CA2341314A1

CA2341314A1 - Method for studying protein interactions in vivo

Info

Publication number: CA2341314A1
Application number: CA002341314A
Authority: CA
Inventors: Aladar A. Szalay; Yubao Wang; Gefu Wang-Pruski
Original assignee: Individual
Current assignee: Loma Linda University
Priority date: 1998-09-03
Filing date: 1999-09-02
Publication date: 2000-03-16
Also published as: JP2002524087A; CN1160470C; AU752675B2; CN1323353A; WO2000014271A1; EP1109931A1; EP1109931A4; AU5805699A

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 flurophore 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

WO 00/14Z~1 PCTIUS99/ZOZ07 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' stud~ie~i using° immuno-precipitation, the yeast two hybrid method; and ~-gal complementation method.
ZO 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 molcxules other than proteins.
SUMMARY

WO 00/14271 PCTNS99nOZ07

2 method for determining whether a first protein interacts with a second pmtein within a living cell. The method comprises providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluomphore 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 iuciferase 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 compiexed 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 det~don of acceptor fluorophore fluorescence is performed using spectrofluoronlettry.
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 {L.RET). 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 fluomphore.
The efficiency of luminescence resonance energy transfer is dependent on the

3 Generally, significant energy ttnnsfers occur only where the donor hicifcrase and acceptor fluorophore are less than about 80 A of each other. This short distance is considerably less than the distance between for optical resolution between two entities using conventional microscopy. Therefore, detecting luminescence resonance energy transfer between a donor hiciferase and an acceptor fluorophore indicates that the donor luciferase and acceptor fluorophore have come within the distance noeded 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 interacEion 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 fast protein and the complexed second protein in the cell under conditions suitable for an interaction between We 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 resona~e 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 nd 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 e~rgy transfer to detect the interaction between two proteins accordiwg to the present invention. First, the specific labeling of the proteins in living cells can be achieved through generic 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

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 fluomphore 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 b) and insulin-like growth factor II (IGR
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 cDNA
and the fusion proteins were expressed. Cell extracts were produced and mixed.
The wo oon4Zm rc~rn~s~nozo~
s 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 IGP'BP-6 cDNA to Rtnilla Lucifierase cDNA; Fused IGF
II
S cDNA to 'hmnanized' 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 MG90S4, encoded IGFBP-6, SEQ ID N0:2, which was used as the first protein.
Renilla luciferase cDNA, SEQ ID N0:3, GenBank accession number M63S01, encoded Renilla luciferase, SEQ II3 N0:4, which was used as the donor luciferase. IGF-II cDNA, SEQ ID NO:S, encoded IGF-II, SEQ ID N0:6, which was used as the second protein.
'Humanized' GFP cDNA, SEQ ID N0:7, GenBank accession numberUS0963, encoded 'humanized' GFP, SEQ ID N0:8,which was used as the acceptor fluorophore.
Insulin 1S cDNA, SEQ ID NO:9, accession number AH002$44, encoded insulin, SEQ ID
NO;10.
Insulin, fused to 'humanized' GFP, was used as a control protein because insulin is homologous to IGF-II, but it does not bind to IGFBP-6. The IGFBP-6 cDNA, SEQ
ID
NO:1, IGF-II cDNA, SEQ ID NO:S, and insulin cDNA, SEQ ID N0:9, were modified using PCR as follows. --First, the cDNA of prepro-IGF-1I carried on an EcoRI fragment was cloned into pBluescript KS (+) II vector. The insert was sequenced using T7 and T3 primers and confirmed to contain the known cDNA sequence of prepro-IGF-IT. The S' end of the IGF-II
precursor was connected to the T7 promoter in the pBlueseript KS (+) II
vector. An IGF-II
3' primer was designed to ge~rate a Notice of Allowance restriction site, to remove the D
2S and E domains of prepro-IGF-II, and to maintain the Notice of Allowance fragment of the 'humanized' GFP in frame with the open reading frame of IGF-Q.
Next, the IGF-II fragment was amplif:ed with PCR using the T7 promoter primer and the IGF-II 3' primer. The PCR-amplified IGF-II fragment was digested by EcoRI and Not I and cloned into pCDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, US) producing pCDNA-IGF-II. Then, the Notice of Allowance fragment of the 'humanized' The eDNA for precursor of insulin, which contained a signal peptide the B, C
and A domains, was modified in a manner corresponding to the IGF-II fragment, above.
The 'humanized' GFP cDNA was then linked to the 3' end of the modified insulin cDNA to produce pC-INS-GFP.
Finally, IGFBP 6 cDNA was amplified by PCR from a plasmid named Rat-tagged human IGFBP6. The stop codon of IGFBP 6 was removed and the open reading frame of IGFBP 6 was in frame with Renilla luciferase cDNA from pCEP4-RUC
(Mayerhofer R, Langridge WHR, Cormier MG and Szalay AA. Expression of recombin~urt Renilla luciferase. in transgereic plants results in high levels of light emission. The Plant Journal 1995;7;1031-$). 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 ail 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 Calccium Phosphate Precipitation Method:
Next, msunmalian cells were transfected with the clod fusion DNAs. First, COS-7 cells (African green monkey kidney cell, American Type Culture Collection CRL
_ 1651) were grownat 37 C in Dulbecco's Modified Eagle Medium (DMEM) with L-Glutamine supplemented with 1096 fetal bovine serum and antibiotic antimycotic solution containing a final concentration of penicillin 100 unit/ml, streptomycin 100 mglmi and amphotericin B 250 ng/ml (Sigma-Aldrich Co., St. Louis, MO, US) in 5% C02.
Groups of 1x106 of these cells were plated the day before transfection and were approximately 5096 to 60 k 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 mamnnalian 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 transfeeted cells and cells transfected with a WO 00/14271 PCTNS99/ZOZO~

C) Confirmation of Expression of ~~sion 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 thmugh 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 Spedrofluorometry:
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 IO seconds using a Fisher Model 550 Sonic Dismembrator (Fisher Scient~c, Pittsburgh, PA, US) to produce cell extracts.
Next, the cell extracts containing IGF-II-GFP and IGFBP 6-RUC were mixed and 0.1 pg of coelenterazinc was immediately added. Spectrofluorometry was performed using a SPEX FluoroMaxm (Institunents 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 spectrofluommetry, the mixtures were kept at room temperature for 30 minutes and the spectra wore traced again after fresh coelenterazine was added. The trace at 30 minutes showed two peaks with emission maxiimum at 471 nm and wo oon4z~~ Pcnus~nozo~
s 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 thmugh 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.

wo oon4z7mcrnJS99noZO~
ssQvs~ca LIST=xa <110> 8salay~ Aladar A.
Ifaag, Y'ubao ~iaag-Pruaki, aelu Loata Liada D'niversity <1Z0> Method !or Studyiag Protein Iateractioaa is vivo <130> 11785-3PCT
<140> filed herewith <141> 1999-09-OZ
<150> 60/135.835 <151> 1999-05-~4 <150> b0/099,Ob8 <151> 1998-09-03 <160> 10 <170> PateatIn Ver. Z.0 <Z10> 1 <Z11> 918 <ZlZ> DMA
<Z13> hcm~o aapiena <ZZO>
<~Zly CD8' , , ... . . ,.._ . .
<~ZZ> (3)..(719) <400> 1 tg tgc acc cac agg ctg ctg aca cag ctg ctg ctg atg ota get ctg 47 Cys pro His l~rg Leu Leu Pro pro Leu Leu Leu Leu Leu Ala Leu ctg ctc get gcc agc cca gga ggc gcc ttg gcg cgg tgc cca ggc tgc 95 Leu Leu ltla 111a Ssr Pro Oly Oly lrla Leu 111a Arg Cys Pro aly Cye ggg can ggg gtg cag gcg ggt tgt cca ggg ggc tgc gtg gag gag gag 143 aly ala (ily Val Ola 111a Lily Cyra Pro aly Oly Cya Val Olu Gtlu alu gat ggg ggg tcg cca gcc gag ggc tgc gcg gaa get gag ggc tgt ctc 191 Aap aly aly 8er Pro Ala Glu oily Cys Al.a Olu Jlla alu Aly Cya Leu wo oon4zm pc~rius99nozo7 agg agg gag ggg cag gag tgc ggg gtc tac acc cct aac tgc gcc cca Z39 Arg Arg Glu Gly Gln Glu Gys Gly Val Tyr Thr pro Asn Cys Ala Pro gga ctg cag tgc cat ccg cac ug gaa gac gag gcg cct ttg cgg gcg Z87 Gly Leu Gln Cys 8ie pro Pro Lys Asp Asp Glu Ala Pro Leu Arg Ala ctg ctg ctc ggc cga ggc cgc tgc ctt ccg gcc cgc gcg cct get gtt 335 Leu Leu Lsu Gly Arg Gly Arg Gys Leu Pro Ala Arg Ala Pro Ala Val gca gag gag aat cct aag gag agt aaa ccc caa gca ggc act gcc cgc 383 Ala Glu Glu Asa Pro Lys Glu Ser Lys Pro Gla Ala Gly Thr Ala Arg 115 1~0 125 cca cag gat gtg aac cgc aga gac caa cag agg aat cca ggc acc tct s31 Pro Gln Asp Val Ass Arg Arg Asp Gln Gln Arg Asn Pro Giy Thr Bar acc acg ccc tcc cag cec aat tct gcg ggt gtc aaa gac act gag atg 4?9 Thr Thr Pro Ser Gln Pro Asa Sex Ala filly Val Gin Asp Thr Giu list ggc cca tgc cgt aga cat ctg gac tca gtg ctg cag caa ctc cag act 5Z7 Gly Pro.Cys Arg Arg 83s Lau Asp 8sr Vai Leu Gln Gia Leu Gla Thr gag~gtc=tac-cga ggg get caa aca atc tac gtg ccc aat tgt gac cat 575 Glu Val Tyr Arg Gly Ala Gla Thr Leu Tyr Val Pro Asa Cys Asp 8is i80 185 190 cga ggc ttc tac cgg aag cgg cag tgc cgc tcc tcc cag ggg cag agc 6Z3 Arg Aly Phe Tyr Arg Lys Arg Gla Cys Arg Ssr Ser Gla Oly Gia Arg cga ggt ccc tgc tgg tgt gtg gat cyg atg ggc aag tcc ctg cca ggg 691 Arg Gly Pro Cys Trp Cys Val Asp Arg lLst Giy Lys 8er Lau Pro Gly tct cca gat ggc aat gga agc tcc tcc tgc ccc act ggg agt agc ggc 719 Ser pro Asp Gly Asn Oly Sar 9er 8er Cys Pro Thr Gly Ser Ser Gly taaagctggg ggatagaggg gctgcwgggc cactggaagg aacatggagc tgtcatcact 979 caacaaaaaa ccgaggccct caatccaact tcaggaaccg ccccatgggc ccctcaccgc 839 tggttggaaa gagtgttggt gttggctggg gtgtcaataa agctgtQctt ggggtcgctg 899 aaaaaaaaaa aaaaaaaaa 918 <210> Z
<Z11> Z39 <Z12> PRT
<Z13> Homo Sapiens <400> Z
Cys Dro 81s Arg Leu Leu Pro pro Leu Leu L~u Leu Iilu Ala Leu L1u i.au Ala Ala Her Pro Gly Gly Ala Leu Ala Arg Cys pro Gly Cys Gly a0 Z5 30 Gin Gly Val Gin Ala Gly Cys pro Gly Gly Cys Val Glu Glu Olu Asp Gly Gly Her pro Ala Glu Aly G~ra Ala Glu Ala Giu Gly Cys Leu Arg Arg Glu Gly Gla Glu Cye Gly Val Tyr Tier Pro Asn Cys Ala Pro Gly Leu Gla Cyrs His Pro pro Lys Asp Asp Olu 11a Bro Zreu Arg Ala Leu Leu Leu G1y Arg Giy Arg Cys Leu pro Ala Arg Ala 8ro Ala Val Ala Glu Glu Asa pro Lys Glu Bar Lys Pro A1n Ala Gly Thr Ala Arg pro 115 1Z0 1~5 Gln Asp val Asn Arg Arg Asp Gla Gln Arg Asn Hro Gly Thr Her Thr Thr Pro $er Gln pro Asn Ser Ala Gly Val Gla Asp Thr Glu flat Gly 145 150 i55 160 Bro Cya Arg Arg Isis Leu Asp Her Val Lau Gla Gln Leu Gla Ttsr Glu Val Tyr Arg Gly Ala Gln Thr Lsu Tyr Val pro Asn Gars Asp.His Arg WO 00/14271 PCTNS99n0207 Giy phe Tyz Arg Lys Arg Gla Cys Arg Ser Ser Gla Aly Gln Arg Arg Gly pro Cys Trp Cys Val Asp Arg list Gly Lys Ser Leu pro Oly Ser pro Asp Gly Asa Gly Ssr Ser Ser Cys pro Thr Gly Bar Ser Gly <Z10> 3 <all> 1196 <21Z> DNA
<213> Renilla reniformis <aso>
<221> CDS
<zaa> (10)..(945) <400> 3 agcttaaag atg act tcg aaa gtt tat gat eca gaa eaa agg aaa cgg stg 51 lSet Thr Ser Lys Val Tyr Asp pro Glu Gla Arg Lys Arg llet ata act ggt ccg cag tgg tgg gcc sga tgt aaa caa atg aat gtt ctt 99 Ile Thr Gly Pro Gln Trp Trp Ala Arg Cys Lye Gla stet Asa Val Leu gat tea ttt att aat tat tat gat tea gaa aaa cat gca gas aat get 147 -Asp 8er phe Ile Asa Tyr Tyr Asp Ser Glu Lye His Ala Glu Asn Ala gtt att ttt tta cat ggt aac gcg gcc tct tct tat tta tgg cga cat 195 Val Ile phe Leu His Gly Asn Ala Ala Ser Ser Tyr Leu Trp Arg 81s gtt gtg cca cat att gag cca gta gcg cgg tgt att ata cca gat ctt Z43 Val Val pro His Ile Olu pro Val Ala Arg Cys Ile Ile Pro Asp Lau att ggt atg ggc aaa tca ggc aaa tct ggt ast ggt tct tat agg tta z91 Ile Gly Met Gly Lys Ser Gly Lys Ssr Gly Asa Gly Ser Tyr Arg Leu ctt gat cat tac aaa tat ctt act gca tgg ttt gaa ctt ctt aat tta 339 Lau Asp His Tyr Lys Tyr Leu Thr Ala Trp phe Glu Leu Leu Asn Leu ".. _,_..~"""~,.~,....w...~.""..,~,.,...... ._ ._..~....,"....",,.",.~".~...._..

wo oon427i rc~rius~no2o7 cca aag sag atc att ttt gtc ggc cst gat tgg ggt get tgt ttg gca 387 Pro Lys Lys Ile Ile Phs Val Gly 8is lisp Trp Gly Ala Cps Leu 111a 115 1Z0 ls5 ttt cat tat agc tat gag cat caa gat aag atc aaa gca ata gtt cac 835 Pho 8is Tyr 8sr Tyr Glu His Ola Asp Lys Ile Lys Ala Ile Val 81s get gaa agt gta gta gat gtg att gaa tea tgg gat gaa tgg cct gat 883 Ala Glu Ser Val Val Asp Val Ile Olu 8sr Trp Asp Glu Trp Pro Asp att gaa gaa gat att gcg ttg atc aaa tct gaa gas gga gaa aaa atg 531 Ile Glu Glu Asp Ile Ala Leu Its Lys 8er Glu Glu Gly Glu Lye llet gtt ttg gag aat aac ttc ttc gtg gaa acc atg ttg cca tca aaa atc 579 Val Leu Glu Asn Asn Phe Phe Val Qlu Thr lLet Leu Pro 8er Lys Ile atg aga aag tta gaa cca gaa gaa ttt gca gca tat ctt gaa cca ttc 6Z7 lset Arg Lys Leu Glu Pro Olu Glu phe Ala Ala Tyr Leu Glu Pro Phe aaa gag aaa ggt gaa gtt cgt cgt cca aca tta tca tgg cct cgt gaa 675 Lys Glu Lys Gly Glu Val Arg Arg pro Thr Leu 8er Trp Pro Arg Glu atc ccg tta gta aaa ggt ggt asa cct gac gtt gta caa att gtt agg 7Z3 ... Ile Pro Leu Val Lys Gly Oly Lys Pro Asp Val Val Gla Ile Val Asg aat tat aat get tat cta cgt gca agt gat gat tta cca aaa atg ttt 771 Asa Tyr Asa Ala Tyr Leu Arg Ala 8er Asp Asp Leu Pro Lys tlet Phe att gaa tcg gat cca gga ttc ttt tcc aat get att gtt gaa ggc gcc 819 Ile Glu 8er Asp Pro Gly Phe Phe 8er Asa Ala Ile Val Glu Gly Ala aag aag ttt cct sat act gaa ttt gtc aaa gta aaa ggt ctt cat ttt 867 Lys Lys Phe Pro Asn Thr Glu Phe Val Lys Val Lys Gly Lau gis Phe tcg caa gaa gat gca cct gat gaa atg gga aaa tat atc aaa tcg ttc 915 Ser Gln Glu Asp Ala Pro Asp Glu ltet Aly Lys Tyr Ile Lys Ser Phe gtt gag cga gtt ctc aaa aat gaa caa taa ttactttggt tttttattta 965 Val Glu l~rg Val Leu Lya 7lan Alu Gln catttttccc gggtttaata atataaatgt cattttcaac aattttattt taactgaata 1025 tttcacaggg aacattcata tatgttgatt aatttagctc gaactttact ctgtcatatc 1085 attttggaat attacctctt tcaatgaaac tttataaaca gtggttcaat taattaatat 11l5 atattataat tacatttgtt atgtaataaa ctcggtttta ttataaaaaa a 1196 <Z10> 4 <Z11> 311 <al2> pRT
<213> Renilla reaitormis <~100> !
Dset Thr Ser Lys Val Tyr 7lap pro Glu Gln ~lrg Lys llrg list Ile Thr Gly Pro Gla Trp Trp l~la 7lrg Cya Lys Gla ltet flan Val Leu llsp 8er Hhe Its Aaa Tyr Tyr 7lap 8er Glu Lys 8ia Ala Glu 7lsa Ala Val Ile 35 !0 !5 phe Lou 8is Gly llsn Ala ~11a 8er 8er Tyr Leu Trp llrg 8is Val Val Pro 8is Ile Glu pro Val Ala 7lrg Cys Ile Ila pro Asp Leu Ile Gly llet Gly Lya 8er Gly Lys Ssr Aly Asn Aly 8er Tyr llrg Leu Leu llsp 81s Tyr Lys Tyr Lau Thr hla Trp phe Glu Leu Leu llsn Leu Pro Lys Lys Ile Ile Phe Val Gly 8ia llsp Trp Gly 111a Gys Lsu Ala Phe 8ia 115 1Z0 ls5 Tyr Ser Tyr Glu 81s Gln hsp Lya Ile Lys Ala Ile Val 81s Ala Glu 130 135 1!0 Sar Val Val Aap Val Ila Alu 8er Trp flap Glu Trp pro Asp Ile Alu wo oon4i~~ rcrius99noZO~

Alu Asp Ile Ala Leu Ile Lys Ser Alu Olu Aly A1u Lys list Val Leu c~lu Asa Ara Phe Phs Val Olu Thr llst Lsu Pro 8sr Lys ils list Arg Lys Leu Alu Pro Alu Glu Phe Ala Ala Tyr Leu Alu Pro phs Lys Alu Lys Aly Alu Val Arg Arg pro Thr Leu Ser Trp Pro ArQ Alu ile Pro Z10 115 Z~0 Leu Val Lys Oly Aly Lys Pro Asp Val Val Ala Ile Val Arg Asn Tyr Z~5 Z90 i35 140 Asn Ala Tyr Lau Arg Ala Sar Asp Asp Lsu Pro Lys lsat Phe Ile Alu Bar Asp Pro Aly Phs Phe Bar Asn Ala ile Val Alu Aly Ala Lys Irys Z60 Z6s Z70 Phe Pro Asa Thr Alu Phe Val Lys Val Lys Aly Leu His Phe Ssr Aln Alu Asp Ala Pro Asp Alu llet Aly Lys Tyr Ile Lys Ser Phe Val Alu Arg Val Leu Lys Asa Gilu Aln <a10> 5 <Z11> 543 cZl2> DNA
<~13> Homo sapisas <ZZO>
<aal> cDs <222> il)..(543) <400> 5 atg gga atc cca atg ggg aag tcg atg ctg gtg ctt ctc acc ttc ttg 48 liet Gly Tla Pro lief Aly Lys Bar ldst Lsu Val Leu Leu Thr Phe Leu gcc ttc gcc tcg tgc tgc att get get tac cgc ccc agt gag acc ctg 96 wo oo~iar~i Pccrtrs~9noZO7 Ala Phe Ala 8er Cys Cys Zle Ala Ala Tyr Arg Pro 8er Glu Thr Lau tgc ggc ggg gag ctg gtg gac acc ctc cag ttc gtc tgt ggg gac cgc 144 Cya lily Lily Glu Leu Yai Asp Thr Leu Oln phe Vai Cys Oly Asp Arg ggc ttc tac ttc agc agg acc gca agc cgt gtg agc cgt cgc agc cgt 192 lily Phe Tyr Phe 8er Arg pro Ala 8er Arg Yal 8er Arg Arg 8er Arg ggc atc gtt gag gag tgc tgt ttc cgc agc tgt gac ctg gcc ctc ctg a40 aly Ile Val Glu Glu Cys Cys phe Arg 8er Cyr Asp Leu Ala Leu Leu gag acg tac tgt get acc cec gec aag tec gag agg gac gtg leg acc Z88 Cilu Thr Tyr Cys Ala Thr Pro Ala Lys 8er flu Arg Asp Val 8er Thr cct ccg acc gtg ctt ccg gac aac ttc ecc aga tac ccc gtg ggc aag 336 Pro Pro Thr Val Leu pro Asp Asa Phe Pro Arg Tyr pro Val Qly Lys lao ios 110 ttc ttc caa tat gac acc tgg aag cag tcc acc cag cgc ctg cgc agg 384 Phe Phe liln Tyr Asp Thr Trp Lys Gla 8er Thr Aln Arg Lsu Arg Arg 115 1Z0 1~5 ggc ctg cct gcc ctc ctg cgt gcc cgc cgg ggt cac gtg ctc gcc sag 43~
aly Leu Pro Ala Leu Leu Arg Ala Arg Arg aly 81s Val Leu Ala Lys gag ctc gag gcg ttc agg gag gce aaa cgt cac cgt ccc ctg alt get 480 Qlu Leu Olu Ala Phe Arg Giu Ala Lye Arg 81s Arg Pro Leu Ile Ala cta ccc acc caa gac ccc gcc cac Qgg gga gca cc~c cca gag atg gcc 5Z8 Leu Pro Thz ala Asp Pro Ala 81s fly Qly Ala Pro pro G1u Ilst Ala lb5 170 175 agc aat cgg aag tga 543 8er Asa Arg Lys <Z10>6 <Z11>1B0 <212>PRT

<a13>80~o sapiens W4 00/I4~7I p'C1'1US99/2020~
<400> 6 bet Gly Ile Pro lsat Oly Lys Ssx 1ht Leu Val Leu Leu Thr Phe Leu Ala Phe Ala Ser Cys Cys =le Ala 711a T'yr Arg Pro 8er alu Thr Leu Cys Gly Gly Glu Leu Val llsp Thr Leu Glla Pbe Val Cue Gly flap ~

Gly phi Tyr Phe Ssr Arg Pro 111a Ssr Arg Val Ser Arg Arg Ser Arg Oily I1e val Giu Glu Cys Cys Phe Arg Sar Cys Asp Lsu Ala Leu Lau Glu Thr Tyr Cps Ala Thr Pro Aia Irys 8er Glu Arp Asp Val Ser Thr Pro Pro Thr Val Leu Pro Asp Asn Phe Pro ArQ Tyr Pro Val Gly Lys Phe Phe Ola Tyr Asp Thr Trp Lys Ola Ser Thr Gln Asg Leu Arg ArQ
115 iS0 1~5 Gly Leu Pro Ala Lsu Leu Arg 11"7.a ~ ArQ Gly 8is Val Lsu Ala Lys Glu Lau Glu Ala Phe Arg Olu 111a Lys Arg 8is Arg Pro Leu =le Ala Leu pro Thr Gla Asp Pro Ala 81s Gly A1y Ala Pro Pro Giu taet Ala Ser Asa Arg Lys <Z10> 7 <211> 717 <~12> aNA
<213> Artificial Sequeace <ZZO>
<Z21> CD8 <2Z2> (1)..(717) <ZZO>
<Z23> Description of Artificial Sequences humanised green fluorescent protein eDI~A
<400> 7 atg agc aag ggc gag gaa ctg ttc act ggc gtg gtc cca aft ctc gtg ~8 Met Ser Lys Gly Glu Glu Leu Phe Thr Oly Val Val Pro Ile Lsu Val gaa ctg get ggc get gtg eat ggg cac aaa ttt tat gte agc gga gag 96 Glu Leu Asp Giy Asp Val Asn Gly 81s Lye Phs Ser Val Ser Aly Glu ggt gaa ggt get gcc sca tac gga aag ctc acc ctg aaa ttc atc tgc 144 Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys acc act gga aag cte cct gtg eca tgg cca ace etg gte act ace tte 192 Thr Thr Gly Lys Leu Pro Val pro Trp Pro Thr Leu Val Thr Thr Phe tct tat gge gtg cag tge ttt tcc age tac eca gac cat atg aag cag 380 Ser Tlrr Gly Val Gln Cys Phe Ser Arg Tyr pro Asp His leaf Lys Gla cat gac ttt tte aag age gcc atg ecc gag ggc tat gtg cag gag age 288 His Asp Phs Phe Lye Ser Ala llat Pro Olu Qly Tyr Val Oln Olu Arg ace ate ttt tte aas get gac ggg aac tae aag aec age get gaa gtc 336 ,, Thr Ile Phe Phe Lys Asp Asp Aly Asn Tyr Lya Thr Arg Ala Glu Val aag ttc gaa ggt gee ace ctg gtg eat age ate gag ctg aag ggc aft 38~
Lys Pha Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile 115 120 1~5 gac ttt aag gag get gga aac att ate gge cac aag etg gaa tae aac 432 Asp Phe Lys Glu Asp Gly Asa Ile Leu Gly His Lys Lsu Glu Tyr Asa tat aac tcc cac sat gtg tae ate atg gcc gac aag caa aag eat ggc 880 err Asn Ser 81s Asn Val Tyr Ile Met Ala Asp Lys Gia Lys Asn Gly ate aag gtc aac ttc aag ate age cac aac aft gag get gga tcc gtg 528 ile Lys Val Asn Phe Lys ile Arg His Asn Ile Glu Asp Gly Bar Vsl WO 00/14Z'11 PCT/US99/20Z87 cag ctg gcc gac cat tat caa cag aac act cca atc ggc gac ggc cct 576 Gln Leu Ala Asp 81s Tyr Gla Gla Jlsa Thr Pro Ile Gly Asp Gly pro gtg ctc ctc cca gac aac eat tac ctg tcc acc eag tct gec ctg tet 6Z4 Val Lau Leu Pro flap llsn 81s Tyr Lau 8er Thr Gln Ser 111a Leu Set aaa gat ccc aac gaa aag aga Qac cac atg gtc ctg ctg gag ttt gtg 693 Lys Asp Hro hsn Glu Lys llrg lisp 8is tt~t Val Lsu Leu Glu Phe Val ace get get ggg ate aca cat ggc atg gac gag etg tae aag tga 719 Thr Ala Ala Gly ile Thr 8is Gly ltet Asp Glu Leu Tyr Lys <310> 8 <311> 338 <312> PRT
<313> Artificial Sequence <400> 8 lcet 8er Lys Gly Glu Glu Lsu phe Thr Gly Val Val pro Ile Leu Val Glu Leu hsp Gly ~lsp Val llsa Gly 8is Lys phe 8sr Val Ser Gly Glu ... O~.y Glu Gay Asp ~11a Thr Tyr Gly Lys Leu Thr ~Leu Lys pha Ila GSrs Thr Thr Gly Lys Leu Pro Val pro Txp Pro Thr Leu Val Thr Thr Phe Ser Tyr Giy Val Gla Cys phs Ser llrg Tyr Pro lisp 8is flet Lyr Gln 8is hsp phe phe Lys Ser Ala ttat pro G1u Gly Tyr Val Gla Glu llrg Thr Ile Phe phe Lys lisp Asp Gly 1ua Tyr Lys Thr ~lrg Ala Glu Vai Lys phe Glu Gly Asp Thr Leu Val Asa J~rg Ila Giu Leu Lys Gly Ile 115 120 i35 wo oonaZm Pcrnrs99noZO~
Asp Phe Lys Glu Asp Gly Asa Ile Leu Aly 8is Lys Leu Alu Tyr Aaa Tyr Aaa Ser 8is Aaa Val Tyr =le ttat Ala Asp Lys Oln Lys Asa Oly Ile Lys Val Asa Phs Lys =le Arg 8is Asa Ile Olu Asp Aly 8er Dal lb5 190 175 Gla Leu Ala Asp 8is Tyr Ala Ola Asa Thr Pro ile Oly Asp Oly Pro Val Leu Leu Pro Asp Asa 8is Tyr Leu Ser Tbr Ola 8er A1a Leu Ser Lys Asp Pro Asa Glu Lys Arg Asp 8is lfat Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr 8is Oly llet Asp Olu Lsu Tyr Lye <Z10>9 <~11>333 <slz>a~A

<Z13>8amao sapieas <ZZO>
<Zal> cas <asZ> (1) .. (333) <400> 9 atg gcc ctg tgg atg cgc ctc ctg ccc ctg atg gcg ctg ctg gcc ctc ~18 Diet Ala Leu Trp llet Arg Lsu Leu Pro Leu Leu Ala Leu Leu Ala Leu tgg gga cct gac cca gcc gca gcc ttt gtg aaa caa cac ctg tgc ggc 96 Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asa Gala 8is Leu Cys Oly tca cac ctg gtg gaa get ctc tac cta gtg tga ggg gaa cga ggc ttc 1~4 8er His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Oly Phe 35 '10 ~5 ttc tac aca ccc aag acc cgc cgg gag gca gag gac ctg csg gtg qgg 19a Phe.Tyr Thr Pro Lys Thr Arg Arg Glu Ala Olu Asp Leu G1a Vai Gly caQ gtg gag ctg gge ggg Qgc cct ggt Qca ggc agc ctg eag cca ttg Z40 Oln Val Olu Lau Oly Oly Oly Pro Oly ,illa Oly Ber Leu 01a pro Leu gcc ctg Qag gQg tcc ctg cag aay agt ggc att gtg gaa aaa tgc tgt Z88 Ala Leu Olu Oly eer i.eu Ola Lya Arg Oly Ile Val 31u Ola C~ra Cys acc agc atc tpc tcc ctc tac cag ctg Qag aac tac tgc aac taQ 333 Ths 8er I1e Cys Sar Lsu Tyr 01a teu Olu Aaa Tyr Cys llsn <Z10>10 <Z11>110 <~la>PRT

<a13>80~o aapiena <<00> 10 let 711a Leu Trp llet Jlrg Leu Lau pro Leu Leu 111a leu Leu 711a Leu 1 S 10 i5 Trp Oly pro leap pro Jlls 111a Ai.a phe Val ~laa Ola 8ie l.eu Cye Oly 8er 8ia teu Val Olu 111a lau Tyr Leu Val CYs Oly Olu Arg Oly phe Phe Tyr Thr pro Lya Thr ~ Arg Alu 111a Olu hap Leu Ola Val 01y 50 55 60 , Gln VaI 01u Leu Oly Oly Oly Pro Oly ~11a Oly 8er Leu Ola pro Leu l~la Leu Olu Aly 8sr Leu 01a Lya Arg 01y Ile Val 01u Ola Cars Cya Thr Ser Ile Cya 8er Leu Tyr Ola beu 01u llsn Tyr Cya 7lsa

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.

b. 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 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 Renitta 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:

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;

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 flurophore 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.