AU752675B2

AU752675B2 - Method for studying protein interactions (in vivo)

Info

Publication number: AU752675B2
Application number: AU58056/99A
Authority: AU
Inventors: Aladar A. Szalay; Yubao Wang; Gefu Wang-Pruski
Original assignee: Loma Linda University
Current assignee: Loma Linda University
Priority date: 1998-09-03
Filing date: 1999-09-02
Publication date: 2002-09-26
Anticipated expiration: 2019-09-02
Also published as: CN1323353A; WO2000014271A1; AU5805699A; CN1160470C; EP1109931A1; CA2341314A1; JP2002524087A; EP1109931A4

Description

WO 00/14271 PCT/US99/20207 1 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 [-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 WO 00/14271 PCT/US99/20207 2 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.

WO 00/14271 PCT/US99/20207 3 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 WO 00/14271 PCTIUS99/20207 4 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 WO 00/14271 PCT/US99/20207 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:1, 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 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:1, 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.

WO 00/14271 PCT/US99/20207 6 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 transgenic 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% CO 2 Groups of 1x106 of these cells were plated the day before transfection and were approximately 50% to 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.

WO 00/14271 PCT/US99/20207 7 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 NaCI, 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 Ag of coelenterazine was immediately added. Spectrofluorometry was performed using a SPEX FluoroMax® (Instruments 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 WO 00/14271 PCT/US99/20207 8 spectral pattern did not change over time.

Control cell extract mixtures from cells transfected with pC-INS-GFP and pC-IGFBP 6-RUG 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.

EDITORIAL NOTE 58056/99 SEQUENCE LISTING PAGES 1 TO 13 FOLLOW PAGE 8 OF THE

SPECIFICATION.

WO 00/14271 WO 00/ 4271PCT/US99/20207 SEQUENCE LISTING <110> Szalay, Aladar A.

Wang, Yubao Wang-Pruski, Gefu Loma Linda University <120> Method for Studying Protein interactions in Vivo <130> 11785-3PCT <140> filed herewith <141> 1999-09-02 <150> 60/135,835 <151> 1999-05-24 <150> 60/099,068 <151> 1998-09-03 <160> <170> Pateritln Ver. <210> 1 <211> 918 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (719) <400> 1 tg tgc ccc cac agg Cys Pro His Arg 1 ctg ctg cca ccg ctg ctg ctg ctg cta gct ctg Leu Leu Pro Pro Leu Leu Leu Lau Leu Ala Leu 5 10 is ctg ctc gct gcc agc cca gga ggc gcc ttg gcg cgg tgc cca ggc tgc Leu Leu Ala Ala Ser Pro Gly Gly Ala Leu Ala Arg Cys Pro Gly Cys 25 ggg caa ggg gtg cag gcg ggt tgt cca ggg ggc tgc gtg gag gag gag Gly Gin Gly Val Gin Ala Gly Cys Pro Gly Gly Cys Val Glu Glu Glu 40 47 143 gat ggg ggg tcg cca gcc gag ggc tgc gcg gaa gct gag ggc tgt ctc 191 Asp Gly Gly Ser Pro Ala Glu Gly Cys Ala Glu Ala Glu Gly Cys Leu WO 00/14271 WO 0014271PCT/US99/20207 agg agg Arg Arg gag ggg cag gag Glu Gly Gin Glu ggg gtc tac acc Gly Val Tyr Thr cct.

Pro aac tgc gcc cca Asn Cys Ala Pro gga Gly ctg cag tgc cat Leu Gin Cys His ccg Pro 85 ccc aag gac gac Pro Lys Asp Asp gcg cct. ttg cgg Ala Pro Leu Arg gcg Ala ctg ctg ctc ggc Leu Leu Leu Gly ggc cgc tgc ctt Gly Arg Cys Leu ccg Pro 105 gcc cgc gcg cct gct gtt Ala Arg Ala Pro Ala Val gca gag gag Ala Glu Glu cca cag gat Pro Gin Asp 130 aat Asn 115 cct aag gag agt Pro Lys Glu Ser ccc caa gca. ggc Pro Gin Ala Gly act gcc cgc Thr Ala Arg 125 ggc acc tct Gly Thr Ser gtg aac cgc aga Val Asn Arg Arg gac Asp 135 caa cag agg aat Gin Gin Arg Asn cca Pro 140 acc acg Thr Thr 145 ccc tcc cag ccc Pro Ser Gln Pro aat Asn 150 tct gcg ggt gtc Ser Ala Gly Val caa Gin 155 gac act gag atg Asp Thr Glu Met ggc Gly 160 cca tgc cgt aga Pro Cys Arg Arg ctg gac tca gtg Leu Asp Ser Val ctg Leu 170 cag caa ctc cag Gin Gin Leu Gin 479 527 575 gag gtc tac cga Glu Val Tyr Arg ggg Gly 180 gct caa aca ctc Ala Gin Thr Leu tac Tyr 185 gtg ccc aat tgt Val Pro Asn Cys gac cat Asp His 190 cga ggc ttc Arg Gly Phe cga ggt ccc Arg Gly Pro 210 cgg aag cgg cag Arg Lys Arg Gin tgc Cys 200 cgc tcc tcc cag Arg Ser Ser Gin ggg cag cgc Gly Gin Arg 205 ctg cca ggg Leu Pro Gly tgc tgg tgt gtg Cys Trp Cys Val cgg atg ggc aag Arg Met Gly Lys tct cca Ser Pro 225 gat ggc aat gga Asp Gly Asn Gly agc Ser 230 tcc tcc tgc ccc Ser Ser Cys Pro act Thr 235 ggg agt agc ggc Gly Ser Ser Gly taaagctggg ggatagaggg gctgcagggc cactggaagg aacatggagc tgtcatcact 779 WO 00/1427 1 PCTIUS99/20207 caacaaaaaa ccgaggccct caatccacct tcaggccccg ccccatgggc ccctcaccgc 839 tggttggaaa gagtgttggt gttggctggg gtgtcaataa agctgtgctt ggggtcgctg 899 aaaaaaaaaa aaaaaaaaa 918 <210> 2 <211> 239 <212> PRT <213> Homo sapiens <400> 2 Cys Pro His 1 Leu Ala Ala Gin Gly Val Gly Gly Ser Arg Ser Gin Leu 5 Pro Leu Pro Pro Leu Leu Leu Leu Ala Leu Leu Gly Gly Ala Arg Cys Pro Ala Gly Cys Pro 40 Cys Gly Cys Val Glu Gly Giy Cys Gly Glu Glu Asp Cys Leu Arg Pro Ala Glu Gly Gly Ala Glu Ala Glu Asn Arg Leu Gly Gin Glu Val Tyr Thr Cys Ala Pro Gly Leu Gin Cys His Pro Gly Lys Asp Asp Glu Al a Pro Leu Arg Ala Leu Leu Gly Glu Giu Asn 115 Gin ABD Val Arg Cys Leu Pro 105 Pro Arg Ala Pro Lys Glu 3cr Gin Ala Gly Thr 125 Gly Ala Val Ala 110 Ala Arg Pro Thr Ser Thr Asn Arg Arg Gin Arg Asn 130 Thr Pro Pro 140 Asp Ser Gin Pro 145 Pro Asn 150 Leu Ala Gly Vai Gin 155 Thr Glu Met Gly 160 Cys Arg Arg Asp Ser Val Leu Gin 170 Val Pro Gin Leu Gin Thr Glu 175 Asn Cys Asp.His Arg 190 Val Tyr Arg Giy 180 Ala Gin Thr Leu Tyr 185 WO 00/14271 WO 0014271PCT[US99/20207 Gly Phe Tyr 195 Gly Pro Cys 210 Arg Lys Arg Gin Cys 200 Arg Ser Ser Gin Gly 205 Gin Arg Arg Trp Cys Val Arg Met Gly Lys Ser 220 Lau Pro Gly Ser Pro Asp Gly Asn Gly Ser 225 230 Ser Ser Cys Pro Thr 235 Gly Ser Ser Giy <210> 3 <211> 1196 <212> DNA <213> Renilia reniformis <220> <221> CDS <222> (10)..(945) <400> 3 agcttaaag atg act tcg aaa gtt tat gat cca gaa caa. agg aaa cgg atg 51 Met Thr Ser Lys Val Tyr Asp Pro Glu Gin Arg Lys Arg Met ata Ile act ggt ccg cag Thr Gly Pro Gin tgg Trp 20 tgg gcc aga tgt aaa caa atg sat gtt Trp Ala Arg Cys Lys Gin Met Asn Val gat tca ttt att Asp Ser Phe Ile aat tat tat gat tca gaa Asn Tyr Tyr Asp Scr Glu 40 aaa cat gca gaa aat gct Lys His Ala Giu Asn Ala 147 gtt att ttt Val Ile Phe gtt gtg cca Val Val Pro cat ggt aac gcg His Gly Asn Ala gcc Ala tct tct tat tta Ser Ser Tyr Leu cat att gag cca His Ile Giu Pro gta Val 70 gcg cgg tgt att Ala Arg Cys Ile ata Ile tgg cga cat Trp Arg His cca gat ctt Pro Asp Leu tat agg tta Tyr Arg Leu att ggt Ile Gly atg ggc aaa tca Met Giy Lys Ser 9gc Gly 85 aaa tct ggt aat Lys Ser Gly Asn ggt tct Gly Ser 291 339 ctt Leu gat cat tac aaa.

Asp His Tyr Lys ctt act gca tgg Leu Thr Ala Trp, ttt Phe 105 gaa ctt ctt aat Glu Leu Leu Asn tta Leu 110 WO 00/14271 WO 0014271PCTIUS99/20207 cca aag aag atc Pro Lys Lys Ile ttt gtc ggc cat Phe Val Gly His gat Asp 120 tgg ggt gct tgt Trp Gly Ala Cys ttg gca Leu Ala 125 387 ttt cat tat Phe His Tyr gct gaa agt Ala Glu Ser 145 agc Ser 130 tat gag cat caa Tyr Glu His Gin aag atc a aa gca Lys Ile Lys Ala ata gtt cac Ile Val His 140 tgg cct gat Trp Pro Asp 435 483 gta gta gat gtg Val Val Asp Val att Ile 150 gaa tca tgg gat Giu Ser Trp Asp att gaa Ile Giu 160 gaa gat att gcg Giu Asp Ile Ala atc aaa tct gaa.

Ile Lys Ser Giu gaa Glu 170 gga gaa aaa atg Gly Giu Lys Met gtt Val 175 atg Met ttg gag aat aac Leu Giu Asn Asn aga aag tta gaa Arg Lys Leu Giu 195 t tc Phe 180 ttc gtg gaa acc Phe Val Giu Thr ttg cca tca aaa Leu Pro Ser Lys cca gaa, gaa ttt Pro Glu Glu Phe gca Ala 200 gca tat ctt gaa Ala Tyr Leu Glu cca ttc Pro Phe 205 aaa gag aaa.

Lys Glu Lys atc ccg tta Ile Pro Leu 225 ggt Gly 210 gaa gtt cgt cgt Glu Val Arg Arg cca Pro 215 aca tta tca tgg Thr Leu Ser Trp cct cgt gaa Pro Arg Giu 220 att gtt agg Ile Val Arg gta aaa ggt ggt Val Lys Giy Gly aaa Lys 230 cct gac gtt gta Pro Asp Val Val caa Gin 235 aat tat Asn Tyr 240 aat gct tat cta Asn Ala Tyr Leu cgt Arg 245 gca agt gat gat Ala Ser Asp Asp tta cca aaa atg ttt Leu Pro Lys Met Phe 250 att gtt gaa ggc gcc Ile Val Glu Gly Ala att Ile 255 gaa. tcg gat cca Glu Ser Asp Pro gga Gly 260 ttc ttt tcc aat Phe Phe Ser Asn 771 819 867 aag aag ttt cct Lys Lys Phe Pro act gaa ttt gtc Thr Glu Phe Val aaa Lys 280 gta aaa ggt ctt Val Lys Gly Leu cat ttt His Phe 285 tcg caa gaa Ser Gin Glu gat Asp 290 gca cct gat gaa Ala Pro Asp Glu atg gga Met Gly 295 aaa tat atc Lys Tyr Ile aaa tcg ttc Lys Ser Phe 300 WO 00/14271 WO 0014271PCTIUS99/20207 gtt gag cga gtt ctc aaa sat gaa caa taa ttactttggt tttttattta Val Glu Arg Val Leu Lys Asri Giu Gin 305 310 catttttccc gggtttaata atataaatgt cattttcaac aattttattt taactgaata tttcacaggg aacattcata tatgttgatt aatttagctc gaactttact ctgtcatatc attttggaat attacctctt tcaatgaaac tttataaaca gtggttcaat taattaatat atattataat tacatttgtt atgtaataaa ctcggtttta ttataaaaaa a <210> 4 <211> 311 <212> PRT <213> Renilia reniformis <400> 4 965 1025 1085 1145 1196 Met 1 Gly Phe Thr Ser Lys Pro Gin Trp Ile Asn Tyr Val 5 Trp Tyr Asp Pro Glu Arg Lys Arg Met Ile Thr Ala Arg Cys Lys 25 Lys Met Asn Val Tyr Asp Ser His Ala Giu Phe Leu His Asn Arg Leu Asp Ser Ala Val Ile His Val Val Giy Asn Ala Ser Tyr Leu His Trp Pro Pro Met Ile Giu Pro Val 70 Lys Arg Cys Ile Ile Ser Asp Leu Ile Giy Lys Ser Ser Gly Asn Gly Glu Tyr Arg Leu Leu Asp His Tyr Lys Lye Ile Ile 1is Tyr Ser Tyr Tyr 100 Phe Thr Aia Trp Leu Leu Asn Val Gly His Asp 120 Lys Gly Ala Cys Leu 125 Val Leu Pro Lye 110 Ala Phe His His Ala Giu Giu His Gin 130 Ser Val Val Asp Val Ile Asp 135 Glu Ile Lys Ala Ile 140 Trp Ser Trp Asp Giu Pro Asp Ile Giu WO 00/14271 WO 0014271PCTIUS99/20207 Giu Asp Ile Ala Leu 165 Ile Lys Ser Giu Gly Giu Lys Met Val Leu 175 Glu Asn Asn Lys Leu Glu 195 Phe 180 Phe Val Giu Thr Met 185 Leu Pro Ser Lys Ile Met Arg 190 Phe Lys Giu Pro Giu Giu Phe Ala Tyr Leu Giu Pro 205 Lys Gly 210 Giu Val Arg Arg Pro 215 Thr Leu Ser Trp Pro 220 Arg Giu Ile Pro Val Lys Gly Gly Lys 230 Pro Asp Val Val Gin 235 Ile Val Arg Asn Asn Ala Tyr Leu Arg 245 Ala Ser Asp Asp Leu 250 Pro Lys Met Phe Ile Giu 255 Ser Asp Pro Phe Pro Asn 275 Gly 260 Phe Phe Ser Asn Ala 265 Ile Vai Giu Gly Ala Lys Lys 270 Phe Ser Gin Thr Giu Phe Val Val Lys Gly Leu His 285 Giu Asp 290 Ala Pro Asp Giu Met 295 Giy Lys Tyr Ile Lys 300 Ser Phe Val Glu Vai Leu Lye Asn Giu Gin 310 <210> <211> 543 <212> DNA <213> HOMO sapiens <220> <221> CDS <222> <400> atg gga atc cca atg Met Gly Ile Pro Met ggg aag tcg atg Giy Lys Ser Met c tg Leu gtg ctt, ctc acc Val Lau Leu Thr ttc ttg Phe Leu gcc ttc gcc tcg tgc tgc att. gct gct tac cgc ccc agt. gag acc Ctg 96 WO 00/14271 WO 0014271PCTIUS99/20207 Ala Phe Ala tgo ggc ggg Cys Gly Gly Ser Cys Cys Ile Ala Tyr Arg Pro Ser Glu Thr Leu ggg gao cgc Gly Asp Arg gag ctg gtg gac Glu Leu Val Asp acc Thr 40 etc cag ttc gtc Leu Gin Phe Val tgt Cys 144 ggc ttC Gly Phe tac ttc ago agg Tyr Phe Ser Arg gca ago egt gtg Ala Ser Arg Val agc Ser egt cgo ago cgt Arg Arg Ser Arg ggo Gly ate gtt gag gag Ile Val Glu Glu tgt ttc ego ago Cys Phe Arg Ser gao etg goo etc Asp Leu Ala Leu o tg Leu gag aeg tao tgt Glu Thr Tyr Cys got Ala ace ccc gee aag Thr Pro Ala Lys too Ser 90 gag agg gao gtg Glu Arg Asp Val tog ace Ser Thr cot cog ace Pro Pro Thr ttc ttc caa Phe Phe Gin 115 ott cog gao aao Leu Pro Asp Asn coo aga tao ccc Pro Arg Tyr Pro gtg ggc aag Val Gly Lys 110 otg oge agg Leu Arg Arg 336 384 tat gao ace tgg Tyr Asp Thr Trp cag tee ace eag Gin Ser Thr Gin oge Arg 125 ggc otg Gly Leu 130 cot gee etc etg Pro Ala Leu Leu gee Cc egg ggt Ala Arg Arg Gly eac His 1.40 gtg etc gee aag Val Leu Ala Lys etc gag geg tte Leu Glu Ala Phe agg Arg 150 gag gee aaa egt Glu Ala Lys Arg egt 000 otg att Arg Pro Leu Ile got Al a 160 eta ccc ace caa.

Leu Pro Thr Gln gao ceo Asp Pro 165 goc eac ggg ggc goc eec eca gag Ala His Gly Gly Ala Pro Pro Glu 170 atg gee Met Ala 175 age aat egg Ser Asn Arg aag tga Lys 180 <210> 6 <211> 180 <212> PRT <213> Homo sapiens WO 00/14271 WO 0014271PCTIUS99/20207 <400> 6 Met Gly Ile Pro Met 1 5 Gly Lys Ser Met Leu 10 Val Leu Leu Thr Phe Leu.

Ala Phe Ala Cys Gly Gly Cys Cys Ile Ala Al a Tyr Arg Pro Ser Giu Thr Leu Gly Asp Arg Giu Leu Val Asp Thr 40 Leu. Gin Phe Val Cys Gly Phe Tyr Phe Ser Arg Pro Ala Ser Arg Val Arg Arg Ser Arg Gly Ile Val Giu Giu Cys Phe Arg Ser Asp Leu Ala Leu Giu Thr Tyr Cys Al a Thr Pro Ala Lys Ser 90 Glu Arg Asp Val Ser Thr Pro Pro Thr Phe Phe Gin 115 Leu. Pro Asp Asn Phe 105 Pro Arg Tyr Pro Val Gly Lys 110 Leu Arg Arg Tyr Asp Thr Trp, Lys 120 Gin Ser Thr Gin Arg 125 Gly Leu 130 Pro Ala Leu, Leu,Arg 135 Ala Arg Arg Gly His 140 Val Leu Ala Lys Glu 145 Leu Leu Glu Ala Phe Pro Thr Gin Asp 165 Arg 150 Glu Ala Lys Arg Arg Pro Leu Ile Ala 160 Pro Ala His Gly Gly 170 Ala Pro Pro Glu Met Ala 175 Ser Asn Arg Lys 180 <210> 7 <211> 717 <212> DNA <213> Artificial Sequence <220> <221> <222>

CDS

(717) WO 00/14271 WO 0014271PCTIUS99/20207 <220> <223> Description of Artificial Sequence: humanized green fluorescent protein cDNA <400> 7 atg Met

I

agc aag ggc Ser Lys Gly gaa ctg ttc act Giu Leu Phe Thr gtg gtc cca. att Val Val Pro Ile ctc gtg Leu Val gaa. ctg gat Glu Leu Asp ggt gaa ggt Gly Glu Gly ggc Gly gat gtg aat ggg Asp Val Asn Gly cac His 25 aaa ttt tct. gtc Lys Phe Ser Val agc gga gag Ser Gly Glu ttc atc tgc Phe Ile Cys gat gcc aca tac Asp Ala Thr Tyr aag ctc acc ctg Lys Leu Thr Leu acc act Thr Thr gga aag ctc cct Gly Lys Leu Pro gtg Val 55 cca tgg cca aca Pro Trp, Pro Thr ctg Leu.

gtc act acc ttc Val Thr Thr Phe tc t Ser tat ggc gtg cag Tyr Gly Val Gln ttt tcc aga tac Phe Ser Arg Tyr gac cat atg aag Asp His Met Lys cag Gin so cat gac ttt ttc His Asp Phe Phe aag Lys agc gcc atg ccc Ser Ala Met Pro ggc tat gtg cag Gly Tyr Val Gin gag aga Giu Arg acc atc ttt Thr Ile Phe ttC Phe 100 aaa gat gac ggg Lys Asp Asp Gly aac Asn 105 tac aag acc cgc gct gaa. gtc Tyr Lys Thr Arg Ala Glu Val 110 aag ttc gas.

Lys Phe Giu 115 ggt gac acc ctg Gly Asp Thr Leu aat aga atc gag Asn Arg Ile Giu ctg Leu 125 aag ggc att Lys Gly Ile gac ttt Asp Phe 130 aag gag gat gga Lys Glu. Asp Gly aac Asn 135 att ctc ggc cac Ile Leu Gly His aag Lys 140 ctg gaa tac aac Leu Glu Tyr Asn tat Tyr 145 aac tcc cac sat Asn Ser His Asn gtg Val 150 tac atc atg gcc Tyr Ile Met Ala gac Asp 155 aag caa aag aat Lys Gin Lys Aen ggc Gly 160 432 480 528 atc aag gtc aac Ile Lys Val Asn ttc Phe 165 aag atc aga cac Lys Ile Arg His aac Asn 170 att gag gat gga Ile Giu Asp Gly tcc gtg Ser Val 175 WO 00/14271 WO 0014271PCT/US99/20207 cag ctg gcc Gin Leu Ala gtg ctc ctc Val Leu Leu 195 gac Asp 180 cat tat caa cag His Tyr Gin Gin aac Asn 185 act cca atc ggc Thr Pro Ile Gly gac ggc cct Asp Gly Pro 190 gcc ctg tct Ala Leu. Ser 576 624 cca gac aac cat Pro Asp Asn His tac Tyr 200 ctg tcc acc cag Leu, Ser Thr Gin tc t Ser 205 aaa gat Lys Asp 210 ccc aac gaa aag Pro Asn Glu Lys gac cac atg gtc Asp His Met Val c tg Leu 220 ctg gag ttt gtg Leu Giu Phe Val 672 717 acc Thr 225 gct gct ggg atc Ala Ala Gly Ile ac a Thr 230 cat ggc atg gac His Gly Met Asp ctg tac aag tga Leu Tyr Lys <210> 8 <211> 238 <212> PRT <213> Artificial Sequence <400> 8 Met Ser 1 Lys Gly Qiu Giu Leu Phe Thr 5 Val Val Pro Ile Leu Val Glu Leu. Asp Gly Glu Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu Phe Ile Cys Asp Ala Thr Tyr Gly 40 Lys Leu Thr Leu Lys Thr Thr Gly Lys Leu Pro Pro Trp Pro Thr Leu.Val Thr Thr Phe Tyr Gly Val Gin Cys Phe Ser Arg Tyr Asp His Met Lys Gin His Asp Phe Phe Lys Ser Ala Met Pro Giu 90 Gly Tyr Val Gin Glu Arg Thr Ile Phe Phe 100 Lys Asp Asp Gly Tyr Lys Thr Arg Ala Giu Val 110 Lys Phe Glu 115 Gly Asp Thr Leu Val 120 Asn Arg Ile Giu Leu 125 Lys Gly Ile WO 00/14271 WO 0014271PCT/US99/20207 Asp Phe 130 Lys Giu Asp Gly Ile Leu Gly His Lys Leu Giu Tyr Asn 140 Lys Gin Lys Asn Gly Tyr 145 Asn Ser His Asn Val 150 Tyr Ile Met Ala Asp 155 Ile Lys Val Aen Phe 165 Lys Ile Arg His Asn 170 Ile Glu Asp Gly Ser Val 175 Gin Leu Ala Val Leu Leu 195 Asp 180 His Tyr Gin Gin Asn 185 Thr Pro Ile Gly Asp Gly Pro 190 Ala Leu Ser Pro Asp Asn His Tyr 200 Leu Ser Thr Gin Lys Asp 210O Pro Aen Giu Lys Asp His Met Vai Leu 220 Leu Giu Phe Val Thr 225 Aia Ala Gly Ile His Giy Met Asp Leu Tyr Lye <210> 9 <211> 333 <212> DNA <213> <220> <221> <222> Homo sapiens CDs <400> 9 atg gcc Met Ala ctg tgg atg cgc ctc ctg ccc Leu Trp Met Arg Leu Leu Pro ctg gcg ctg ctg Leu Ala Leu Leu gcc ctc 48 Ala Leu tgg gga cct Trp Gly Pro tca cac ctg Ser His Leu gac Asp cca. gcc gca gcc Pro Ala Ala Ala ttt Phe 25 gtg aac caa. cac Val Aen Gin His ctg tgc ggc Leu Cys Gly gtg gaa gct ctc Val Giu Ala Leu tac Tyr 40 cta gtg tgc ggg Leu Val Cys Gly gaa. cga ggc ttc 144 Giu Arg Giy Phe ctg cag gtg ggg 192 Leu Gin Val Giy ttc tac Phe Tyr aca ccc aag acc Thr Pro Lys Thr cgg gag gca. gag Arg Giu Ala Giu gac Asp WO 00/14271 WO 0014271PCTIUS99/20207 cag gtg gag ctg ggc ggg Gin Val Glu Leu Gly Gly 70 ggc cot ggt gca Giy Pro Gly Ala ggc Gly 75 agc ctg cag ccc Ser Leu Gin Pro ttg 240 Leu so gcc ctg gag ggg Ala Leu Giu Gly ctg cag aag cgt Leu Gin Lys Arg ggc Gly att gtg gaa caa Ile Val Giu Gin tgc tgt Cys Cys acc agc atc Thr Ser Ile tcc ctc tac cag Ser Leu Tyr Gin gag aac tac tgc Glu Asn Tyr Cys aac tag An 110 <210> <211> 110 <212> PRT <213> Homo sapiens <400> Met Ala Leu Trp Met 1 5 Arg Leu Leu Pro Leu 10 Lau Ala Leu Leu Ala Lou Trp Gly Pro Ser His Leu Pro Ala Ala Ala Val Asn Gin His Leu Cys Gly Arg Giy Phe Val Glu Ala Leu Tyr 40 Leu Val Cys Gly Glu Phe Tyr Thr Pro Lys Thr Arg Giu Ala Glu Leu Gin Vai Giy Val Giu Leu Gly Gly Gly Pro Gly Ala Gly Ser Leu Gin Pro Aia Leu Giu Gly Ser Leu Gin Lys Arg Gly 90 le Val Giu Gin Cys Cys Thr Ser Ile Cys 100 Ser Leu Tyr Gin Leu 105 Giu Asn Tyr Cys An 110

Claims

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.

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: WO 00/14271 PCT/US99/20207 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.

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 WO 00/14271 PCT/US99/20207 11 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.

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.

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 WO 00/14271 PCT/US99/20207 12 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.

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.