WO2000014271A1 - METHOD FOR STUDYING PROTEIN INTERACTIONS $i(IN VIVO) - Google Patents

METHOD FOR STUDYING PROTEIN INTERACTIONS $i(IN VIVO) Download PDF

Info

Publication number
WO2000014271A1
WO2000014271A1 PCT/US1999/020207 US9920207W WO0014271A1 WO 2000014271 A1 WO2000014271 A1 WO 2000014271A1 US 9920207 W US9920207 W US 9920207W WO 0014271 A1 WO0014271 A1 WO 0014271A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
complexed
acceptor fluorophore
cell
luciferase
Prior art date
Application number
PCT/US1999/020207
Other languages
French (fr)
Inventor
Aladar A. Szalay
Yubao Wang
Gefu Wang-Pruski
Original Assignee
Loma Linda University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Loma Linda University filed Critical Loma Linda University
Priority to AU58056/99A priority Critical patent/AU752675B2/en
Priority to CA002341314A priority patent/CA2341314A1/en
Priority to EP99945460A priority patent/EP1109931A4/en
Priority to JP2000569011A priority patent/JP2002524087A/en
Publication of WO2000014271A1 publication Critical patent/WO2000014271A1/en

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/10Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034Isolating an individual clone by screening libraries
    • C12N15/1055Protein x Protein interaction, e.g. two hybrid selection
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide

Definitions

  • the method could be used with a wide variety of proteins and in a wide variety of living cells. Also preferably, the method could be used to determine the interactions between molecules other than proteins.
  • a method for determining whether a first protein interacts with a second protein within a living cell comprises providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore within the cell.
  • the donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore when the first protein is in proximity to the second protein.
  • the complexed first protein and the complexed second protein are allowed to come into proximity to each other within the cell.
  • any fluorescence from the acceptor fluorophore is detected. Fluorescence of the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase to acceptor fluorophore the indicates that the first protein has interacted with the second protein.
  • providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore comprises genetically engineering DNA and transferring the genetically engineered DNA to the living cell causing the cell to produce the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore.
  • the cell which is provided with the first protein complexed to a donor luciferase and the cell which is provided with the second protein complexed to an acceptor fluorophore are mammalian cells.
  • the donor luciferase provided is Renilla luciferase.
  • the acceptor fluorophore provided is an Aequorea green fluorescent protein.
  • the detection of acceptor fluorophore fluorescence is performed using spectrofluorometery.
  • the present invention includes a method for determining whether a first protein interacts with a second protein in a living cell using luminescent resonance energy transfer (LRET).
  • LRET luminescent resonance energy transfer results from the transfer of excited state energy from a donor luciferase to an acceptor fluorophore.
  • LRET luminescent resonance energy transfer
  • the efficiency of luminescence resonance energy transfer is dependent on the distance separating the donor luciferase and the acceptor fluorophore, among other variables. Generally, significant energy transfers occur only where the donor luciferase and acceptor fluorophore are less than about 80 A of each other.
  • the present invention utilizes luminescence resonance energy transfer to determine whether an interaction takes place between a first protein and a second protein in a living cell. This is accomplished by complexing a first protein to the donor luciferase and complexing the second protein to the acceptor fluorophore and placing the complexed first protein and the complexed second protein in the cell under conditions suitable for an interaction between the first protein and the second protein to take place. If the first protein interacts with the second protein, the donor luciferase will come close enough to the acceptor fluorophore for luminescence resonance energy transfer to take place and the acceptor fluorophore will fluoresce.
  • this method allows for the detection of interaction between the first protein and the second protein even though the interaction cannot be detected by optical methods such as conventional microscopy.
  • the specific labeling of the proteins in living cells can be achieved through genetic engineering methods where the introduction of fluorescent dyes into living cells is very difficult. Further, fluorescent dyes photobleach quickly while light emission of a luciferase such as Renilla luciferase originates from an enzymatic reaction that is relatively stable if substrate and oxygen are supplemented.
  • complexing a first protein to the donor luciferase refers to joining the donor luciferase to the first protein in a manner that the donor luciferase and the first protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein.
  • complexing a second protein to the acceptor fluorophore refers to joining the acceptor fluorophore to the second protein in a manner that the acceptor fluorophore and the second protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein.
  • Such complexing can be done, for example, by genetically engineering the cell to produce a fusion protein containing the donor luciferase and first protein, and the acceptor fluorophore and the second protein.
  • the present invention uses Renilla luciferase as the donor luciferase and "humanized" Aequorea green fluorescent protein ('humanized' GFP) as the acceptor fluorophore.
  • Renilla luciferase is a 34 kDa enzyme purified from Renilla reniformis. The enzyme catalyzes the oxidative decarboxylation of coelenterazine in the presence of oxygen to produce blue light with an emission wavelength maximum of 471 nm.
  • Renilla luciferase was used as the donor luciferase because it requires an exogenous substrate rather than exogenous light for excitation. This, advantageously, eliminates background noise from an exogenous light source and from autofluorescence, and allows easy and accurate quantitative determination of light production.
  • 'Humanized' GFP is a 27 kDa protein fluorophore that has an excitation maximum at 480 nm. It has a single amino acid difference from wild-type Aequorea green fluorescent protein. 'Humanized' GFP was chosen as the acceptor fluorophore because its excitation spectrum overlaps with the emission spectra of Renilla luciferase. Additionally, emissions from 'humanized' GFP can be visualized in living cells. Further, 'humanized'
  • GFP is expressed well in the mammalian cells transfected with 'humanized' GFP cDNA that were used to demonstrate this method.
  • IGFBP 6 insulin-like growth factor binding protein 6
  • IGF- II insulin-like growth factor II
  • the Renilla luciferase cDNA was fused to IGFBP 6 cDNA and 'humanized' GFP cDNA was fused to IGF-II cDNA.
  • Living cells were transfected with the fused cDNAs and the fusion proteins were expressed. Cell extracts were produced and mixed.
  • the substrate for the Renilla luciferase moiety of the fused Renilla luciferase-IGFPB 6 protein was added. Finally, fluorescence from the 'humanized' GFP moiety of the fused 'humanized' GFP-IGF-II protein was detected. Demonstration one method according to the present invention will now be described in greater detail.
  • IGFBP-6 cDNA SEQ ID NO: l, GenBank accession number M69054, encoded IGFBP-6, SEQ ID NO:2, which was used as the first protein.
  • Renilla luciferase cDNA SEQ ID NO:3, GenBank accession number M63501, encoded Renilla luciferase, SEQ ID NO:4, which was used as the donor luciferase.
  • Insulin cDNA SEQ ID NO:9, accession number AH002844, encoded insulin, SEQ ID NO: 10.
  • Insulin, fused to 'humanized' GFP was used as a control protein because insulin is homologous to IGF-II, but it does not bind to IGFBP-6.
  • the cDNA of prepro-IGF-II carried on an EcoRI fragment was cloned into pBluescript KS (+) II vector.
  • the insert was sequenced using T7 and T3 primers and confirmed to contain the known cDNA sequence of prepro-IGF-II.
  • the 5' end of the IGF-II precursor was connected to the T7 promoter in the pBluescript KS (+) II vector.
  • An IGF-II 3' primer was designed to generate a Notice of Allowance restriction site, to remove the D and E domains of prepro-IGF-II, and to maintain the Notice of Allowance fragment of the 'humanized' GFP in frame with the open reading frame of IGF-II.
  • the IGF-II fragment was amplified with PCR using the T7 promoter primer and the IGF-II 3' primer.
  • the PCR-amplified IGF-II fragment was digested by EcoRI and Not I and cloned into pCDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, US) producing pCDNA-IGF-II.
  • the Notice of Allowance fragment of the 'humanized' GFP was inserted into the Not I site of pCDNA-IGF-II producing pC-IGF-II-GFP.
  • the cDNA for precursor of insulin which contained a signal peptide the B, C and A domains, was modified in a manner corresponding to the IGF-II fragment, above.
  • the 'humanized' GFP cDNA was then linked to the 3' end of the modified insulin cDNA to produce pC-INS-GFP.
  • IGFBP 6 cDNA was amplified by PCR from a plasmid named
  • Rat-tagged human IGFBP6 Rat-tagged human IGFBP6.
  • the stop codon of IGFBP 6 was removed and the open reading frame of IGFBP 6 was in frame with Renilla luciferase cDNA from pCEP4-RUC (Mayerhofer R, Langridge WHR, Cormier MG and Szalay AA. Expression of recombinant Renilla luciferase in trans genie plants results in high levels of light emission. The Plant Journal 1995 ;7; 1031-8).
  • the linking of the Renilla luciferase cDNA to the 3' end of modified IGFBP 6 cDNA produced pC-IGFBP 6-RUC.
  • COS-7 cells African green monkey kidney cell, American Type Culture Collection CRL 1651
  • DMEM Dulbecco's Modified Eagle Medium
  • streptomycin 100 mg/ml antibiotic antimycotic solution containing a final concentration of penicillin 100 unit/ml, streptomycin 100 mg/ml and amphotericin B 250 ng/ml (Sigma-Aldrich Co., St. Louis, MO, US) in 5% CO 2 .
  • Groups of 1x10° of these cells were plated the day before transfection and were approximately 50% to 60% confluent at the time of transfection. Forty mg of each plasmid fusion DNA were precipitated and resuspended into
  • fusion proteins IGF-II-GFP and IGFBP 6-RUC having the expected molecular weights of about 36 kDa and 56 kDa, respectively, were detected using immunoblot analysis. This confirmed the presence of both fusion proteins in the transiently transfected cells.
  • cell extracts from these transiently transfected cells were used to carry out a protein binding assay based on energy transfer between the Renilla luciferase and 'humanized' GFP moieties of the fusion proteins.
  • the COS cells were washed twice with PBS and harvested using a cell scraper in luciferase assay buffer containing 0.5 M NaCl, 1 mM EDTA and 0.1 M potassium phosphate at a pH 7.5.
  • the harvested cells were sonicated 3 times for 10 seconds with an interval of 10 seconds using a Fisher Model 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, US) to produce cell extracts.
  • the cell extracts containing IGF-II-GFP and IGFBP 6-RUC were mixed and 0.1 ⁇ g of coelenterazine was immediately added.
  • Spectrofluorometry was performed using a SPEX FluoroMax ® (Instruments S.A., Inc., Edison, NJ). The spectrum showed a single emission peak at 471 nm, which corresponds to the known emission of Renilla luciferase.
  • the spectrofluorometry of the cell extracts was carried out at a longer time, but the spectral pattern did not change over time.
  • Control cell extract mixtures from cells transfected with pC-INS-GFP and pC-IGFBP 6-RUC were made similarly and their spectra traced.
  • the traces showed only one peak at 471 nm, which corresponds to the emission peak of Renilla luciferase.
  • the spectral pattern did not change over time.
  • protein-protein interactions were also detected by the detection of LRET using corresponding methods in E. coli cells and mammalian cells which were co-transformed.

Abstract

A method for determining whether a first protein interacts with a second protein within a living cell. The method comprises providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore within the cell. The complexed first protein and the complexed second protein are allowed to come into proximity to each other within the cell. Then, any fluorescence from the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase is detected, where fluorescence from the acceptor fluorophore indicates that the first protein has interacted with the second protein.

Description

METHOD FOR STUDYING PROTEIN INTERACTIONS IN VIVO
BACKGROUND
The study of interactions between proteins in living cells is often necessary to understand the proteins' functions and their mechanisms of action. These interactions are currently studied using immuno-precipitation, the yeast two hybrid method, and β-gal complementation method. However, these methods are associated with several disadvantages. For example, these methods are associated with false positives. Second, they do not permit the determination of quantitative information regarding the interactions. Further, they do not allow for in vivo real time monitoring of the interactions.
Therefore, it would be advantageous to have another method of studying interactions between proteins in vivo, which does not have these disadvantages. Further preferably, the method could be used with a wide variety of proteins and in a wide variety of living cells. Also preferably, the method could be used to determine the interactions between molecules other than proteins.
SUMMARY According to one embodiment of the present invention, there is provided a method for determining whether a first protein interacts with a second protein within a living cell. The method comprises providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore within the cell. The donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore when the first protein is in proximity to the second protein. Then, the complexed first protein and the complexed second protein are allowed to come into proximity to each other within the cell. Next, any fluorescence from the acceptor fluorophore is detected. Fluorescence of the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase to acceptor fluorophore the indicates that the first protein has interacted with the second protein.
In a preferred embodiment, providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore comprises genetically engineering DNA and transferring the genetically engineered DNA to the living cell causing the cell to produce the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore. In a particularly preferred embodiment, the cell which is provided with the first protein complexed to a donor luciferase and the cell which is provided with the second protein complexed to an acceptor fluorophore are mammalian cells.
In another preferred embodiment, the donor luciferase provided is Renilla luciferase. In yet another preferred embodiment, the acceptor fluorophore provided is an Aequorea green fluorescent protein.
In a particularly preferred embodiment, the detection of acceptor fluorophore fluorescence is performed using spectrofluorometery.
DESCRIPTION The present invention includes a method for determining whether a first protein interacts with a second protein in a living cell using luminescent resonance energy transfer (LRET). Luminescence resonance energy transfer results from the transfer of excited state energy from a donor luciferase to an acceptor fluorophore. In order for LRET to occur, there must be an overlap between the emission spectrum of the donor luciferase and the excitation spectrum of the acceptor fluorophore. The efficiency of luminescence resonance energy transfer is dependent on the distance separating the donor luciferase and the acceptor fluorophore, among other variables. Generally, significant energy transfers occur only where the donor luciferase and acceptor fluorophore are less than about 80 A of each other. This short distance is considerably less than the distance needed between for optical resolution between two entities using conventional microscopy. Therefore, detecting luminescence resonance energy transfer between a donor luciferase and an acceptor fluorophore indicates that the donor luciferase and acceptor fluorophore have come within the distance needed for LRET to occur, that is less than about 80 A of each other.
The present invention utilizes luminescence resonance energy transfer to determine whether an interaction takes place between a first protein and a second protein in a living cell. This is accomplished by complexing a first protein to the donor luciferase and complexing the second protein to the acceptor fluorophore and placing the complexed first protein and the complexed second protein in the cell under conditions suitable for an interaction between the first protein and the second protein to take place. If the first protein interacts with the second protein, the donor luciferase will come close enough to the acceptor fluorophore for luminescence resonance energy transfer to take place and the acceptor fluorophore will fluoresce. Detection of fluorescence from the acceptor fluorophore will, thereby, indicate that the first protein has interacted with the second protein. Advantageously, this method allows for the detection of interaction between the first protein and the second protein even though the interaction cannot be detected by optical methods such as conventional microscopy.
There are several advantages of using luminescent resonance energy transfer to detect the interaction between two proteins according to the present invention. First, the specific labeling of the proteins in living cells can be achieved through genetic engineering methods where the introduction of fluorescent dyes into living cells is very difficult. Further, fluorescent dyes photobleach quickly while light emission of a luciferase such as Renilla luciferase originates from an enzymatic reaction that is relatively stable if substrate and oxygen are supplemented.
As used in this disclosure, "complexing a first protein to the donor luciferase" refers to joining the donor luciferase to the first protein in a manner that the donor luciferase and the first protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein. Similarly, "complexing a second protein to the acceptor fluorophore" refers to joining the acceptor fluorophore to the second protein in a manner that the acceptor fluorophore and the second protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein. Such complexing can be done, for example, by genetically engineering the cell to produce a fusion protein containing the donor luciferase and first protein, and the acceptor fluorophore and the second protein.
In a preferred embodiment, the present invention uses Renilla luciferase as the donor luciferase and "humanized" Aequorea green fluorescent protein ('humanized' GFP) as the acceptor fluorophore. Renilla luciferase is a 34 kDa enzyme purified from Renilla reniformis. The enzyme catalyzes the oxidative decarboxylation of coelenterazine in the presence of oxygen to produce blue light with an emission wavelength maximum of 471 nm. Renilla luciferase was used as the donor luciferase because it requires an exogenous substrate rather than exogenous light for excitation. This, advantageously, eliminates background noise from an exogenous light source and from autofluorescence, and allows easy and accurate quantitative determination of light production.
'Humanized' GFP is a 27 kDa protein fluorophore that has an excitation maximum at 480 nm. It has a single amino acid difference from wild-type Aequorea green fluorescent protein. 'Humanized' GFP was chosen as the acceptor fluorophore because its excitation spectrum overlaps with the emission spectra of Renilla luciferase. Additionally, emissions from 'humanized' GFP can be visualized in living cells. Further, 'humanized'
GFP is expressed well in the mammalian cells transfected with 'humanized' GFP cDNA that were used to demonstrate this method.
The method for determining whether a first protein interacts with a second protein according to the present invention was demonstrated as follows. In summary, insulin-like growth factor binding protein 6 (IGFBP 6) and insulin-like growth factor II (IGF- II) were selected as the first protein and second protein. IGFBP 6 is a protein known to have a marked binding affinity for IGF-II.
The Renilla luciferase cDNA was fused to IGFBP 6 cDNA and 'humanized' GFP cDNA was fused to IGF-II cDNA. Living cells were transfected with the fused cDNAs and the fusion proteins were expressed. Cell extracts were produced and mixed. The substrate for the Renilla luciferase moiety of the fused Renilla luciferase-IGFPB 6 protein was added. Finally, fluorescence from the 'humanized' GFP moiety of the fused 'humanized' GFP-IGF-II protein was detected. Demonstration one method according to the present invention will now be described in greater detail.
A) The Cloning of Fused IGFBP-6 cDNA to Renilla Luciferase cDNA; Fused IGF-II cDNA to 'humanized' GFP cDNA; and Fused Insulin cDNA to 'humanized' GFP cDNA: First, three fused cDNAs were produced: 1) fused IGFBP-6 cDNA and Renilla luciferase cDNA; 2) fused IGF-II cDNA and 'humanized' GFP cDNA; and 3) fused insulin cDNA and 'humanized' GFP cDNA. IGFBP-6 cDNA, SEQ ID NO: l, GenBank accession number M69054, encoded IGFBP-6, SEQ ID NO:2, which was used as the first protein. Renilla luciferase cDNA, SEQ ID NO:3, GenBank accession number M63501, encoded Renilla luciferase, SEQ ID NO:4, which was used as the donor luciferase. IGF-II cDNA, SEQ ID NO:5, encoded IGF-II, SEQ ID NO:6, which was used as the second protein. 'Humanized' GFP cDNA, SEQ ID NO:7, GenBank accession numberU50963, encoded 'humanized' GFP, SEQ ID NO: 8, which was used as the acceptor fluorophore. Insulin cDNA, SEQ ID NO:9, accession number AH002844, encoded insulin, SEQ ID NO: 10. Insulin, fused to 'humanized' GFP, was used as a control protein because insulin is homologous to IGF-II, but it does not bind to IGFBP-6. The IGFBP-6 cDNA, SEQ ID NO:l, IGF-II cDNA, SEQ ID NO:5, and insulin cDNA, SEQ ID NO:9, were modified using PCR as follows. First, the cDNA of prepro-IGF-II carried on an EcoRI fragment was cloned into pBluescript KS (+) II vector. The insert was sequenced using T7 and T3 primers and confirmed to contain the known cDNA sequence of prepro-IGF-II. The 5' end of the IGF-II precursor was connected to the T7 promoter in the pBluescript KS (+) II vector. An IGF-II 3' primer was designed to generate a Notice of Allowance restriction site, to remove the D and E domains of prepro-IGF-II, and to maintain the Notice of Allowance fragment of the 'humanized' GFP in frame with the open reading frame of IGF-II.
Next, the IGF-II fragment was amplified with PCR using the T7 promoter primer and the IGF-II 3' primer. The PCR-amplified IGF-II fragment was digested by EcoRI and Not I and cloned into pCDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, US) producing pCDNA-IGF-II. Then, the Notice of Allowance fragment of the 'humanized' GFP was inserted into the Not I site of pCDNA-IGF-II producing pC-IGF-II-GFP. The cDNA for precursor of insulin, which contained a signal peptide the B, C and A domains, was modified in a manner corresponding to the IGF-II fragment, above. The 'humanized' GFP cDNA was then linked to the 3' end of the modified insulin cDNA to produce pC-INS-GFP. Finally, IGFBP 6 cDNA was amplified by PCR from a plasmid named
Rat-tagged human IGFBP6. The stop codon of IGFBP 6 was removed and the open reading frame of IGFBP 6 was in frame with Renilla luciferase cDNA from pCEP4-RUC (Mayerhofer R, Langridge WHR, Cormier MG and Szalay AA. Expression of recombinant Renilla luciferase in trans genie plants results in high levels of light emission. The Plant Journal 1995 ;7; 1031-8). The linking of the Renilla luciferase cDNA to the 3' end of modified IGFBP 6 cDNA produced pC-IGFBP 6-RUC.
The sequences of the insert DNA fragments from all the constructs were verified by DNA sequencing analysis. Qiagen Maxi Plasmid Kit (Qiagen, Inc., Valencia, CA) was used for the purification of plasmid DNA. B) Transient Transfection of Mammalian Cells With pC-IGF-II-GFP, pC-INS-GFP and pC-IGFBP 6-RUC Using the Calcium Phosphate Precipitation Method:
Next, mammalian cells were transfected with the cloned fusion DNAs. First, COS-7 cells (African green monkey kidney cell, American Type Culture Collection CRL 1651) were grown at 37 C in Dulbecco's Modified Eagle Medium (DMEM) with L-Glutamine supplemented with 10% fetal bovine serum and antibiotic antimycotic solution containing a final concentration of penicillin 100 unit/ml, streptomycin 100 mg/ml and amphotericin B 250 ng/ml (Sigma-Aldrich Co., St. Louis, MO, US) in 5% CO2. Groups of 1x10° of these cells were plated the day before transfection and were approximately 50% to 60% confluent at the time of transfection. Forty mg of each plasmid fusion DNA were precipitated and resuspended into
Dulbecco's Phosphate Buffered Saline Solution and the plasmid fusion DNAs was introduced into mammalian cells using the standard calcium phosphate precipitation method. Transfection efficiency was estimated by fluorescence microscopy after 24 hours. The number of green fluorescent cells per plate were comparable in plates of pC-IGF-II-GFP DNA transfected cells, pC-INS-GFP DNA transfected cells and cells transfected with a plasmid DNA containing GFP only, which was used as a positive control. C) Confirmation of Expression of Fusion Proteins:
Twenty-four hours after DNA transfection using DNA calcium phosphate precipitation method, individual plasmid DNA transfected COS-7 cells were visualized using fluorescence microscopy by detection of GFP fluorescence. pC-IGF-II-GFP and pC-INS-GFP transfected cells showed similar fluorescence patterns typical of secretory protein translocated through ER to Golgi. The pC-IGFBP 6-RUC transfected cells did not fluoresce. However, the pC-IGFBP 6-RUC transfected cells did show luminescence using a low light imaging system after the addition of coelenterazine.
Further, the presence of fusion proteins IGF-II-GFP and IGFBP 6-RUC, having the expected molecular weights of about 36 kDa and 56 kDa, respectively, were detected using immunoblot analysis. This confirmed the presence of both fusion proteins in the transiently transfected cells.
D) Detection of Protein Interactions by Spectrofluorometry:
Having confirmed the presence of the expected fusion proteins IGF-II-GFP and IGFBP 6-RUC, and the function of the donor luciferase and acceptor fluorophore, cell extracts from these transiently transfected cells were used to carry out a protein binding assay based on energy transfer between the Renilla luciferase and 'humanized' GFP moieties of the fusion proteins. Forty-eight hours after calcium transfection, the COS cells were washed twice with PBS and harvested using a cell scraper in luciferase assay buffer containing 0.5 M NaCl, 1 mM EDTA and 0.1 M potassium phosphate at a pH 7.5. The harvested cells were sonicated 3 times for 10 seconds with an interval of 10 seconds using a Fisher Model 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, US) to produce cell extracts.
Next, the cell extracts containing IGF-II-GFP and IGFBP 6-RUC were mixed and 0.1 μg of coelenterazine was immediately added. Spectrofluorometry was performed using a SPEX FluoroMax® (Instruments S.A., Inc., Edison, NJ). The spectrum showed a single emission peak at 471 nm, which corresponds to the known emission of Renilla luciferase.
Following the first spectrofluorometry, the mixtures were kept at room temperature for 30 minutes and the spectra were traced again after fresh coelenterazine was added. The trace at 30 minutes showed two peaks with emission maximum at 471 nm and
503 nm. The spectrofluorometry of the cell extracts was carried out at a longer time, but the spectral pattern did not change over time.
Control cell extract mixtures from cells transfected with pC-INS-GFP and pC-IGFBP 6-RUC were made similarly and their spectra traced. The traces showed only one peak at 471 nm, which corresponds to the emission peak of Renilla luciferase. The spectral pattern did not change over time.
Therefore, these data demonstrated that IGFBP 6 and IGF-II interacted but that insulin and IGFBP 6 did not interact.
In addition to the above disclosed examples, protein-protein interactions were also detected by the detection of LRET using corresponding methods in E. coli cells and mammalian cells which were co-transformed.
Although the present invention has been discussed in considerable detail with reference to certain preferred embodiments, other embodiments are possible. For example, the interaction between molecules other than proteins could be studied by corresponding methods. Such other molecules could be provided to the living cell by diffusion, infusion, and incorporation or by other means. Further, fusion proteins produced from genetically engineered living cells could have post translational changes, such as the addition of sugar moieties, before their interactions are studied. Also, living cells can be visualized using these methods by spectrofluorometry by low light image analysis in cells, colonies and tissues. Additionally, high through put screening of colonies can be accomplished using the present methods combined with cell sorting and low light video analysis of micro titre dishes or multiple array detection. Therefore, the spirit and scope of the appended claims should not be limited to the description of preferred embodiments contained herein.

Claims

WHAT IS CLAIMED IS:
1. A method for determining whether a first protein interacts with a second protein within a living cell, the method comprising: a) providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore within the cell; b) placing the complexed first protein and the complexed second protein in proximity to each other within the cell; and c) detecting any fluorescence from the acceptor fluorophore; where the donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore when the first protein is in proximity to the second protein; and where fluorescence of the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase indicates that the first protein has interacted with the second protein.
2. The method of claim 1, where providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore comprises genetically engineering DNA and transferring the genetically engineered DNA to the living cell causing the cell to produce the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore.
3. The method of claim 1 , where the cell provided with the first protein complexed to a donor luciferase is a mammalian cell.
4. The method of claim 1, where the cell provided with the second protein complexed to a acceptor fluorophore is a mammalian cell.
5. The method of claim 1, where the donor luciferase provided is Renilla luciferase.
6. The method of claim 1, where the acceptor fluorophore provided is a green fluorescent protein.
7. The method of claim 1, where the acceptor fluorophore provided is an Aequorea green fluorescent protein.
8. The method of claim 1, where detecting any fluorescence from the donor luciferase is performed using spectrofluorometery.
9. A method for determining whether a first molecule interacts with a second molecule within a living cell, the method comprising: a) providing the first molecule complexed to a donor luciferase and the second molecule complexed to an acceptor fluorophore within the cell; b) placing the complexed first molecule and the complexed second molecule in proximity to each other within the cell; and c) detecting any fluorescence from the acceptor fluorophore; where the donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore when the first molecule is in proximity to the second molecule; and where fluorescence of the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase indicates that the first molecule has interacted with the second molecule.
10. The method of claim 9, where the first molecule is a first protein and where the second molecule is a second protein; and where providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore comprises genetically engineering DNA and transferring the genetically engineered DNA to the living cell causing the cell to produce the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore.
11. The method of claim 10, where the cell provided with the first protein complexed to a donor luciferase is a mammalian cell.
12. The method of claim 10, where the cell provided with the second protein complexed to a acceptor fluorophore is a mammalian cell.
13. The method of claim 9, where the donor luciferase provided is Renilla luciferase.
14. The method of claim 9, where the acceptor fluorophore provided is a green fluorescent protein.
15. The method of claim 9, where the acceptor fluorophore provided is a Aequorea green fluorescent protein.
16. The method of claim 9, where detecting any fluorescence from the donor luciferase is performed using spectrofluorometery.
17. A method for determining whether a first protein interacts with a second protein, the method comprising: a) providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore; b) placing the complexed first protein and the complexed second protein in proximity to each other; and c) detecting any fluorescence from the acceptor fluorophore; where the donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore when the first protein is in proximity to the second protein; and where fluorescence of the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase indicates that the first protein has interacted with the second protein.
18. The method of claim 17, where providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore comprises genetically engineering DNA and transferring the genetically engineered DNA to a living cell causing the cell to produce the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore.
19. The method of claim 18, where the cell provided with the first protein complexed to a donor luciferase is a mammalian cell.
20. The method of claim 18, where the cell provided with the second protein complexed to a acceptor fluorophore is a mammalian cell.
21. The method of claim 17, where the donor luciferase provided is Renilla luciferase.
22. The method of claim 17, where the acceptor fluorophore provided is a green fluorescent protein.
23. The method of claim 17, where the acceptor fluorophore provided is an Aequorea green fluorescent protein.
24. The method of claim 17, where detecting any fluorescence from the donor luciferase is performed using spectrofluorometery.
25. A method for determining whether a first molecule interacts with a second molecule, the method comprising: a) providing the first molecule complexed to a donor luciferase and the second molecule complexed to an acceptor fluorophore; b) placing the complexed first molecule and the complexed second molecule in proximity to each other; and c) detecting any fluorescence from the acceptor fluorophore; where the donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore when the first molecule is in proximity to the second molecule; and where fluorescence of the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase indicates that the first molecule has interacted with the second molecule.
26. The method of claim 25, where the first molecule is a first protein and where the second molecule is a second protein; and where providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore comprises genetically engineering DNA and transferring the genetically engineered DNA to a living cell causing the cell to produce the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore.
27. The method of claim 26, where the cell provided with the first protein complexed to a donor luciferase is a mammalian cell.
28. The method of claim 26, where the cell provided with the second protein complexed to a acceptor fluorophore is a mammalian cell.
29. The method of claim 25, where the donor luciferase provided is Renilla luciferase.
30. The method of claim 25, where the acceptor fluorophore provided is a green fluorescent protein.
31. The method of claim 25, where the acceptor fluorophore provided is a Aequorea green fluorescent protein.
32. The method of claim 25, where detecting any fluorescence from the donor luciferase is performed using spectrofluorometery.
PCT/US1999/020207 1998-09-03 1999-09-02 METHOD FOR STUDYING PROTEIN INTERACTIONS $i(IN VIVO) WO2000014271A1 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
AU58056/99A AU752675B2 (en) 1998-09-03 1999-09-02 Method for studying protein interactions (in vivo)
CA002341314A CA2341314A1 (en) 1998-09-03 1999-09-02 Method for studying protein interactions in vivo
EP99945460A EP1109931A4 (en) 1998-09-03 1999-09-02 METHOD FOR STUDYING PROTEIN INTERACTIONS $i(IN VIVO)
JP2000569011A JP2002524087A (en) 1998-09-03 1999-09-02 How to study protein interactions in vivo

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US9906898P 1998-09-03 1998-09-03
US60/099,068 1998-09-03
US13583599P 1999-05-24 1999-05-24
US60/135,835 1999-05-24

Publications (1)

Publication Number Publication Date
WO2000014271A1 true WO2000014271A1 (en) 2000-03-16

Family

ID=26795497

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/020207 WO2000014271A1 (en) 1998-09-03 1999-09-02 METHOD FOR STUDYING PROTEIN INTERACTIONS $i(IN VIVO)

Country Status (6)

Country Link
EP (1) EP1109931A4 (en)
JP (1) JP2002524087A (en)
CN (1) CN1160470C (en)
AU (1) AU752675B2 (en)
CA (1) CA2341314A1 (en)
WO (1) WO2000014271A1 (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1533619A2 (en) 2003-11-20 2005-05-25 F. Hoffmann-La Roche Ag Specific markers for metabolic syndrome
US7771952B2 (en) 2002-06-26 2010-08-10 Abott Laboratories Modulators and modulation of the interaction between RGM and Neogenin
WO2011083147A1 (en) 2010-01-08 2011-07-14 Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh Wave1 inhibition in the medical intervention of inflammatory diseases and/or infections caused by a pathogen
WO2011131626A1 (en) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 encodes for tetrahydrobiopterin-dependent alkylglycerol monooxygenase activity
US8647887B2 (en) 2009-01-29 2014-02-11 Commonwealth Scientific And Industrial Research Organisation Measuring G protein coupled receptor activation
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US9175075B2 (en) 2009-12-08 2015-11-03 AbbVie Deutschland GmbH & Co. KG Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein
US10415960B2 (en) 2015-04-06 2019-09-17 Worldvu Satellites Limited Elevation angle estimating system and method for user terminal placement
US11579149B2 (en) 2017-11-01 2023-02-14 Queen's University At Kingston Hippo pathway bioluminescent biosensor

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1920021B (en) * 2005-08-24 2010-05-05 中国医学科学院基础医学研究所 Preparation method of activated insulin-like growth factor-II mediated by insulin-like growth factor binding protein-6
CN101620233B (en) * 2009-05-27 2012-10-31 华中科技大学 Method for detecting interaction of proteins
CN102798717B (en) * 2012-06-15 2014-11-26 杭州师范大学 Method for detecting activity of O6-methylguanine-DNA (Deoxyribose Necleic Acid) methyltransferase
CN103616502B (en) * 2013-09-12 2016-05-25 西北农林科技大学 Based on the method for bacterial luciferase BRET technology for detection protein interaction
CN110794129B (en) * 2018-08-01 2020-12-01 清华大学 Method for detecting interaction between biological molecules and regulating factor thereof in cell and used reagent

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4318707A (en) * 1978-11-24 1982-03-09 Syva Company Macromolecular fluorescent quencher particle in specific receptor assays
US4604364A (en) * 1974-01-04 1986-08-05 Kosak Kenneth M Bioluminescent tracer composition and method of use in immunoassays
US5418155A (en) * 1989-12-29 1995-05-23 University Of Georgia Research Foundation, Inc. Isolated Renilla luciferase and method of use thereof
US5491084A (en) * 1993-09-10 1996-02-13 The Trustees Of Columbia University In The City Of New York Uses of green-fluorescent protein
US5683888A (en) * 1989-07-22 1997-11-04 University Of Wales College Of Medicine Modified bioluminescent proteins and their use
US5811238A (en) * 1994-02-17 1998-09-22 Affymax Technologies N.V. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5866348A (en) * 1993-02-10 1999-02-02 Parkard Instrument Company, Inc. Bioluminescence measurement system
US5891646A (en) * 1997-06-05 1999-04-06 Duke University Methods of assaying receptor activity and constructs useful in such methods
US5976796A (en) * 1996-10-04 1999-11-02 Loma Linda University Construction and expression of renilla luciferase and green fluorescent protein fusion genes

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES8607563A1 (en) * 1983-10-13 1986-06-01 Univ Georgia Res Found Bioluminescent immunoassays.
WO1999066324A2 (en) * 1998-06-16 1999-12-23 Biosignal Packard Inc. A bioluminescence resonance energy transfer (bret) system and its use

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4604364A (en) * 1974-01-04 1986-08-05 Kosak Kenneth M Bioluminescent tracer composition and method of use in immunoassays
US4318707A (en) * 1978-11-24 1982-03-09 Syva Company Macromolecular fluorescent quencher particle in specific receptor assays
US5683888A (en) * 1989-07-22 1997-11-04 University Of Wales College Of Medicine Modified bioluminescent proteins and their use
US5418155A (en) * 1989-12-29 1995-05-23 University Of Georgia Research Foundation, Inc. Isolated Renilla luciferase and method of use thereof
US5866348A (en) * 1993-02-10 1999-02-02 Parkard Instrument Company, Inc. Bioluminescence measurement system
US5491084A (en) * 1993-09-10 1996-02-13 The Trustees Of Columbia University In The City Of New York Uses of green-fluorescent protein
US5811238A (en) * 1994-02-17 1998-09-22 Affymax Technologies N.V. Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
US5976796A (en) * 1996-10-04 1999-11-02 Loma Linda University Construction and expression of renilla luciferase and green fluorescent protein fusion genes
US5891646A (en) * 1997-06-05 1999-04-06 Duke University Methods of assaying receptor activity and constructs useful in such methods

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1109931A4 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7771952B2 (en) 2002-06-26 2010-08-10 Abott Laboratories Modulators and modulation of the interaction between RGM and Neogenin
EP2053409A1 (en) 2003-11-20 2009-04-29 F. Hoffmann-La Roche Ag Specific markers for metabolic syndrome
EP1533619A2 (en) 2003-11-20 2005-05-25 F. Hoffmann-La Roche Ag Specific markers for metabolic syndrome
US8906864B2 (en) 2005-09-30 2014-12-09 AbbVie Deutschland GmbH & Co. KG Binding domains of proteins of the repulsive guidance molecule (RGM) protein family and functional fragments thereof, and their use
US8962803B2 (en) 2008-02-29 2015-02-24 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM A protein and uses thereof
US9605069B2 (en) 2008-02-29 2017-03-28 AbbVie Deutschland GmbH & Co. KG Antibodies against the RGM a protein and uses thereof
US8647887B2 (en) 2009-01-29 2014-02-11 Commonwealth Scientific And Industrial Research Organisation Measuring G protein coupled receptor activation
US9175075B2 (en) 2009-12-08 2015-11-03 AbbVie Deutschland GmbH & Co. KG Methods of treating retinal nerve fiber layer degeneration with monoclonal antibodies against a retinal guidance molecule (RGM) protein
WO2011083147A1 (en) 2010-01-08 2011-07-14 Cemm-Forschungsinstitut Für Molekulare Medizin Gmbh Wave1 inhibition in the medical intervention of inflammatory diseases and/or infections caused by a pathogen
WO2011131626A1 (en) 2010-04-19 2011-10-27 Medizinische Universität Innsbruck Tmem195 encodes for tetrahydrobiopterin-dependent alkylglycerol monooxygenase activity
US9102722B2 (en) 2012-01-27 2015-08-11 AbbVie Deutschland GmbH & Co. KG Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration
US9365643B2 (en) 2012-01-27 2016-06-14 AbbVie Deutschland GmbH & Co. KG Antibodies that bind to repulsive guidance molecule A (RGMA)
US10106602B2 (en) 2012-01-27 2018-10-23 AbbVie Deutschland GmbH & Co. KG Isolated monoclonal anti-repulsive guidance molecule A antibodies and uses thereof
US10415960B2 (en) 2015-04-06 2019-09-17 Worldvu Satellites Limited Elevation angle estimating system and method for user terminal placement
US11579149B2 (en) 2017-11-01 2023-02-14 Queen's University At Kingston Hippo pathway bioluminescent biosensor

Also Published As

Publication number Publication date
EP1109931A4 (en) 2004-12-15
CA2341314A1 (en) 2000-03-16
CN1160470C (en) 2004-08-04
JP2002524087A (en) 2002-08-06
AU752675B2 (en) 2002-09-26
CN1323353A (en) 2001-11-21
AU5805699A (en) 2000-03-27
EP1109931A1 (en) 2001-06-27

Similar Documents

Publication Publication Date Title
EP1109931A1 (en) METHOD FOR STUDYING PROTEIN INTERACTIONS $i(IN VIVO)
JP4459058B2 (en) Photoprotein with improved bioluminescence
WO2019174633A1 (en) Fluorescent probe for branched chain amino acids and use thereof
US20140329718A1 (en) Nicotinamide Adenine Dinucleotide Gene Encoding Fluorescent Probe, Preparation Method Therefor and Application Thereof
CN113603756B (en) Corynebacterium glutamicum membrane protein Ncgl2775, surface display system and construction method thereof
JP2007508841A5 (en)
Wang et al. A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP
US8552151B2 (en) Mutant blue fluorescent protein and method of using the same for fluorescence energy transfer and blue fluorescent fish
CN111269324B (en) Fusion protein of Gauss luciferase and digoxin single-chain antibody and application thereof
CN113801211B (en) Corynebacterium glutamicum protein Ncgl0717, surface display system and construction method thereof
AU2012227790B2 (en) Probe for analyzing biological tissue and method for utilizing same
JP2011211983A (en) Protein molecule pair, gene encoding protein molecule pair, and cell producing gene transfer vector and protein molecule pair
JP6051438B2 (en) Calcium sensor protein using red fluorescent protein
EP1840213A1 (en) Target physiological function inactivator using photosensitizer-labeled fluorescent protein
CN104277120B (en) NAD gene code fluorescence probe and its preparation method and application
CN116068198B (en) PPI in-situ detection method and carrier, diagnostic reagent, kit and application thereof
KR20200137596A (en) Composition for diagnosis acute kidney transplant rejection reaction and diagnostic kit containing the same
CN102181465B (en) Fluorescent clone screening vector and preparation and application thereof
KR102210877B1 (en) Flavin mononucleotide binding protein variants derived from Arabidopsis thaliana with enhanced fluorescence intensity
WO2021253126A1 (en) Cleavable linkers for protein translation reporting
CN117551208A (en) L-2-hydroxyglutarate biosensor based on cyclic rearrangement fluorescent protein and application thereof
WO2024056560A1 (en) Novel phagocytosis assay combining a synthetic cell death switch and a phagocytosis reporter system
ES2445018B1 (en) VARIANTS OF THE CALCIUM SENSITIVE FUSION PROTEIN tdTOMATO-AEQUORINA
JP2005287327A (en) Method for synthesis of new protein labeled with site-specific fluorescence
NZ614600B2 (en) Probe for analyzing biological tissue and method for utilizing same

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 99811958.X

Country of ref document: CN

AK Designated states

Kind code of ref document: A1

Designated state(s): AE AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM EE ES FI GB GD GE GH GM HR HU ID IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SL SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
WWE Wipo information: entry into national phase

Ref document number: 09786377

Country of ref document: US

ENP Entry into the national phase

Ref document number: 2000 569011

Country of ref document: JP

Kind code of ref document: A

WWE Wipo information: entry into national phase

Ref document number: 58056/99

Country of ref document: AU

WWE Wipo information: entry into national phase

Ref document number: 1999945460

Country of ref document: EP

WWP Wipo information: published in national office

Ref document number: 1999945460

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

ENP Entry into the national phase

Ref document number: 2341314

Country of ref document: CA

Ref document number: 2341314

Country of ref document: CA

Kind code of ref document: A

WWG Wipo information: grant in national office

Ref document number: 58056/99

Country of ref document: AU

WWW Wipo information: withdrawn in national office

Ref document number: 1999945460

Country of ref document: EP