the cell or the sensor polypeptide of the fluorescent indicator or circularly permuted fluorescent protein. Therefore, a cell containing such a nucleic acid sequence is a composition provided by the present invention The invention can be used in screening assays to determine whether a compound (e.g., a drug, a chemical or a biologic) alters the activity of a particular protein, i.e., the sensor polypeptide (e.g., ligand binding to a receptor). In one embodiment, the assay is performed on a sample containing the chimeric protein in vitro. A sample containing a known amount of activity, such as an enzymatic activity, is mixed with a fluorescent indicator substrate of the invention, with the co- factors required for activity, and with a test compound. The amount of the enzyme activity in the sample is then determined by measuring the amount of an optical property, such as a fluorescent property, at least a first and second time after contact between the sample, the chimeric protein substrate of the invention, and any co- factors or components required to conduct the reaction, and the test compound. Then the amount of activity per mole of enzyme, for example, in the presence of the test compound is compared with the activity per mole of enzyme in the absence of the test compound. A difference indicates that the test compound alters the activity of the enzyme. In general a change in the optical parameter by any measurable amount between activity in the presence of the test compound as compared with the activity in the absence of the test compound, is indicative of activity.
In another embodiment, the ability of a compound to alter the activity of a particular protein (i.e., a sensor polypeptide) in vivo is determined. In an in vivo assay, cells transfected with a expression vector encoding a substrate of the invention are exposed to different amounts of the test compound, and the effect on the optical parameter, such as fluorescence, in each cell can be determined. Typically, the difference is calibrated against standard measurements to yield an absolute amount of protein activity. This provides a method for screening for compounds which affect cellular events (e.g., receptor-ligand binding, protein-protein interactions or protein kinase activation). In a given cell type, any measurable change between activity in the presence of the test compound as compared with the activity in the absence of the test compound, is indicative of activity.
The materials of the invention are ideally suited for a kit for determining the presence of an activity in a sample. Such a kit may contain a container containing a chimeric protein comprising an optically active polypeptide having operatively inserted therein a sensor polypeptide, or fragment thereof, which is affected by a change in a parameter or the env ironment, wherein optical properties of the sensor are altered in response to the change In another embodiment, a kit of the invention contains an isolated nucleic acid sequence which encodes a chimeric protein comprising an optically active polypeptide having operatively insereted therein a sensor polypeptide, or fragment thereof, which is affected by a change in a parameter or the environment, wherein optical properties of the sensor are altered in response to the change The nucleic acid sequence of the later kit may be contained in a host cell, preferably stably transfected The cell could optionally be transiently transfected Thus, the cell acts as an indicator kit in itself
Transgenic Animals
In another embodiment, the present invention relates to transgenic animals that have cells that express an optically active polypeptide having operatively inserted therein a sensor polypeptide, or fragment thereof, which normally is capable of existing in two or more states, and which causes a change in the optical properties of the optically active polypeptide upon environmental conditions or parameters Transgenic animals expressing high levels of the tagged transgene may be obtained, for example, by over-expression of the transgene with an enhanced promoter and/or with high copy numbers of the transgene The transgenic animal may be heterozygous or homozygous for an ablated or disrupted endogenous indicator gene
The "non-human animals" of the invention comprise any non-human animal having nucleic acid sequence which encodes a fluorescent indicator or circularly permuted fluorescent protein of the invention Such non-human animals include vertebrates such as rodents, non-human primates, sheep, dog, cow, pig, amphibians, reptiles and fish Preferred non-human animals are selected from the rodent family including rat and mouse, most preferably mouse The "transgenic non-human animals" of the invention are produced by introducing "transgenes" into the germhne of the non-human animal Embryonal target cells at various developmental stages can be used to introduce transgenes Different methods are used depending on the stage of development of the embryonal target cell The zygote is the best target for micro- mjection In the mouse, the male pronucleus reaches the size of approximately 20 micrometers in diameter which allows reproducible injection of 1-2 pi of DNA solution The use of zygotes as a target for gene transfer has a major advantage in that in most cases the injected DNA will be incoφorated into the host gene before the first cleavage (Bπnster et al , Proc Natl Acad Sci USA 82 4438-4442, 1985) As a consequence, all cells of the transgenic non-human animal will carry the incoφorated transgene This will in general also be reflected in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene Microinjection of zygotes is the preferred method for incoφorating transgenes in practicing the invention
The term "transgenic" is used to describe an animal which includes exogenous genetic material within all of its cells A "transgenic" animal can be produced by cross-breeding two chimeric animals which include exogenous genetic material within cells used in reproduction Twenty-five percent of the resulting offspring will be transgenic / e , animals which include the exogenous genetic material within all of their cells in both alleles 50% of the resulting animals will include the exogenous genetic material within one allele and 25% will include no exogenous genetic mateπal
Viral infection can also be used to introduce transgene into a non-human animal (e g , retroviral, adenoviral or any other RNA or DNA viral vectors) The developing non-human embryo can be cultured in vitro to the blastocyst stage Dunng this time, the blastomeres can be targets for retro viral infection (Jaenich, R , Proc Natl Acad Sci USA 73 1260-1264, 1976) Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan, et al (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spπng Harbor, N Y ) The viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner, et al , Proc Natl Acad Sci USA 82 6927-6931 , 1985, Van der Putten, et α/ , Proc Natl Acad Sci USA 82 6148-6152, 1985) Transfection is easily and efficiently obtained by cultuπng the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra, Stewart, et al , EMBO J 6 383-388, 1987) Alternatively, infection can be performed at a later stage Virus or virus-producing cells can be injected into the blastocoele (D. Jahner et al , Nature 298 623-628, 1982) Most of the founders will be mosaic for the transgene since incoφoration occurs only in a subset of the cells which formed the transgenic nonhuman animal Further, the founder may contain various retro viral insertions of the transgene at different positions in the genome which generally will segregate in the offspring. In addition, it is also possible to introduce transgenes into the germ line, albeit with low efficiency, by lntrauteπne retro viral infection of the midgestation embryo (D. Jahner et al , supra)
A third type of target cell for transgene introduction is the embryonal stem cell (ES). ES cells are obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J Evans et al Nature 292" 154- 156, 1981 , M O Bradley et al.,
Nature 309 255-258, 1984, Gossler, et al , Proc. Natl. Acad Sci USA 83: 9065-9069, 1986; and Robertson et al , Nature 322 445-448, 1986) Transgenes can be efficiently introduced into the ES cells by DNA transfection or by retro virus-mediated transduction. Such transformed ES cells can thereafter be combined with blastocysts from a nonhuman animal. The ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal. (For review see Jaenisch, R., Science 240 1468-1474, 1988).
"Transformed" means a cell into which (or into an ancestor of which) has been introduced, by means of recombinant nucleic acid techniques, a heterologous nucleic acid molecule. "Heterologous" refers to a nucleic acid sequence that either originates from another species or is modified from either its original form or the form primarily expressed in the cell
"Transgene" means any piece of DNA which is inserted by artifice into a cell, and becomes part of the genome of the organism (i e , either stably integrated or as a stable extrachromosomal element) which develops from that cell Such a transgene may include a gene which is partly or entirely heterologous (i e , foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism Included within this definition is a transgene created by the providing of an RNA sequence which is transcribed into DNA and then incoφorated into the genome. The transgenes of the invention include DNA sequences which encode the fluorescent indicator or circularly permuted fluorescent protein of the invention which may be expressed in a transgenic non-human animal The term "transgenic" as used herein additionally includes any organism whose genome has been altered by in vitro manipulation of the early embryo or fertilized egg or by any transgenic technology to induce a specific gene knockout The term "gene knockout" as used herein, refers to the targeted disruption of a gene in vivo with complete loss of function that has been achieved by any transgenic technology familiar to those in the art. In one embodiment, transgenic animals having gene knockouts are those in which the target gene has been rendered nonfunctional by an insertion targeted to the gene to be rendered non-functional by homologous recombination As used herein, the term "transgenic" includes any transgenic technology familiar to those in the art which can produce an organism carrying an introduced transgene or one in which an endogenous gene has been rendered non-functional or "knocked out "
Methods For Identifying Insertion Sites A number of methods for identifying insertion sites in fluorescent proteins, such as GFP, YFP and CFP are known in the art, including, for example, site directed mutagenesis, insertional mutagenesis, and deletional mutagenesis Other methods, including that exemplified below and in the Examples, are known or easily ascertained by one skilled in the art (see, for example, Abedi et al , supra).
Sites in, for example, GFP mutants which can tolerate insertions of sensor polypeptides can be identified by generating mutant proteins by manipulating the DNA sequence such that a variety of different insertions are produced and screening the mutants by flow cytometry for mutants which retain fluorescence. Such insertions may include replacement of certain amino acids, as well as the addition of a new sequence without a corresponding deletion or replacement in the sequence of the fluorescent protein. Variants identified in this fashion reveal sites which can tolerate insertions while retaining fluorescence.
Additionally, circularly permutation techniques are also useful in identifying sites in fluorescent proteins which are capable of tolerating insertions while retain the ability to fluoresce Such techniques include are exemplified herein as well as known to those of skill in the art (see, for example, Graf et al , Proc Natl. Acad. Sci USA, 93: 1 1591-1 1596 (October 1996), the disclosure of which is incoφorated herein)
In circular permutations, the original N- and C-terminal amino acids of a fluorescent protein are engineered to be linked by a linker moiety Such linker moieties include those described above, as well as other easily ascertain by one skilled in the art This is typically performed at the nucleic acid level resulting in a polynucleotide sequence wherein the 5' codon encoding the N-terminal ammo acid is linked to the 3' codon encoding the C-terminal amino acid, resulting in a circularized fluorescent protein nucleic acid sequence The circularized sequence is then cleaved with a nuclease to create a linear polynucleotide sequence, the cleavage site corresponding to an ammo acid in of the fluorescent protein The cleavage of the circularized sequence is either random or specific depending on the desired product, nuclease, and desired sequence The linearized polynucleotide, which contains sequence homologous to the starting fluorescent protein sequence, is cloned into an expression vector and expressed. The expressed polypeptide sequence is then screened, for example by flow cytometry, for polypeptides retaining the ability to fluoresce. Accordingly, polypeptides which retain the ability to fluorescence correspondingly, via identification of the cleavage site, identify amino acids which can tolerate insertions without destroying the ability of the fluorescent protein to fluoresce
Methods of performing assays on fluorescent materials are well known in the art and are described in, e g , Lakowicz, J R , Principles of Fluorescence Spectroscopy, New York:Plenum Press (1983), Herman, B , Resonance energy transfer microscopy, in- Fluorescence Microscopy of Living Cells in Culture, Part B, Methods in Cell Biology, vol. 30, ed. Taylor, D.L. & Wang, Y.-L., San Diego- Academic Press (1989), pp. 219-243, Turro, N.J., Modern Molecular Photochemistry, Menlo Park. Benjamin/Cummings Publishing Col, Inc (1978), pp. 296-361 EXAMPLES
Cells and Protein Purification
Bacteria used in this study were BL21(DE3) Gold cells from Stratagene Transformation was performed by electroporating cells suspended in 10%) glycerol directly with a ligation mixture (0 1cm cuvette, 12 5 kV/cm, 200 Ω, 25uFd). For protein expression, cells were grown in LB containing 100 mg/L Ampicil n to an OD6oo of 0.6, at which time they were induced with ImM Isopropylthiogalactoside Bacteria were allowed to express recombinant protein for 6 hours at room temperature and then overnight at 4°C The bacteria were then pelleted by centπfugation, resuspended in 50mM Tπs, 300mM NaCl and lysed with a French Press The bacterial lysates were centπfuged at 30,000xg for 30 minutes, and the supernatants were incubated with NiNTA resin (from Qiagen, used for purifying circularly permuted GFP and Calmodulin Insertions) or Cobalt Talon Resm (from Clontech, used for purifying zinc-finger inserts)
Cloning and Gene Construction-
Yellow GFP mutants (YFPs) with peptide insertions replacing Y145 were made by performing two separate polymerase chain reactions (PCRs) The first PCR amplified the N terminal piece of YFP to include a 5' BamHl site and 3' replacement of Y 145 with the hexapeptide linker GGTGEL (coded for by DNA containing Kpnl and Sacl restriction sites for subsequent cloning). The second PCR amplified the C terminal piece of YFP to include the 5' linker (GGTGEL) replacing Y145 and a 3' EcoRI site. These two PCR products were combined, amplified with N and C terminal YFP primers to yield a full length cDNA containing the insertion. The full length cDNA was restricted with BamHl and EcoRI, ligated and cloned into the BamHl and EcoRI sites of pRSET B (Invitrogen) to yield the plasmid pYFPins. Next, the cDNAs for Xenopus Calmodulin and the first zinc-finger motif from zιf268 were amplified with PCR using primers containing 5' Kpnl sites and 3' Sacl sites and digested with Kpnl and Sacl . Finally, insertions into YFPs were made by cloning cDNAs of inserted proteins in between the Kpnl and Sacl sites of pYFPins (Figure 1) Protein Titrations:
Protein pH titrations were carried out in 125mM KCl, 20mM NaCl, 0.5mM CaCl2, 0.5mM MgCl2, and 50mM buffer. Buffers were chosen to span a wide pH range, and included citrate (pH4-5), MES (pH 5.5-6.5), HEPES (pH 7-8..5), glycine (pH 8.8-10.7), and phosphate (pHl 1.3-13.2). For each pH, a weakly buffered protein solution was mixed with an equal volume of the corresponding buffer solution and analyzed for total fluorescence in triplicate on a microplate fluorescence reader using a 482+/- 10 nm excitation filter and a 532+/- 14 nm emission filter. (Figure 2a).
Calcium titrations of YFP-Calmodulin insertion proteins were done in a cuvette in a fluorescence spectrometer in lOOmM KCl, lOmM MOPS at pH7.5 (buffer was run through a Chelex column to remove traces of calcium). Small aliquots of CaCl2 were added to this cuvette and a full fluorescence emission spectrum was taken after each addition. (Figure 2b, Figure 3).
Zinc titrations of YFP-zinc finger insertions were done in 50mM MOPS, pH 7.0. A fluorescence emission spectrum was taken of the protein in buffer containing 50uM EDTA, and then small aliquots of ZnCl were added, and subsequent spectra were recorded. (Figure 4).
Titration curves were generated by averaging the three intensity values for each pH (for microplate data) or be integrating the total emission intensity (for full spectra), plotting these data versus analyte concentration, and fitting a sigmoidal curve to the data.
YFP containing calmodulin replacing Y145 show an pH-sensitive increase in fluorescence intensity on calcium binding, with an apparent Kd of fluorescence of 7μM. The increase in fluorescence observed at constant pH reflects a shift of 1 pKa unit between the calcium-free and calcium-bound states of the protein, as observed in pH titrations done in the presence of or absence of free calcium (Figure 2). In addition, the absorbance of this protein changes from a predominantly ultraviolet, non-fluorescent band to a predominantly blue, fluorescent band on calcium binding at constant pH. Since other studies suggest that the non-fluorescent, ultraviolet- absorbing band represents the protonated chromophore and the fluorescent, blue- absorbing band represents the deprotonated chromophore, these data show that calcium, binding alters the equilibrium between protonated and deprotonated chromophore states, i e changes the pKa of fluorescence
YFPs containing a zinc-finger motif dem ed from zιf268 also increase in fluorescence on binding zinc The change in fluorescence for Zn-sensmg YFPs is substantially less than that of Calmoduhn-YFPs described above First, as Zn finger motifs contain cysteine residues in close proximity, they can be prone to oxidation, which would prevent zinc binding Second, the change of inserted proteins on substrate binding is responsible for the change in pKa and therefore the change in fluorescence It could be possible that zinc-finger motifs adopt a non-optimal conformation on binding zinc, leaving the chromophore still vulnerable to protonation Last, each of the three reasons listed above concerning the performance of YFP-Calmoduhn Insertions could in principle also apply analogously to the zinc finger insertions As stated above, the zmc-finger inserted YFP reported here is just a first generation prototype of what will likely become a powerful new class of indicators
Circular Permutations
Circular Permutations of GFP mutants with a new N terminus at Y 145 were made by performing two separate PCRs The first PCR amplified the C terminal piece of a GFP mutant (N terminal in the final permuted gene) to have a 5' BamHl site, the mutation Y145M, and a 3' hexapeptide linker (GGTGGS) containing a Kpnl site The second PCR amplified the N terminal piece of the GFP mutant (C terminal in the final permuted gene) to have a 5' hexapeptide linker (GGTGGS) containing a Kpnl site, and a 3' EcoRI site The first PCR product was digested with sequentially with BamHl and Kpnl, the second PCR product was digested sequentially with EcoRI and Kpnl, and the fragments were purified by agarose gel electrophoresis The N and C terminal PCR fragments were then cloned in a three part ligation into the BamHl/EcoRl sites of pRSET B (See Figure 5) To construct a cameleon molecule containing circularly permuted CFP instead of CFP (called YC3.2), the circularly permuted CFP cDNA was amplified with PCR to contain an 5' BamH l site and a 3' Sac l site, digested with BamH l and Sacl and agarose gel purified. Then, a plasmid consisting of the YC3J cDNA cloned into the BamHl and EcoRI sites of pRSETB was digested with BamHl and Sacl, and the 4.2 kb DNA fragment (YC3J X CFP) was agarose gel purified. The circularly permuted CFP cDNA was then ligated to the YC3J ^CFP fragment, and the DNA was transformed into BL21(DE3) Gold cells as descπbed above.
Random Circular Permutations were based on the method of Graf, et al with major modifications because their original conditions were found to be unsuccessful when applied to GFP cDNAs. Through extensive testing, it was found that circular permutation required 1) reducing the concentration of DNA used when circularizing the gene from 300 μg/mL to 5 μg/mL, 2) increasing the amount of DNAse used to relinearize the fragment from 1 U/mg DNA to 100 U/mg DNA, 3) changing the temperature of DNAse incubation from 16 degrees to 22 degrees Celsius, and switching the DNA repair enzyme used from T4 to T7 DNA polymerase. Accordingly, the method of Graf et al , was substantially modified as follows. An expression vector for the random circular permutations was made by ligating an oligonucleotide containing a 5' EcoRV site and three downstream stop codons in each reading frame between the BamHl and EcoRI sites of pRSET B. This vector ("pRSET triple stop") was digested with EcoRV, treated with Alkaline Phosphatase, and purified by agarose gel electrophoresis
To make a library of circular permutations, the circularly permuted GFP gene described above was amplified by PCR with primers that created a final PCR amp con starting and ending at an Xhol site (ctcgag) coding for residues L141 and E142. The PCR product was digested with Xhol and cloned into the Xhol site of pBluescπpt. This plasmid was amplified in bacteria, purified with a Qiagen maxi- prep, digested with Xhol , and the ~730bp fragment was agarose gel-purified to yield a linearized GFP gene. The linear fragment was circularized at a concentration of 5 μg/mL with 8000 U/mL T4 DNA gase (New England Biolabs) overnight at 16°C. After ethanol precipitation, the DNA was digested with DNAse (100 U/mg DNA, Pharmacia) for 15 minutes at room temperature in 50mM Tπs HC1, pH7 5 and ImM MnC12 Digestion was stopped by phenol extraction, then subsequent phenol/chloroform/Isoamyl Alcohol and Chloroform/Isoamyl alcohol extractions The DNA was ethanol precipitated, resuspended in lx synthesis buffer (Stratagene) and incubated with T7 DNA polymerase (Stratagene) and T4 DNA ligase (Stratagene) at room temperature for 1 hour to repair DNA nicks and fill sticky ends (Figure 6)
The linear, repaired, randomly permuted DNA library w as purified by agarose gel electrophoresis, ligated blunt into the pRSET triple stop expression vector, and electro-transformed into BL21 bacteria
LB/agar plates containing ampicillin usually displayed a few thousand colonies per plate and were screened by digital imaging of fluorescence The plates were illuminated with a 150 W xenon arc lamp through a 450-490 nm bandpass filter and a pair of fiber optic light guides (Oriel Instruments) positioned to illuminate the top surface of the agar as evenly as possible The emitted fluorescence was selected by a 510-550 nm bandpass filter and focused by a f/1 2 camera lens (Nikon) onto a cooled charge-coupled-device camera (Photometries) Digital images from the camera were analyzed with Metafluor software (Universal Imaging Coφ ) Out of approximately 25,000 bacterial colonies screened, about 200 became fluorescent after 24 hours at 4°C, and 144 of these were picked for plasmid minipreps and restriction analysis All plasmid minipreps were digested with Hmdlll and Kpnl to analyze the site of permuted termini (Hindlll cuts 3' to the GFP gene in pRSETB, Kpnl cuts at the linker between N and C termini of GFP) Clones which gave restriction fragments of ~750bp or no visible fragments from 100-lOOObp were considered to be regenerations of nearly wild-type sequence and were not investigated further Clones which gave restriction fragments between 100 and lOOObp but not 750 bp, were sequenced at their N and C termini to pinpoint the exact locations of new termini within the GFP sequence
GFP forms a fluorescent circularly permuted protein when its native N and C termini are connected with the hexapeptide linker GGTGGS and new N termini are formed at E142, Y143, Y145, H148, D155, H169, E172, D173, A227, or 1229 (See Table 22). The permuted protein with the N terminus at Y 145 was made and studied for the Cyan, Green, and Yellow mutants of GFP (cpCFP, cpGFP, cpYFP). In each case, the protein had a higher pKa of fluorescence than its native counteφart, although the fluorescence spectra were similar. This suggests that interrupting GFP and its mutants at Y 145 generally increases the chromophore's sensitivity to protonation, which is in agreement with the results obtained from the GFP insertions described above
Table 22 Sequence Summary of Random Circular Permutations
Figure imgf000053_0001
*Startιng Amino Acid is the first amino acid for which is not coded for by the expression vector (which may also have been mutated), e g E142M means that the GFP starts at Position 142, but the glutamate residue has been changed to methionine by the cloning process.
*Endmg Amino Acid is followed by the amino acid sequence added by the expression vector, e g N144LSE means that the GFP sequence ends with asparagine at position 144, but is appended by the C terminal tripeptide LSE N144 means that the protein simply ends with asparagine at position 144 with no addition peptide.
To address cpCFP's orientation in space in fusion proteins, we constructed a cameleon molecule with cpCFP replacing CFP (YC3.2) With this modification, the FRET difference between calcium-free and calcium-bound states of cameleon changes dramatically compared to normal cameleons. This demonstrates that cpCFP maintains a different oπentation in space than CFP, since other factors influencing FRET (lnter-fluorophore distance and spectral overlap) have changed only very little These data further suggest that the above list of possible cpGFP mutants represents a library of GFP mutants for use in FRET applications Since most should have different orientations in space than regular GFP mutants, every FRET-based application of a GFP mutant that fails due to poor orientation could possibly be improved through use of cpGFP mutants (Figure 7)
One obvious use of cpGFP mutants relies purely on their increased pKas Subcellular pH can be measured with appropriately targeted non-permuted GFP mutants, howev er, the dynamic range of pHs over which non-permuted GFPs can measure is limited by the pKas of available mutants Since all circular permutants thus far investigated have higher pKas than their non-permuted counteφarts, they theoretically extend the ability of GFPs to sense pH in more alkaline compartments, and could allow one to investigate relatively neutral compartments with blue-shifted mutants, which was previously not possible
Another possible use of cpGFP mutants is in making novel insertions of GFP into other proteins for use as biosensors GFP, because its termini are close in space, can be inserted into other proteins, but only rarely to date has it been shown to sense a conformational change in such a construct When cpGFP mutants are inserted into a protein, they are topologically similar to the GFP insertion constructs described above, and they might reasonably be expected to have similar sensing properties as GFP insertions (Figure 7)
GFP mutants with peptide insertions replacing Y145 were made by performing two separate polymerase chain reactions (PCRs) The first PCR amplified the N-terminal piece of GFP to include a 5' BAMH1 site and 3' replacement of Y145 wit the hexapeptide linker GGTGEL (coded for by DNA containing Kpnl and Sacl restriction sites for subsequent cloning) The second PCR amplified the C-terminal piece of GFP to include the 5' linker (GGTGEL) replacing Y145 and a 3' EcoRI site These two PCR products were combined, amplified with N- and C-terminal GFP primers to yield a full length fragment, restricted with BamHl and EcoRI, ligated and cloned into the BamHl and EcoRI sites of pRSETB (Invitrogen) Subsequent insertions into this GFP were made by cloning nucleic acid sequences of a desired binding moiety in between the Kpnl and Sacl sites of this plasmid. Any sensor polypeptide can be inserted into a fluorescence protein (e g , GFP, YFP, or CFP) by analogy to the method described above and put in a cell by introducing the cDNA coding for the protein into the cell in a vector that directs protein production The indicator is then visualized using a fluorescence detector
A number of embodiments of the present invention have been described Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS-
1. An isolated nucleic acid sequence which encodes a fluorescent indicator, the indicator comprising a sensor polypeptide which is responsive to a chemical, biological, electπcal or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
2. The nucleic acid of claim 1 , wherein the fluorescent protein moiety is an Aequorea-rdated fluorescent protein moiety
3. The nucleic acid of claim 2, wherein the Aequoreα-τelated protein is GFP, CFP or YFP.
4. The nucleic acid of claim 1, wherein the sensor polypeptide compπses two or more polypeptides operatively linked to each other.
5. The nucleic acid of claim 1 , wherein the sensor polypeptide is selected from the group consisting of calmodulin or binding fragment thereof, a calmoduhn- related protein, recoveπn, a nucleoside diphosphate or tπphosphate binding protein, an ιnosιtol-lJ,5-tπphosphate receptor, a cyclic nucleotide receptor, a nitric oxide receptor, a growth factor receptor, a hormone receptor, a ligand-binding domain of a hormone receptor, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, a cytokine receptor, a growth factor receptor, a neurotransmitter receptor, a hgand-gated channel, a voltage-gated channel, a protein kinase C, a domain of protein kinase C, a cGMP-dependent protein kinase, an inositol polyphosphate receptor, a phosphate receptor, a carbohydrate receptor, an SH2 domain, an SH3 domain, a PTB domain, an antibody, an antigen-binding site from an antibody, a single-chain antibody, a zinc-finger domain, a protein kinase substrate, a protease substrate, a phosphorylation domain, a redox sensitiv e loop, a loop containing at least two cysteines that can form a cyclic disulfide, and a fluorescent protein moiety
6 The nucleic acid of claim 1 , wherein the sensor polypeptide is calmodulin or a calmodulin-related protein moiety
7. The nucleic acid of claim 1, wherein the sensor polypeptide is selected from the group consisting of a calmodulin-binding domain of skMLCKp, smMLCK, CaMKII, Caldesmon, Calspermin, phosphofructokinase calcineuπn, phosphorylase kinase, Ca2+-ATPase 59 kDa PDE, 60 kDa PDE, nitric oxide synthase, type I adenylyl cyclase, Bordetella pertussis adenylyl cyclase, Neuromoduhn, Spectπn, MARCKS, F52, -Adducin, HSP90a, HIV-1 gpl60, BBMHBI, Dilute MHC, Mastoparan, Mehttin, Glucagon, Secretin, VIP, GIP, and Model Peptide CBP2.
8 The nucleic acid of claim 1 , further compπsing a linker moiety linking the N- and C-terminal ends of the sensor polypeptide to the fluorescent protein moiety.
9 The nucleic acid of claim 1, wherein the indicator further comprises a localization sequence.
10 The nucleic acid of claim 2, wherein the sensor polypeptide replaces one or more amino acids between residues 128 - 148, residues 155-160, residues 168- 176 or residues 227-229 of the fluorescent protein moiety.
1 1. The nucleic acid of claim 2, wherein the sensor polypeptide replaces E142, Y143, Y145, H148, D155, H169, E172, D173, A227, or 1229 of the fluorescent protein moiety
12 The nucleic acid of claim 2, wherein the sensor polypeptide replaces residue Y145 of the fluorescent protein moiety
13. The nucleic acid of claim 8, wherein the linker moiety replaces El 42, Y143, Y145, H148, D155, H169, E172, D173, A227, or 1229 of the fluorescence protein moiety.
14. The nucleic acid of claim 8, wherein the linker moiety replaces residue Y145 of the fluorescence protein moiety.
15 An expression vector containing the nucleic acid sequence of claim 1.
16 A transgenic non-human animal comprising a nucleic acid sequence according to claim 1.
17 An expression vector comprising expression control sequences operatively linked to a nucleic acid sequence coding for the expression of a fluorescent indicator, the indicator comprising" a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter; and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide.
18 A host cell transfected with an expression vector comprising an expression control sequence operatively linked to a sequence coding for the expression of a fluorescent indicator, the indicator comprising: a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter; and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
19. The cell of claim 18, wherein the cell is a prokaryote.
20 The cell of claim 19, wherein the cell is E Coli
21 The cell of claim 18, wherein the cell is a eukaryotic cell
22 The cell of claim 21, wherein the cell is a yeast cell
23 The cell of claim 21, wherein the cell is a mammalian cell
24 An isolated polypeptide comprising a fluorescent indicator, the indicator comprising a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide
25 The polypeptide of claim 24, wherein the fluorescent protein moiety is an Aequorea-re atcd fluorescent protein moiety
26 The polypeptide of claim 25, wherein the Aequoreα-x λattd protein moiety is a GFP, CFP or YFP
27 The polypeptide of claim 24, wherein the sensor polypeptide comprises two or more polypeptides operably linked to each other
28 The polypeptide of claim 24, wherein the sensor polypeptide is selected from the group consisting of calmodulin or binding fragment thereof, a calmodu n-related protein, recoveπn, a nucleoside diphosphate or tπphosphate binding protein, an ιnosιtol-l,4,5-tπphosphate receptor, a cyclic nucleotide receptor, a nitric oxide receptor, a growth factor receptor, a hormone receptor, a ligand-binding domain of a hormone receptor, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, a cytokine receptor, a growth factor receptor, a neurotransmitter receptor, a ligand-gated channel, a voltage-gated channel, a protein kmase C, a domain of protein kinase C, a cGMP-dependent protein kinase, an inositol polyphosphate receptor, a phosphate receptor, a carbohydrate receptor, an SH2 domain, an SH3 domain, a PTB domain, an antibody, an antigen-binding site from an antibody, a single-chain antibody, a zinc-finger domain, a protein kinase substrate, a protease substrate, a phosphorylation domain, a redox sensitive loop, a loop containing at least two cysteines that can form a cyclic disulfide, and a fluorescent protein moiety
29 The polypeptide of claim 24, wherein the sensor polypeptide is calmodulin or a calmoduhn-related protein moiety
30 The polypeptide of claim 24, wherein the sensor polypeptide is selected from the group consisting of a calmodulin-bmdmg domain of skMLCKp, smMLCK, CaMKII, Caldesmon, Calspermin, phosphofructokinase calcineuπn, phosphorylase kinase, Ca2+-ATPase 59 kDa PDE, 60 kDa PDE, nitric oxide synthase, type I adenylyl cyclase, Bordetella pertussis adenylyl cyclase, Neuromoduhn, Spectπn, MARCKS, F52, -Adducin, HSP90a, HIV-1 gpl60, BBMHBI, Dilute MHC, Mastoparan, Melittin, Glucagon, Secretin, VIP, GIP, or Model Peptide CBP2
31 The polypeptide of claim 24, further compπsing a linker moiety linking the N- and C-terminal ends of the sensor polypeptide to the fluorescent protein moiety
32 The polypeptide of claim 24, wherein the indicator further compπses a localization sequence
33 The polypeptide of claim 25, wherein the sensor polypeptide replaces one or more amino acids between residues 128 - 148, residues 155-160, residues 168- 176 or residues 227-229 of the fluorescent protein moiety
34 The polypeptide of claim 25, wherein the sensor polypeptide replaces E142, Y143, Y145, H148, D155, H169, E172, D173, A227, or 1229 of the fluorescence protein moiety
35 The polypeptide of claim 25, wherein the sensor polypeptide replaces residue Y145 of the fluorescent protein moiety
36 The polypeptide of claim 31 , wherein the linker moiety replaces residue E142, Y143, Y145, H148, D155, H169, E172, D173, A227, 1229 of the fluorescent protein moiety
37 The polypeptide of claim 31 , wherein the linker moiety replace residue Y145 of the fluorescent protein moiety
38 A fluorescent indicator, the indicator comprising a sensor polypeptide which is responsive to a chemical, biological, electπcal or physiological parameter, and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide resulting in protonation or deprotonation of the chromophore of the fluorescent protein moiety
39 The indicator of claim 38, wherein the fluorescent protein moiety is an Aequorea-x Xat d fluorescent protein moiety
40 The indicator of claim 39, wherein the Aequoreα-related protein is GFP, CFP or YFP
41 The indicator of claim 39, wherein the sensor polypeptide replaces residue Y145 of the fluorescence protein moiety
42. A method for detecting the presence of a response-inducing member in a sample, comprising contacting the sample with a fluorescent indicator, the indicator, comprising: a sensor polypeptide which is responsive to a chemical, biological, electrical or physiological parameter; and a fluorescent protein moiety, wherein the sensor polypeptide is operatively inserted into the fluorescent protein moiety, and wherein the fluorescence of the fluorescent protein moiety is affected by the responsiveness of the sensor polypeptide; and detecting a change in fluorescence wherein a change is indicative of the affect of the parameter on the sensor polypeptide
43. The method of claim 42, wherein the affect is a change in electrical or chemical potential.
44. The nucleic acid of claim 8, wherein the linker moiety is GGTGEL (SEQ ID NOJ ) or FKTRHN (SEQ ID NO:2).
45. The polypeptide of claim 31 , wherein the linker moiety is GGTGEL (SEQ ID NOJ) or FKTRHN (SEQ ID NO:2).
46. An isolated nucleic acid sequence encoding a circularly permuted fluorescent protein moiety comprising: a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.
47. The nucleic acid of claim 46, wherein the fluorescent protein is an Aequorea-i "elated fluorescent protein moiety.
48 The nucleic acid sequence of claim 47, wherein the Aequorea-τelated protein moiety is GFP, CFP or YFP
49 The nucleic acid of claim 46, wherein the linker moiety encodes GGTGEL (SEQ ID NO 1), GGTGGS (SEQ ID NO 63) or FKTRHN (SEQ ID NO 2)
50 The nucleic acid of claim 47, wherein the amino-terminal end or the carboxy terminal end of the indicator has an amino acid corresponding to residues 128 - 148, residues 155-160, residues 168-176 or residues 227 -229 of the fluorescent protein
51 The nucleic acid of claim 47, wherein the amino-terminal end is selected from the group consisting E142, Y143, Y145, H 148, D155, H169, E172, D173, A227 and 1229, and the carboxy-terminal end is selected from the group consisting of N144, N146, N144, N149, K162, K156, N170, 1171, D173, E172, A227, and 1229, of the fluorescent protein, respectively
52 The nucleic acid of claim 46, further comprising a polynucleotide encoding a sensor polypeptide
53 The nucleic acid of claim 52, wherein the sensor polypeptide changes conformation upon interaction with a response-inducing member
54 The nucleic acid of claim 52, wherein the sensor polypeptide is selected from the group consisting of calmodulin or binding fragment thereof, a calmodu n-related protein, recoveπn, a nucleoside diphosphate or tπphosphate binding protein, an inositol- 1 ,A, 5-tπphosphate receptor, a cyclic nucleotide receptor, a nitric oxide receptor, a growth factor receptor, a hormone receptor, a ligand-binding domain of a hormone receptor, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, a cytokine receptor, a growth factor receptor, a neurotransmitter receptor, a ligand-gated channel, a voltage-gated channel, a protein kinase C, a domain of protein kinase C, a cGMP-dependent protein kinase, an inositol polyphosphate receptor, a phosphate receptor, a carbohydrate receptor, an SH2 domain, an SH3 domain, a PTB domain, an antibody, an antigen-binding site from an antibody, a single-chain antibody, a zinc-finger domain, a protein kinase substrate, a protease substrate, a phosphorylation domain, a redox sensitive loop, a loop containing at least two cysteines that can form a cyclic disulfide, and a fluorescent protein moiety
55 The nucleic acid of claim 52, wherein the sensor polypeptide is calmodulin or a calmodu n-related protein moiety
56 The nucleic acid of claim 52, wherein the sensor polypeptide is selected from the group consisting of a calmodulin-binding domain of skMLCKp, smMLCK, CaMKII, Caldesmon, Calspermin, phosphofhictokinase calcineuπn, phosphorylase kinase, Ca2+-ATPase 59 kDa PDE, 60 kDa PDE, nitric oxide synthase, type I adenylyl cyclase, Bordetella pertussis adenylyl cyclase, Neuromoduhn, Spectrm, MARCKS, F52, -Adducin, HSP90a, HIV-1 gpl60, BBMHBI, Dilute MHC, Mastoparan, Me ttin, Glucagon, Secretin, VIP, GIP, and Model Peptide CBP2
57 The nucleic acid of claim 46, wherein the circularly permuted fluorescent protein further comprises a localization sequence
58 An expression vector containing the nucleic acid sequence of claim 46
59 A transgenic non-human animal comprising a nucleic acid sequence according to claim 46
60 An expression vector comprising expression control sequences operatively linked to a circularly permuted fluorescent protein moiety comprising a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety, and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.
61 A host cell transfected with an expression vector compπsing an expression control sequence operatively linked to a sequence coding for the expression of a circularly permuted fluorescent protein moiety compπsing" a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein
62 The cell of claim 61, wherein the cell is a prokaryote.
63 The cell of claim 62, wherein the cell is E coli
64 The cell of claim 61, wherein the cell is a eukaryotic cell.
65 The cell of claim 65, wherein the cell is a yeast cell
66 The cell of claim 65, wherein the cell is a mammalian cell.
67. An isolated polypeptide comprising a circularly permuted fluorescent protein moiety comprising" a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein.
68 The polypeptide of claim 67, wherein the fluorescent protein is an Aequorea-velated fluorescent protein moiety.
69. The polypeptide of claim 68, wherein the Aequoreα-related protein moiety is a GFP, CFP or YFP
70 The polypeptide of claim 67, wherein the linker moiety is GGTGEL (SEQ ID NOJ) GGTGGS (SEQ ID NO:63) or FKTRHN (SEQ ID NO:2).
71. The polypeptide of claim 68, wherein the amino-terminal end or the carboxy terminal end of the indicator has an amino acid of the fluorescent protein, the amino acid corresponding to residues 128 - 148, residues 155-160, residues 168-176 or residues 227-229 of the fluorescent protein
72 The polypeptide of claim 68, wherein the amino-terminal end is selected from the group consisting E142, Y143, Y145, H148, D155, H169, E172, D173, A227 and 1229, and the carboxy-terminal end is selected from the group consisting of N144, N146, N144, N149, K162, K156, N170, 1171, D173, E172, A227, and 1229, of the fluorescent protein, respectively
73. The polypeptide of claim 67, further comprising a sensor polypeptide.
74. The polypeptide of claim 73, wherein the sensor polypeptide is selected from the group consisting of calmodulin or binding fragment thereof, a calmodu n-related protein, recoveπn, a nucleoside diphosphate or tπphosphate binding protein, an inositol- 1,4,5-tπphosphate receptor, a cyclic nucleotide receptor, a nitric oxide receptor, a growth factor receptor, a hormone receptor, a ligand-binding domain of a hormone receptor, a steroid hormone receptor, a ligand binding domain of a steroid hormone receptor, a cytokine receptor, a growth factor receptor, a neurotransmitter receptor, a ligand-gated channel, a voltage-gated channel, a protein kinase C, a domain of protein kinase C, a cGMP-dependent protein kinase, an inositol polyphosphate receptor, a phosphate receptor, a carbohydrate receptor, an SH2 domain, an SH3 domain, a PTB domain, an antibody, an antigen-binding site from an antibody, a single-chain antibody, a zinc-finger domain, a protein kinase substrate, a protease substrate, a phosphorylation domain, a redox sensitive loop, a loop containing at least two cysteines that can form a cyclic disulfide, and a fluorescent protein moiety.
75. The polypeptide of claim 73, wherein the sensor polypeptide is calmodulin or a calmodulin-related protein moiety.
76. The polypeptide of claim 73, wherein the sensor polypeptide is selected from the group consisting of a calmodulin-binding domain of skMLCKp, smMLCK, CaMKII, Caldesmon, Calspermin, phosphofructokinase calcineurin, phosphorylase kinase, Ca2+-ATPase 59 kDa PDE, 60 kDa PDE, nitric oxide synthase, type I adenylyl cyclase, Bordetella pertussis adenylyl cyclase, Neuromodulin, Spectrin, MARCKS, F52, -Adducin, HSP90a, HIV-1 gpl60, BBMHBI, Dilute MHC, Mastoparan, Melittin, Glucagon, Secretin, VIP, GIP, and Model Peptide CBP2.
77. The polypeptide of claim 67, wherein the circularly permuted fluorescent protein further comprises a localization sequence.
78. A circularly permuted fluorescent protein moiety comprising: a linker moiety linking the amino-terminal and carboxy-terminal amino acids of a fluorescent protein, wherein the amino and carboxy termini are linked as internal amino acids in the circularly permuted fluorescent protein moiety; and two terminal ends, wherein the first end is an amino-terminal end and the second end is a carboxy terminal end and wherein the amino and carboxy terminal ends of the circularly permuted fluorescent protein moiety are different from the amino-terminal and carboxy-terminal amino acids of the fluorescent protein; and a sensor polypeptide which is response to a chemical, biological, electrical or physiological parameter. 79 A method of producing a circularly permuted fluorescent nucleic acid sequence, comprising linking a nucleic acid sequence encoding a linker moiety to the 5' nucleotide of a polynucleotide encoding a fluorescent protein, circularizing the polynucleotide with the nucleic acid sequence encoding the linker sequence, and cleaving the circularized polynucleotide with a nuclease, wherein cleavage linearizes the circularized polynucleotide
80 A method of producing a circularly permuted fluorescent protein comprising expressing the nucleic acid sequence produced by the method of claim 79
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