WO1995019374A9 - Bacterial receptor structures - Google Patents
Bacterial receptor structuresInfo
- Publication number
- WO1995019374A9 WO1995019374A9 PCT/SE1995/000034 SE9500034W WO9519374A9 WO 1995019374 A9 WO1995019374 A9 WO 1995019374A9 SE 9500034 W SE9500034 W SE 9500034W WO 9519374 A9 WO9519374 A9 WO 9519374A9
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- proteins
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- receptor
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- receptors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
- C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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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
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/882—Staphylococcus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/822—Microorganisms using bacteria or actinomycetales
- Y10S435/882—Staphylococcus
- Y10S435/883—Staphylococcus aureus
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
- Y10S530/82—Proteins from microorganisms
- Y10S530/825—Bacteria
Definitions
- the present invention relates to new bacterial re ⁇ ceptor structures originating from natural bacterial re- ceptor structures which have been modified in regard to amino acid residues involved in the original interaction function, whereby said original interaction function has been substantially inhibited and replaced by a modified interaction function directed to a desired interaction partner.
- Fc receptors from Gram-positive bacterial pathogens have been isolated and characterized in detail as will be shown below. Most well-characterized are the Fc receptors, named after the capability of binding to the constant Fc part of IgG. Based on binding experiments to IgG from various mammalian sources, and subclasses there- of, Fc receptors have been divided into six types I-VI.
- SPA binds to IgGl, and IgG4 but shows very weak or no interaction with IgG3 [Eliasson, M. et el, 1989 J.Biol.Chem. 9:4323-4327].
- This pseudoimxnune reaction has been used for more than 20 years for the purification and detection of antibodies in diagnostic, research and therapeutic applications.
- CIo- ning, sequencing and Escherichia coli expression of defi ⁇ ned fragments of the SPA gene revealed a highly repetitive organization, with five IgG binding domains [E-D-A-B-C] , a cell wall spanning region and membrane anchoring sequence [XM] [Uhlen, M.
- the structure for a complex between human Fc [IgGl] and a single domain [B] of SPA has been determined by X- ray crystallography at a resolution of 2.8 A [Deisenhofer, J. et al 1981 Biochemistry 20:2361-2370], Based on this structure and additional information from NMR experiments, the B domain can be viewed as a compact structure consis- ting of three anti-parallel ⁇ -helices connected with loops. In the Fc binding, which is of both electrostatic and hydrophobic nature, only side chains of residues from helices 1 and 2 are involved, whilst the third helix is not participating in the binding. Based on this domain B, a synthetic IgG-binding domain [Z] [Nilsson, B.
- Streptococcal strains of serological groups C and G display a binding repertoire for mammalian IgGs, including human IgG3, which is even broader than for the type I re ⁇ ceptor.
- the name protein G was suggested for this type III receptor from group G streptococci.
- Olsson and co- workers reported on the cloning and sequencing of the gene from the serological group G streptococci [G148] [Guss, B. et al, 1987 EMBO J. 5:1567-1575; Olsson, A. et al, 1987 Eur.J.Biochem. 168:319-324].
- SPG In analogy with SPA is SPG a repetitively arranged molecule, comprising an IgG-binding region of three homologous domains [C1,C2,C3], spaced by smaller D-regions (Fig. 2A). Compared to SPA, SPG displays a different binding spectra for immunoglobulins from dif ⁇ ferent species and subclasses thereof.
- the IgG binding domains of protein G are now widely used as an immuno ⁇ logical tool, i.e. in the affinity purification of mono ⁇ clonal antibodies. Production of subfragments constructed by DNA-technology, have shown that an individual C-region is sufficient for full IgG-binding.
- SPG serum albumin binding strength
- the binding strength is species depen- dent, and among the tested samples, SPG binds strongest to serum albumin from rat, man and mouse.
- Production and bin ⁇ ding studies of subfragments of SPG shows that the two binding activities are structurally separated and that the serum albumin binding function is located at the repeti- tive A-B region [Nygren et al 1990 Eur.J.Biochem. 193:143- 148] . This region has been used for several biotechnolo- gical purposes. Recombinant proteins have been produced as fusions to the region which enables the purification by affinity chromatography, where human serum albumin most frequently has been used as immobilized ligand.
- Proteins found to be proteolytically sensitive have been produced as "dual affinity fusions" in which they are flanked by two different affinity tails derived from SPA and SPG, respectively. Purification schemes employing both the N- and C-terminal are thus possible which ensures the recove ⁇ ry of an intact target protein [Hammarberg et al 1989 Proc.Natl.Acad.Sciences USA 86:4367-4371]. The strong and specific binding to serum albumin has also been used for the ⁇ n vivo stabilization of therapeutic proteins.
- the receptor is carried in the circulation (macaque apes) with a half-life which is close to the half-life for serum albumin itself.
- mice with the for HIV/AIDS therapy interesting, but rapidly cleared T-cell receptor CD4 showed that it was substantially sta ⁇ bilized when fused to the serum albumin binding region, when compared with an unfused control protein [Nygren et al 1991 Vaccines 91 Cold Spring Harbor Press 363-368].
- the slow clearance can probably be explained by the complex formation with serum albumin which circumvents elimination by the liver and excretion in the kidney.
- SPG is regarded to be trivalent with regard to binding to serum albumin. Similar to the monovalent IgG-binding domains Z and Cl B2A3 is relatively small and shows high solubility and stability and is therefore a suitable candidate for modification.
- the present invention has for its main purpose to provide new bacterial receptor structures by modifying natural bacterial receptors in regard to their original interaction functions to result in new structures having modified interaction functions.
- Another object of the invention is to provide arti ⁇ ficial bacterial receptor structures which are stable and more resistant to various conditions, such as increased temperatures•
- Yet another object of the invention is to provide artificial bacterial receptor structures, the interaction functions of which have been modified to direct same to other desired interaction partners.
- novel proteins obtainable by mutagenesis of surface- exposed amino acids of domains of natural bacterial re- ceptors said proteins being obtained without substantial loss of basic structure and stability of said natural bacterial receptors.
- Said proteins have preferably been selected from a protein library embodying a repertoire of said novel proteins.
- at least one amino acid residue involved in the interaction fuction of the original bacterial receptor has been made subject to substitution by another amino acid residue so as to result in substantial loss of the original interaction capacity with a modified interaction capacity being created, said substitution being made with ⁇ out substantial loss of basic structure and stability of the original bacterial receptor.
- said bacterial structures origi ⁇ nate from Gram-positive bacteria there may be mentioned Staphylococcus aureus.
- thermophilic bacteria evolved to persist in environments of elevated temperatures.
- Re ⁇ ceptors from species like e.g. Bacillus stearothermo- philus, Thermus aquaticus, Thermococcus l ⁇ toralis and Pyrococcus have the potential of being naturally excep ⁇ tionally stable, thus suitable for providing structural frameworks for protein engineering according to the in ⁇ vention.
- bac ⁇ terial receptors originating from Fc[IgG]receptor type I, type II, type III, type IV, type V and type VI, fibronec- tin receptor, M protein, plasmin receptor, collagen re ⁇ ceptor, fibrinogen receptor or protein L [K light chains] , protein H [human IgG], protein B [human IgA,Al], protein Arp [human IgA] .
- Particularly preferred bacterial receptors originate from the Fc[IgG]receptor type I of staphylococcal protein A or the serum albumin receptor of streptococcal protein G.
- the substi ⁇ tution involving amino acid residues taking part in the interaction function of the original bacterial receptor does not involve more than about 50% of the amino acid residues of the original bacterial receptor. It is parti- cularly preferred that not more than about 25% of the amino acid residues of the original bacterial receptor are made subject to substitution.
- substitution thereof involves not more than substantially all of the amino acid residues taking part in the interaction function of the original bacterial receptor.
- the bacterial receptor according to the pre ⁇ sent invention is comprised of not more than about 100 amino acid residues. It is known from scientific reports that proteins of a relatively small size are fairly resis ⁇ tant to increased temperatures and also to low pH and cer- tain chemicals. For details concerning temperature resis ⁇ tance c.f. the article by Alexander et al. in Biochemistry 1992, 31, pp 3597-3603.
- interaction part ⁇ ners are IGF-I, IGF-II, hGH, Factor VIII, insulin and apolipoprotein and their respective receptors as interac ⁇ tion partners. Furthermore, by selecting new receptor variants with specificity for different folding forms of proteins, affinity resins or analytical tools to facili ⁇ tate the isolation of correctly folded molecules can be produced. Further examples are viral coat proteins, bac ⁇ terial antigens, biotin and cell markers, such as CD 34 and CD 4.
- the present invention is applicable to a variety of natural bacterial receptors the following illustration of the invention more in detail will be directed to the use of the IgG-binding domains Z, Cl and B2A3.
- the concept of the present invention residing in the use of artificial bacterial receptors based on the natural structures of naturally occurring bacterial receptors is associated with several advantages.
- the invention makes it possible to use robust and stable, highly soluble and secretion competent receptors. This is in contrast to previous techniques based on the use of polyclonals and monoclonals, such as for diagnostic purposes, which are not very stable in connection with storage, varying condi ⁇ tions, such as varying temperatures etc.
- the invention makes it possible to modify natural bacterial receptors to obtain desired interaction capacities for specific purposes.
- the phage particle contains packed DNA encoding the phage protein components, a coupling between the spe ⁇ cific variant of the displayed protein and the correspon ⁇ ding genetic information is obtained.
- a coupling between the spe ⁇ cific variant of the displayed protein and the correspon ⁇ ding genetic information is obtained.
- typically 10 phage clones can simultaneously be generated and subjected to panning for screening of de ⁇ sired characteristics.
- the phage display technique can be used for selection of both small peptides as well as more complicated proteins such as antibodies, receptors and hormones.
- the present invention describes the construction of novel proteins based on the mutagene ⁇ sis of surface exposed amino acids of domains derived from bacterial receptors. These artificial bacterial receptors can be selected for different applications using a phage display system.
- the benefits from using bacterial recep ⁇ tors as structural frameworks are several. They have evol ⁇ ved to express a binding function without disturbing the overall structure.
- the bacterial receptors SPA and SPG have been widely used in antibody technology for detection and purification of antibodies from e.g. hybridom supernatants and ascites fluids. However, not all antibodies are recognized by these receptors, depending on species and subclass. For the smaller subfragments of antibodies (Fig. 4), SPA and SPG show a limited binding, and efficient tools for gene- ral purification schemes are lacking. However, from a repertoire of mutant receptors including SPA and SPG, forms displaying a broader affinity for antibodies and subfragments thereof can potentially be selected.
- the complex structural organization of antibodies has a number of consequences for their use in different appli ⁇ cations as well for the production of recombinant deriva ⁇ tives.
- the arrangement of sub- units connected by disulphide bonds can lead to a leakage of released heavy and light chains from columns.
- the re ⁇ quirement of successful docking of the two subunits con ⁇ tributing to the antigen binding site makes the production in bacteria of small subfragments with a low association difficult.
- the folding of the antibody is dependent on the formation of intra- and inter chain disulphidebonds, which are not able to form in the intracellular environment of bacterial cells.
- High-level intracellular expression sys ⁇ tems for recombinant antibodies leads to the formation of inclusion bodies, which have to be renatured to obtain biological activity.
- the CDR regions forming the antigen bidning part of an antibody forms a total surface available for the anti ⁇ gen of approximately 800 A 2 , with typical 10-20 residues from the antibody involved in the binding.
- the binding surface of about 600 A* is of the same order of magnitude as between an antibody and its antigen.
- Immunoglobulins are substantially tetramers built up from so called ⁇ -sheet structures which stabilize the orientation of the antigen- binding loops which in turn consist of continuous peptide sequences.
- the purification of the product is frequently a major problem.
- the hybrid product can effectively and selec ⁇ tively be recovered from the cell lysate or in certain cases from the culture medium by passage through a column containing an immobilized ligand.
- gene fusion systems have been described which are based on the inter- action of a certain protein with a ligand. For industrial applications it is often desirable to clean effectively the columns between the runs to satisfy purity require ⁇ ments by authorities.
- the relatively harsh conditions NaOH, acids, heat
- organic or physical matrises for example in ion exchange chromatography and gel filtration
- the use of new ligands based on stable structures originating from bacterial receptors are of great importance.
- the Z domain from SPA is an excellent example since said domain can be subjected to such difficult conditions as a pH of 1 or heating to 80 0 C without denaturating non-reversibly (see Example 2 below).
- Z variants inte ⁇ resting protein products can be selected for use immobi ⁇ lized on a solid phase for affinity chromatography.
- the high solubility of the said domain enables the use of in ⁇ creased solubility of proteins in either refolding from inclusion bodies or in so called reshuffling of disulphide bridges.
- new forms can be selected having improved properties to facilitate and even make refolding of recombinant proteins possible (cis-acting chaperones) .
- Recently a new unit operation for the purification of recombinant proteins based on ion exchange chromatography in so called expanded bed has been described [Hansson, M. et al 1994 Bio/Technol. in press].
- the present invention provides means for producing and selecting proteins with novel functions. According to the invention this is achieved by extensive mutation of defined residues of stable domains of bacterial receptors. Due to the novel functions of the artificial bacterial re ⁇ ceptors, these can be used as specific binders for thera ⁇ Illustrated, diagnostic, biotechnology or in research.
- the present invention will now be described more in detail by specific examples with reference to the appended drawings.
- Figure 1 A Schematic representation of staphylococcal protein A showing the signal peptide (S), five IgG-binding regions [E-D-A-B-C] , followed by cell wall anchoring region [X-M] .
- FIG 2 A. Schematic representation of streptococcal protein G from the strain G148 showing the signal peptide (Ss), region E (E), the repeti ⁇ tive serum albumin binding A-B region, the spacer region (S), followed by the IgG binding domains Cl through C3, spaced by the D regions and finally the cell wall anchoring region W.
- Figure 3 Schematic representation of the three helix bundle structure of the 58 residue SPA analo ⁇ gue Z. Indicated are some of the side chains proposed to be involved in the binding to Fc with the exception of F30, which stabilizes the helix-helix packing.
- Figure 4 IgG antibody structure, showing the different subfragments Fab,Fd,Fc and the scFv composed of the VH and VL connected by a short (ca 15 aa) linker.
- Figure 5. A. General concept for the gene assembly stra ⁇ tegy used for the generation of the Z gene libraries. For the construction of the library of acid Z derivatives, only residues 9, 11, 14, 27 and 35 were altered using the degenera ⁇ ted oligonucleotides ACID-I, ACID-2. The PCR primers used for the amplification of the as ⁇ Ild library were ZLIB-3 (PCR primer 5' ) and ZLIB-5 (PCR primer 3' ).
- PCR products from the amplification of the assembled library encoding 46 of the 58 residues of the Z-domain can be cloned into phagemid DNA harboring the remaining C-termi- nal part of Z.
- This gene is fused in frame with the gene for protein III of the M13 fami ⁇ ly of E.coli bacteriophages. This enables the display on the phage surface of the repertoire of acid Z-variants.
- FIG. 7 DNA sequences of clones derived from the acid Z protein library. Bold figures indicate amino acid positions in the Z-domain. For clarity the positions of the restriction sites Ace I and Nhe I are shown.
- Figure 8 Result from analysis of the temperature stabi ⁇ lity of an individual Z domain at pH 2.9. The content of ⁇ -helicity in the sample was moni ⁇ tored by measuring the ellipticity at s222 run during a temperature scan.
- Figure 9. Phagemid vector pKNl.
- the library PCR products encoding the variegated helices 1 and 2 was subclo- ned into the phagemid vector, pKNl, containing the gene for residues 44-58 of the wild type Z domain (essentially helix 3), followed by the gene for a 46 residues serum albumin binding region (ABP) derived from streptococcal protein G linked in frame with a truncated version of the M13 phage coat protein 3 gene.
- the phagemid contains the origin of replication derived from plasmid pBR322 as well as the intergenic region ( f1 ori ) required for packing into the phage particles.
- FIG. 10 SDS-PAGE. HSA-affinity purified proteins from the periplasm of Escherichia coli cells produ ⁇ cing the wild type Z domain and two different acid Z-variants as ABP fusion proteins encoded from their respective phagemid vectors were analyzed by SDS/PAGE.
- M molecular weight mar ⁇ ker; lane 1, wild type Z domain; lane 2, clone 10; lane 3, clone 12.
- Figure 11 CD-data. Overlay plot of CD spectra obtained for the wild type Z domain and two variants of the Z-protein library. The signals of the pro ⁇ teins were obtained after subtraction of the CD signal contribution of the ABP tail, present during the analysis.
- FIG. 12 Ion exchange chromatography.
- the two acid Z- variant proteins no. 10 and no. 12 together with the wild type Z-domain (produced as ABP fusion proteins) were each subjected to analy ⁇ sis at pH 5.5, employing an anion exchange chromatography column. Elution of the proteins from the column was obtained by a NaCl gradi ⁇ ent.
- FIG. 13 Z-domain structure.
- the structure of helices one and two are from the co-crystal structure be ⁇ tween domain B of SPA and Fc (Deisenhofer, (1981) Biochemistry, 20, 2361-2370).
- the third helix was built based on the secondary structu ⁇ re assignments from NMR spectroscopy (Gouda et al., (1992) Biochemistry, 31, 9665-9672).
- Non- hydrogen atoms of side-chains of residues that were mutated in the construction of the combi ⁇ natorial library are displayed as ball-and- stick models.
- the display was generated by the program MOLSCRIPT (Kraulis (1991) J.Appl.
- Figure 14 Amino acid sequences. Result from DNA-sequen- cing of 31 randomly chosen Z-variants from the library. The residues subjected to the mutage ⁇ nesis are boxed. Horisontal lines indicate nu ⁇ cleotide identity with the wild type Z sequence listed at the top. Indicated are the clones that were expressed and characterized as fusion proteins to the ABP-tail.
- Figure 15 Aminoacid distribution. Result from the statis ⁇ tical analysis of the deduced amino acids at the mutated positions. In total, 13 residues from 31 clones (403 codons) were included in the calculation. The ratios between observed and expected frequencies are shown for all 20 amino acids as well for the only termination signal (TAG) included in the NNG/T degeneracy profile.
- TAG termination signal
- FIG. 16 SDS-PAGE analysis. HSA-affinity purified pro ⁇ teins from the periplasm of E.coli cells produ ⁇ cing the wild type Z domain and four different Z-variants as ABP fusion proteins encoded from their respective phagemid vectors were analyzed by SDS/PAGE. Lanes 1-5: Reduced conditions.
- Lanes 6 and 7 Non-reduced conditions. Lane 1, wild type Z domain; lane 2, clone 16; lane 3, clone 21; lane 4, clone 22; lane 5, clone 24; M, molecular weight marker; lane 6, clone 16 and lane 7, clone 22.
- Figure 17 CD-data. Overlay plot of CD spectra obtained for the wild type Z domain and four variants of the ⁇ c-helical protein surface library. The sig ⁇ nals of the variants were obtained after sub ⁇ traction of the CD signal contribution of the ABP tail, present during the analysis.
- FIG. 18 Biosensor assay. An overlay plot of sensorgrams obtained from the BIA-coreTM analysis of the wild type Z domain and four different variants (no. 16,21,22,24; Figure 4) fused to the ABP tail. The IgG-binding activities of the diffe ⁇ rent proteins were analyzed using a sensor chip coated with approx. 5000 RU human polyclonal
- oligonucleotides (Fig. 6) were purchased from Scandinavian Gene Synthesis (Sweden), and phosphorylated where indicated according to [Maniatis et al (1988) Mole ⁇ cular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press].
- ZLIB-I was biotinylated in the 5'-end enabling immobilization on paramagnetic beads M-280 Strep- tavidin purchased from Dynal A/S (Norway). Washing/binding buffer was 1 M NaCl, 10 mM Tris-HCl, pH 7.5, 1 mM EDTA (ethylenediamine tetraacetic acid).
- the annealing/ligation buffer was 30 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 0.2 mM ATP, 1 mM 1.4 dithiothreitol (DTT).
- DNA ligase were from Boehringer Mannheim, Germany.
- 10 x PCR buffer contained 20 mM MgCl 2 , 2 mM dNTPs, 100 mM Tris-HCl, pH 8.3, 50 mM KCl, 1% Tween 20.
- Taq DNA polymerase was from Cetus Inc., USA.
- the thermal cycler was a Perkin-Elmer 9600.
- the synthetic 58 residue SPA analogue Z [Nilsson et al, Prot.Eng] was subjected to a mutagenesis approach to construct new variants with an altered pi, in order to produce fusion partners for recombinant proteins to be pu- rified by ion-exchange chromatography.
- the PCR mix (50 ⁇ l) contained one pmole each of PCR primers ZLIB-3 and ZLIB-5, 5 ⁇ l each of the ligation mix, 10 x PCR buffer and 10 x CHASE, 1 unit of Taq polymerase and sterile water to 50 ⁇ l.
- the temperature cycling programme was: 96 0 C, 1 min, 60 0 C, 1 min and 72 0 C, 2 min, repeated for 35 cycles.
- Ana ⁇ lysis by 1% agarose gel electrophoresis showed a band of the expected size of 179 bp, showing the feasibility of the assembly concept.
- the 179 bp band from the PCR of the Z(Acid)-library was cut out from the gel and purified (GenecleanTM, Bio 101, Inc.
- the temperature stability of the Z conformation was determined by following the ellipticity at 222 nm by cir ⁇ cular dichroism (CD) spectroscopy through a temperature scan. This wavelength is used to monitor the presence of oc-helicity of Z [Cedergren et al. 1993 Prot. Eng. 6:441- 448] .
- the experiment was performed at a rather low pH (approximately 2.9) in order to destabilize the molecule since the mid-point of temperature denaturation (Tm) is »95 0 C at neutral pH (data not shown), which is outside the range that can be determined by a complete scan through the transition under normal atmospheric pressure.
- the experiment shows (Fig. 4) that the Tm (as defined by the inflexion point of the temperature scan) of the Z domain is as high as 71 0 C at pH 2.9. This demonstrates the ex- treme temperature stability of the ⁇ -helices of the Z molecule.
- the experiment was performed in a J-720 spectro- polarimeter (JASCO, Japan) and the temperature was con ⁇ trolled by circulating water through the cuvette holder from a NESLAB water bath. The temperature was monitored in the cuvette through a micro sensor device (JASCO, Japan).
- the buffer was 50 mM acetic acid, pH 2.9.
- the protein was domain Z [Cedergren et al 1993 Prot. Eng. 6:441-448] at a protein concentration of 50 ⁇ g/mL and the cuvette cell path length was 1 cm.
- the temperature scan speed in the experiment was 50°C/h.
- Example 3 Characterization of proteins derived from the acid Z-library.
- Two protein variants derived from the acid Z-library were expressed in Escherichia coli. Du ⁇ fied and characterized using SDS-PAGE, circular dichroism and ion exchange chromatography.
- the PCR products from a solid phase gene assembly were restricted with 45 U Esp 31 (Labassco AB, Sweden) and 50 U Nhe I (Pharmacia, Sweden) in 200 ⁇ l buffer (33 mM Tris-acetate, pH 7.9, 10 mM Mg- acetate, 66 mM potassium-acetate, 0.5 mM DTT and 0.1 mg/ml BSA). The mix was overlaid with mineral oil and incubated at 37 0 C over night.
- the restricted fragments (approximately 5 ⁇ g) were purified by phenoL/chloroform/isoamylaJcohol extraction followed by additional washing with chloroform and later ethanol precipitated before ligation at 15 0 C over night to MIu l-Nhe I cleaved pKLNl vector (1 ⁇ g) (see below) using 13.5 Weiss units of T4 DNA ligase.
- the ligation mixture was heat-treated at 7O 0 C for 20 min, extracted with phenol/chloroform/isoamylalcohol followed by washing with chloroform, ethanol precipitated and redissolved in 20 ⁇ l of sterile water.
- the phagemid vector pKNl (figure 9) was constructed in several steps as follows.
- a double stranded linker encoding the invariant residues 44-58 of the Z-domain was formed from oligonucleotides ZLIB-6 and ZLIB-7 and cloned as a M hi l-Xho I fragment into phagemid pKP986 (A kind gift from Dr. Lars Abrahmsen. Pharmacia BioScience Center, Sweden), resulting in pKN.
- Plasmid pKP9S6 encodes the E. coli Omp A leader peptide followed by residues 249-406 of fd filamentous phage coat protein 3 (Lowman et al.
- a gene fragment encoding a monovalent serum albumin binding region derived from streptococcal protein G was amplified by PCR from the plasmid pB2T (Eliasson et al., Molecular Immunol, 28, 1055- 1061), using primers ABP- I and ABP-2 (which contain XJw I and Sal I recognition sites, respectively) and cloned into Xho I restricted plasmid pKN, yielding pKNl.
- This phagemid vector thus encodes for the Omp A bignal peptide, the third helix of the wild type Z domain followed by a 46 residue albumin binding protein (ABP) linked to residues 249-406 of fd phage protein III and is adapted for insertion of Esp 3l/Nhe I-digested PCR products encoding variegated helices one and two of the Z domain.
- ABC albumin binding protein
- the solid-phase DNA sequencing of the PCR products was performed employing the FITC labeled sequencing primers NOKA-3 (for the immobilized strand) and ABP-2 (for the eluted strand) on a robotic workstation (BiomekTM 1000, Beckman Instruments, Fullerton, CA) and an Automated Laser Fluorescent (A. L. F.) DNA SequencerTM (Pharmacia Biotech, Sweden) as described by Hultman and coworkers (Hultman et ai, ( 1989) Nucleic acids Research, 17, 4937-4946).
- the periplasmic content from the cells was subjected to affinity chromatography on HSA-Sepharose as described by Nygren and coworkers (Nygren et ai, (1988) J. MoL Recognit., 1, 69-74) and apalyzed by SDS/PAGE on a homogeneous 12% slab gel (BioRad Inc., USA), which was stained with Coomassie Brilliant Blue R-250.
- SDS- PAGE analysis Figure 10 of purified proteins suggest that the acid Z variants analyzed are stably expressed in E. coli.
- CD signals for the derivatives were obtained by subtracting the signal obtained for the ABP tail, after adjustments for differences in protein concentrations, followed by normalization for amino acid contents.
- the proteins Z(wild 5 type) and the acid variants no. 10 and no. 12 were each (5 ⁇ g) dissolved in 300 ⁇ l of 20 mM Piperazine buffer (pH 5.5) and separately applied at 100 ⁇ l/min on a MonoQ, PC 1.6/5 column (Pharmacia, Sweden). Elution of the proteins were performed by applying a NaCl gradient in Piperazine buffer (pH 5.5) (Sigma, USA) ranging from 0-50 % NaCl in 20 min.
- the series cf experiments performed on the two acid Z- variant proteins shows that the expression behaviour, proteolytic stability and secondary structure content of the variants were unchanged when compared to the native Z-domain. Furthermore, a novel functions were introduced into the two Z-variants by the substitution of surface located positions with acid amino acids.
- the two acid variants can be used e.g. as
- a library of Z-variants was assembled using a solid-phase gene assembly strategy (see example 1 ). Most of the amino acid residues suggested to take part in the binding
- the chosen NNK degeneracy includes 32 codons covering all 20 amino acids, including the TAG (amber) termination signal. 5
- Oligonucleotide ZLIB- I was synthesized with a 5' biotin group to enable robust anchoring onto streptavidin-coated paramagnetic beads used as solid support during the gene assembly.
- This ZLIB- I oligonucleotide, together with its complementary sequence (ZLIB-2) encodes residues 1-8 of the Z domain, preceeded by the first six O residues of region E of protein A which were included to facilitate the E. coli secretion of the Z variants (Abrahmsen et al, (1986) EMBO J., 4, 3901-3906).
- the oligonucleotides DEGEN- I and DEGEN-2 (Table I) encode the two mutated helices of the Z domain, respectively, normally involved in Fc-binding. Theoretically, full and simultaneous NNK degeneracy at the 13 selected positions would yield a ⁇ combinatorial library of appr. 8* 10 ⁇ protein variants encoded by 3.7» 1019 different DNA sequences. However, here the assembly of the library was initiated by the immobilization of appr. 15 pmole of prehybridized oligonucleotides ZLIB-I and ZLIB-2 ( Figure 6), which limits the theoretical size of the Z-library to appr. 0.9*10 13 different DNA sequences encoding appr. 2 # 10 ⁇ Z variants.
- oligonucleotides ZLEB-3 and ZLIB-5 containing the recognition sequences for the endonucleases Esp 3 I and Nhe I respectively, were used as primers for PCR amplification of the assembled constructs using one tenth of the bead-immobilized ssDNA as template (theoretically corresponding to 2 » 10 ⁇ protein variants).
- oligonucleotides ZLIB-2, BRIDGE and ZLIB-5 were first eluted with alkali. The resulting PCR product was analysed by agarose gel electrophoresis and found to be homogenous and of the expected size, 179 bp.
- the PCR product was subcloned into the pKLN l pbagemid vector containing the gene for residues 44-58 of the wild type Z domain in frame with a truncated version of the fd phage coat protein 3 gene for surface display on phage particles upon helper phage superinfection of phagemid transformed E. coli cells (Lowman et ai, ( 1991 ) Biochemistry, 30, 10832-10S44) ( Figure 9).
- the phagemid vector contains an interspaced in-frame cassette encoding a 5 kDa (46 aa) serum albumin binding region (denoted ABP) derived from streptococcal protein G (Nygren et ai, (1988) J. MoI. Recognit., 1, 69-74; Nilsson et ai, (1994) Eur. J. Biochem., 224, 103-108), enabling efficient affinity purification of produced Z variants devoid of their native Fc-binding activity.
- ABP serum albumin binding region
- the serum albumin binding activity can potentially be used for pre-selection of phage particles carrying recombinant molecules, prior to the panning for Z variants with new binding functions, to decrease the background originating from unspecifically bound non-recombinant phage particles.
- PCR screening using the oligonucleotides RIT-27 and NOKA- 2 of 25 clones showed that over 95% (24/25) of the clones contained an insert of the expected length, suggesting that the gene assembly procedure was carried out with high efficiency.
- Fortyfive transformants were randomly selected and subjected to direct solid phase DNA sequencing (see Example 3) in order to further analyze the quality and heterogeneity of the library. Approximately 69% of the clones were correct, containing wild type and degenerate codons at expected positions. The remaining clones had spurious discrepancies which in part can be attributed to the oligonucleotide synthesis or errors introduced during PCR.
- Soluble proteins from the periplasm of IPTG-induced cultures were subjected to HSA-affinity chromatography employing the ABP-tail for general and efficient recovery (Nygren et ai, ( 1988) J. MoI. Recognit., 1, 69-74). For all proteins appr. 1.5-2.5 mg/L culture could be recovered, indicating similar production and secretion efficiencies for the variants and the wild type domain.
- the results from a SDS-PAGE analysis ( Figure 16) of purified proteins suggest that the four Z variants analyzed are stably expressed in E. coli.
Abstract
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US4879213A (en) * | 1986-12-05 | 1989-11-07 | Scripps Clinic And Research Foundation | Synthetic polypeptides and antibodies related to Epstein-Barr virus early antigen-diffuse |
US5084559A (en) * | 1987-03-27 | 1992-01-28 | Repligen Corporation | Protein a domain mutants |
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US9321832B2 (en) | 2002-06-28 | 2016-04-26 | Domantis Limited | Ligand |
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CA2181042A1 (en) | 1995-07-20 |
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NZ278991A (en) | 1997-04-24 |
WO1995019374A1 (en) | 1995-07-20 |
JP4373461B2 (en) | 2009-11-25 |
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ATE279439T1 (en) | 2004-10-15 |
PT739353E (en) | 2005-01-31 |
AU696186B2 (en) | 1998-09-03 |
EP0739353A1 (en) | 1996-10-30 |
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