CN1323353A - Method for studying protein interactions i(invivo) - Google Patents
Method for studying protein interactions i(invivo) Download PDFInfo
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- CN1323353A CN1323353A CN 99811958 CN99811958A CN1323353A CN 1323353 A CN1323353 A CN 1323353A CN 99811958 CN99811958 CN 99811958 CN 99811958 A CN99811958 A CN 99811958A CN 1323353 A CN1323353 A CN 1323353A
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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
Background
Research to the interphase interaction of viable cell internal protein is often very necessary for understanding protein function and mechanism of action thereof.These interact and adopt immunoprecipitation, yeast two-hybrid method and β-gal complementarity method to study at present.
Yet these methods have some defectives.For example, these methods have false positive.The second, these methods cannot be measured about interactional quantification information.In addition, they can not monitor interaction in the body in real time.
Therefore, obtain the method for the another kind of research body internal protein interphase interaction that does not have these shortcomings with highly beneficial.More preferably, present method can be used for protein widely in diversified viable cell.Equally preferably, present method can be used for measuring isolating protein other intermolecular interaction in addition.
General introduction
According to one embodiment of the invention, provide the interior first kind of protein of a kind of mensuration viable cell and second kind of protein whether interactional method takes place.Present method is included in to provide in the cell with first kind of protein of donor luciferase compound with acceptor fluorescence rolls into a ball second kind of protein of compound.When first kind of protein donor luciferase when second kind of protein can pass to acceptor fluorescence group with luminous resonance energy.First kind of protein of compound and second kind of protein of compound allow close mutually in cell then.Any fluorescence of subsequent detection from acceptor fluorescence group.Pass to acceptor fluorescence group by the luminous resonance energy of donor luciferase and cause acceptor fluorescence group to produce fluorescence, show that interaction has taken place for first kind of protein and second kind of protein.
In a preferred embodiment, provide with first kind of protein of donor luciferase compound with acceptor fluorescence and rolled into a ball second kind of protein of compound, comprise genetically engineered DNA and genetically engineered DNA transferred in the viable cell, this cause cell produce with first kind of protein of donor luciferase compound and with second kind of protein of acceptor fluorescence group's compound.In an especially preferred embodiment, contain with first kind of proteinic cell of donor luciferase compound and contain and be mammalian cell with second kind of proteinic cell of acceptor fluorescence group's compound.
In another preferred embodiment, the donor luciferase that is provided is the Renilla luciferase.And in another kind of embodiment preferred, the acceptor fluorescence group that is provided is a kind of jellyfish (Aequorea) egfp.
In an especially preferred embodiment, use spectrofluorimetry to detect the fluorescence that acceptor fluorescence group is sent.
Describe
The present invention includes interior a kind of protein of the luminous resonance energy transmission of a kind of utilization (LRET) mensuration viable cell and another kind of protein whether interactional method takes place.Luminous resonance energy transmission is because the transmission that the excited state of donor luciferase energy is rolled into a ball to acceptor fluorescence.For the generation of LRET, between the excitation spectrum that the emmission spectrum and the acceptor fluorescence of donor luciferase are rolled into a ball, must have overlapping.
The efficient of luminous resonance energy transmission especially depends on the distance of separating donor luciferase and acceptor fluorescence group in all multivariates.In general, significantly transmission ofenergy only just takes place when donor luciferase and acceptor fluorescence are rolled into a ball each other less than 80 .This short range is significantly smaller than with the required optical resolution distance of two entities of conventional microscopic examination.Therefore, detect between donor luciferase and acceptor fluorescence group luminous resonance energy transmission take place, show donor luciferase and acceptor fluorescence group near and be in LRET and take place within the required distance, promptly each other approximately less than 80 .
Whether the present invention uses luminous resonance energy transmission to measure between interior a kind of protein of viable cell and the another kind of protein and interacts.Finishing of it is to rely on first kind of protein with the donor luciferase is compound and it is compound that second kind of protein and acceptor fluorescence are rolled into a ball, and first kind of protein of compound and second kind of protein of compound put into cell and be in suitable first kind of protein and second kind of protein takes place under the interactional condition.If first kind of protein and second kind of protein interaction, thus luminous resonance energy transmission takes place with close enough acceptor fluorescence group in the donor luciferase, and acceptor fluorescence group will send fluorescence.Thus, the fluorescence that detects from acceptor fluorescence group will show that first kind of protein with second kind of protein interaction has taken place.Advantageously, though this method also can be used for detecting use-case such as the microscopical optical means of routine can't detected first kind of protein and second kind of protein between interaction.
Use luminous resonance energy transmission to detect that interactional method has several advantages between two kinds of protein according to the present invention.At first, the specific mark of viable cell internal protein can obtain by engineered method, and fluorescence dye is imported very difficulty of viable cell.In addition, if for substrate and oxygen are arranged, enzymatic reaction causes as the luciferase of Renilla luminous relatively stable, the then rapid photobleaching of fluorescence dye.
As disclosing used method herein, " first kind of protein and donor luciferase is compound " be meant donor luciferase and first kind of protein combined in such a manner, makes that donor luciferase and first kind of protein are gone up close equally mutually substantially when first kind of protein and second kind of protein interaction.Similarly, " group is compound with second kind of protein and acceptor fluorescence " be meant acceptor fluorescence group combined in such a manner with second kind of protein, makes when first kind of protein and second kind of protein interaction acceptor fluorescence roll into a ball and second kind of protein is gone up close equally mutually substantially.For example, can make cells produce comprise donor luciferase and first kind of proteinic fusion rotein and comprise acceptor fluorescence group and second kind of proteinic fusion rotein carries out this compound by genetically engineered.
In a preferred embodiment, the present invention uses the Renilla luciferase as the donor luciferase, uses " humanized " jellyfish green fluorescent protein (humanization GFP) as acceptor fluorescence group.The Renilla luciferase is the enzyme of a kind of 34kDa that is purified into from Renilla reniformis.The oxidative decarboxylation of this enzyme catalysis coelentenazine during aerobic, producing the emission wavelength maximum value is the blue light of 471nm.The Renilla luciferase is because it needs the external source substrate to activate except that external source light as the donor luciferase.Can advantageously remove background noise like this, thereby allow the light that quantitative assay easily and exactly produced from external source light source and autofluorescence generation.
" humanization " GFP is the protein fluorescence group of a kind of excitation maximum at the 27kDa of 480nm fluorescence.It has compared an amino acid difference with wild-type jellyfish green fluorescent protein.Why selecting " humanization " GFP is because its excitation spectrum and the emmission spectrum overlaid of Renilla luciferase as acceptor fluorescence group.In addition, the light that sends of " humanization " GFP is visible in viable cell.And " humanization " GFP is being used for proving that " humanization " GFP cDNA mammalian cells transfected of this method can obtain good expression.
Be used to measure a kind of protein and another kind of protein whether interactional method shows below according to the present invention.In brief, IGFBP6 (IGFBP6) and IGF-(IGF-II) are elected to be first kind of protein and second kind of protein.Known IGFBP6 is a kind of protein that the IGF-II is had obvious binding affinity.
The cDNA of Renilla luciferase merges mutually with the cDNA of IGFBP6, and " humanization " GFP cDNA merges mutually with IGF-II cDNA.The cDNA transfection viable cell that merges, and expressed fusion protein.Produce and the cell mixing extract.The substrate that adds the Renilla luciferase part in Renilla luciferase-IGFPB 6 protein that merge.Finally, detect the fluorescence that partly sends by " humanization " GFP in " humanization " GFP-IGF-II protein that merges.A kind of method that shows according to the present invention is described now in further detail.A) IGFBP-6 cDNA-Renilla luciferase cDNA, IGF-II cDNA-" humanization " the GFP cDNA of fusion of clone's fusion and Regular Insulin cDNA-" humanization " GFPcDNA of fusion:
At first, IGFBP-6 cDNA and the Renilla luciferase cDNA that the merges cDNA:1 of three kinds of fusions of production); 2) the IGF-II cDNA of Rong Heing and " humanization " GFP cDNA; With 3) the Regular Insulin cDNA and " humanization " GFP cDNA that merge.IGFBP-6 cDNA, SEQ NO:1, the Genbank number of including M69054, coding is as the IGFBP-6 of first kind of proteinic SEQNO:2.Renilla luciferase cDNA, SEQ NO:3, the Genbank number of including M63501, coding is as the Renilla luciferase of the SEQ NO:4 of donor luciferase.IGF-II cDNA, SEQ NO:5, coding is as the IGF-II of second kind of proteinic SEQNO:6." humanization " GFP cDNA, SEQ NO:7, the Genbank number of including is U50963, coding is as " humanization " GFP of the SEQ NO:8 of acceptor fluorescence group.Regular Insulin cDNA, SEQ NO:9, the Genbank number of including is AH002844, the Regular Insulin of coding SEQNO:10.The Regular Insulin that merges with " humanization " GFP contrasts as protein, because Regular Insulin and IGF-II homology, but do not combine with IGFBP-6.The Regular Insulin cDNA of the IGFBP-6 cDNA of SEQ NO:1, the IGF-II cDNA of SEQ NO:5 and SEQ NO:9 modifies with PCR is following.
At first, will clone into pBluescript KS (+) II carrier by preceding former-IGF-II cDNA that EcoR I fragment is carried.Insert fragment by T
7And T
3Primer checks order, and determines to comprise former before known-IGF-II cDNA sequence.T in 5 of IGF-II precursor ' end and pBluescriptKS (+) the II carrier
7Promotor links to each other.Design IGF-II 3 ' primer is to produce a permission perceptible (Notice of Allowance) restriction site, thereby the D and the E structural domain of former before removing-IGF-II keep the perceptible fragment of permission of " humanization " GFP to be in the IGF-II open reading frame.
Secondly, IGF-II fragment is used T
7Promoter primer and IGF-II 3 ' primer carry out pcr amplification.The IGF-II fragment of pcr amplification is cloned into pCDNA3.1 (+) carrier (Invitrogen, Carlsbad, CA, the U.S.) by EcoR I and the digestion of Not I, produces the pCDNA-IGF-II.Then, the Not I site that the perceptible fragment of the permission of " humanization " GFP is inserted the pCDNA-IGF-II produces pC-IGF-II-GFP.
The cDNA that comprises B, C and A structural domain signal propeptide Regular Insulin modifies corresponding to the segmental mode of IGF-II according to above.3 ' the end that then cDNA of " humanization " GFP is connected to the Regular Insulin cDNA of modification links to each other, and produces pC-INS-GFP.
At last, utilize the PCR IGFBP6 cDNA that from the people IGFBP6 plasmid of a rat mark by name, increases.The terminator codon of IGFBP6 is removed, and its open reading frame meets framework (Mayerhofer R, Langridge WHR, Cormier MG and the Szalay AA from the Renilla luciferase cDNA of pCEP4-RVC.The expression of reorganization Renilla luciferase in transgenic plant causes high level of light reflection.The plant magazine, 1995; 7; 1031～8).Renilla luciferase cDNA is connected to 3 ' end of the IGFBP-6 cDNA of modification, produces pC-IGFBP6-RUC.
The sequence of inserting dna fragmentation in all constructs confirms by the DNA tests analysis.(Valencia CA) is used for plasmid DNA purification to Qiagen Maxi plasmid kit for Qiagen, Inc..B) with the calcium phosphate precipitation method with pC-IGF-II-GFP, pC-INS-GFP and pC-IGFBP6-RUC transient transfection mammalian cell:
And then, with the fusion dna transfection mammalian cell of cloning.At first, COS-7 cell (African green monkey kidney cell, American type culture collection CRL 1651) be penicillin 100u/ml, Streptomycin sulphate 100mg/ml and amphotericin B 250ng/ml (Sigma-Aldrich Co. containing L-glutaminate, 10% foetal calf serum and comprising final concentration, St.Louis, in the Dulbecco ' s improvement Eagle substratum (DMEM) of Vanobid solution MO, U.S.) 37 ℃ in 5%CO
2Grow under the condition.Transfection the day before yesterday is with every group 1 * 10
6The cell bed board has 50%～60% compile approximately during transfection.
Each plasmid fusion dna 40mg post precipitation is resuspended with Dulbecco ' s phosphate buffered saline(PBS), and the calcium phosphate precipitation method with standard imports mammalian cell with the plasmid fusion dna then.Estimate transfection efficiency with fluorescent microscope after 24 hours.Each dull and stereotyped green fluorescence cell number is similar at the dull and stereotyped of the flat board of pC-IGF-II-GFP DNA transfectional cell, pC-INS-GFP DNA transfectional cell and in as the flat board of the plasmid DNA transfection that only comprises GFP of positive control.C) confirmation of expressing fusion protein
After 24 hours, can be observed the single COS-7 cell of plasmid DNA transfection with DNA calcium phosphate precipitation method transfection DNA by detection GFP fluorescence with fluorescent microscope.PC-IGF-II-GFP and pC-INS-GFP cells transfected demonstrate similar fluorescence mode, and promptly typical secretory protein shifts to golgi body from ER.The pC-IGFBP6-RUC cells transfected does not fluoresce.Yet, show that with the low light imaging system pC-IGFBP 6-RUC cells transfected is luminous behind the adding Coelenterazine.
Further, the expection molecular weight is about fusion rotein IGF-II-GFP of 36kDa and 56kDa respectively and IGFBP6-RUC can detect with the immunoblotting assay method.Can prove conclusively the existence of two kinds of fusion roteins in the transient transfection cell like this.D) detect protein interaction by spectrofluorimetry:
Proved conclusively the existence of expection fusion rotein IGF-II-GFP and IGFBP 6-RUC, and after the function of donor luciferase and acceptor fluorescence group, the protein bound that the cell extract of these transient transfection cells can be used for carrying out based on transmission ofenergy between fusion rotein Renilla luciferase part and " humanization " GFP part is tested.After the calcium transfection 48 hours, the COS cell is washed 2 times with PBS, then with cell scraper with cell harvesting in the luciferase analysis buffer that comprises 0.5M NaCl, 1mM EDTA and 0.1M potassiumphosphate (pH7.5).The cell of results with Fisher 550 type Ultrasonic Cell Disruptors (Fisher Scientific, Pittsburgh, PA, the U.S.) with carried out at interval in 10 seconds 3 times 10 seconds ultrasonic, to produce cell extract.
Then, mix the cell extract that comprises IGF-II-GFP and IGFBP 6-RUC, add 0.1 μ g coelenterazine immediately.Use SPEX Fluoro Max
(Edison NJ) carries out spectrofluorimetry for instrument S.A., Inc..Spectrum shows at the 471nm place single emission peak, and it is corresponding to known Renilla luciferase emission spectrum.
After first time spectrofluorimetry, mixture room temperature preservation 30 minutes, and add fresh Coelenterazine and repeat spike spectrum.Spike is presented at 471nm and there are 2 emission maximum peak values at the 503nm place after 30 minutes.The spectrofluorimetry of cell extract carries out the longer time, but spectral patterns does not change in time.
The extract mixtures of pC-INS-GFP in contrast and pC-IGFBP 6-RUC transfectional cell is by the similar approach preparation and carry out the spectrum spike.Spike shows only a peak at the 471nm place, it is corresponding to the emission peak of Renilla luciferase.Spectral patterns does not change in time.
Therefore, these data show that IGFBP-6 and IGF-II interact and Regular Insulin and IGFBP 6 do not interact.
Except above disclosed embodiment, protein-protein interaction also can use corresponding method to detect by detecting LRET in the intestinal bacteria of cotransformation and mammalian cell.
Although the present invention has quite at large discussed relevant preferred embodiment, other embodiment is feasible equally.For example, the molecular interaction beyond the isolating protein can be studied with corresponding method.Other molecule can be by diffusion, import and mix or alternate manner offers viable cell.In addition, the fusion rotein of genetically engineered viable cell production can carry out posttranslational modification before research interacts, as adding sugar moieties.Equally, use these methods in cell, colony and tissue, to observe viable cell by spectrofluorimetry low light imaging analysis.In addition, cell sorting art and the low light imaging analysis of the inventive method can be used, high-throughout colony screening can be finished in conjunction with droplet hole or many array detection.Therefore, the description of the marrow of additional claim and the scope preferred embodiment that should not be limited to comprise here.
Sequence table＜110〉Szalay, Aladar A.
Wang,Yubao
Wang-Pruski,Gefu
Loma Linda University＜120〉for interactional method in the Study on Protein body＜130〉11785-3PCT＜140〉in this submission＜141〉1999-09-02＜150〉60/135; 835＜151〉1999-05-24＜150〉60/099,068＜151〉1998-09-03＜160〉10＜170〉PatentIn Ver.2.0＜210〉1＜211〉918＜212〉DNA＜213〉people＜220〉＜221〉CDS＜222〉(3) .. (719)＜400〉1tg tgc ccc cac agg ctg ctg cca ccg ctg ctg ctg ctg cta gct ctg 47 Cys Pro His Arg Leu Leu Pro Pro Leu Leu Leu Leu Leu Ala Leu
1 5 10 15ctg?ctc?gct?gcc?agc?cca?gga?ggc?gcc?ttg?gcg?cgg?tgc?cca?ggc?tgc 95Leu?Leu?Ala?Ala?Ser?Pro?Gly?Gly?Ala?Leu?Ala?Arg?Cys?Pro?Gly?Cys
20 25 30ggg?caa?ggg?gtg?cag?gcg?ggt?tgt?cca?ggg?ggc?tgc?gtg?gag?gag?gag 143Gly?Gln?Gly?Val?Gln?Ala?Gly?Cys?Pro?Gly?Gly?Cys?Val?Glu?Glu?Glu
35 40 45gat?ggg?ggg?tcg?cca?gcc?gag?ggc?tgc?gcg?gaa?gct?gag?ggc?tgt?ctc 191Asp?Gly?Gly?Ser?Pro?Ala?Glu?Gly?Cys?Ala?Glu?Ala?Glu?Gly?Cys?Leu
50 55 60agg?agg?gag?ggg?cag?gag?tgc?ggg?gtc?tac?acc?cct?aac?tgc?gcc?cca 239Arg?Arg?Glu?Gly?Gln?Glu?Cys?Gly?Val?Tyr?Thr?Pro?Asn?Cys?Ala?Pro
65 70 75gga?ctg?cag?tgc?cat?ccg?ccc?aag?gac?gac?gag?gcg?cct?ttg?cgg?gcg 287Gly?Leu?Gln?Cys?His?Pro?Pro?Lys?Asp?Asp?Glu?Ala?Pro?Leu?Arg?Ala?80 85 90 95ctg?ctg?ctc?ggc?cga?ggc?cgc?tgc?ctt?ccg?gcc?cgc?gcg?cct?gct?gtt 335Leu?Leu?Leu?Gly?Arg?Gly?Arg?Cys?Leu?Pro?Ala?Arg?Ala?Pro?Ala?Val
100 105 110gca?gag?gag?aat?cct?aag?gag?agt?aaa?ccc?caa?gca?ggc?act?gcc?cgc 383Ala?Glu?Glu?Asn?Pro?Lys?Glu?Ser?Lys?Pro?Gln?Ala?Gly?Thr?Ala?Arg
115 120 125cca?cag?gat?gtg?aac?cgc?aga?gac?caa?cag?agg?aat?cca?ggc?acc?tct 431Pro?Gln?Asp?Val?Asn?Arg?Arg?Asp?Gln?Gln?Arg?Asn?Pro?Gly?Thr?Ser
130 135 140acc?acg?ccc?tcc?cag?ccc?aat?tct?gcg?ggt?gtc?caa?gac?act?gag?atg 479Thr?Thr?Pro?Ser?Gln?Pro?Asn?Ser?Ala?Gly?Val?Gln?Asp?Thr?Glu?Met
145 150 155ggc?cca?tgc?cgt?aga?cat?ctg?gac?tca?gtg?ctg?cag?caa?ctc?cag?act 527Gly?Pro?Cys?Arg?Arg?His?Leu?Asp?Ser?Val?Leu?Gln?Gln?Leu?Gln?Thr160 165 170 175gag?gtc?tac?cga?ggg?gct?caa?aca?ctc?tac?gtg?ccc?aat?tgt?gac?cat 575Glu?Val?Tyr?Arg?Gly?Ala?Gln?Thr?Leu?Tyr?Val?Pro?Asn?Cys?Asp?His
180 185 190cga?ggc?ttc?tac?cgg?aag?cgg?cag?tgc?cgc?tcc?tcc?cag?ggg?cag?cgc 623Arg?Gly?Phe?Tyr?Arg?Lys?Arg?Gln?Cys?Arg?Ser?Ser?Gln?Gly?Gln?Arg
195 200 205cga?ggt?ccc?tgc?tgg?tgt?gtg?gat?cgg?atg?ggc?aag?tcc?ctg?cca?ggg 671Arg?Gly?Pro?Cys?Trp?Cys?Val?Asp?Arg?Met?Gly?Lys?Ser?Leu?Pro?Gly
210 215 220tct?cca?gat?ggc?aat?gga?agc?tcc?tcc?tgc?ccc?act?ggg?agt?agc?ggc 719Ser?Pro?Asp?Gly?Asn?Gly?Ser?Ser?Ser?Cys?Pro?Thr?Gly?Ser?Ser?Gly
225 230 235taaagctggg ggatagaggg gctgcgggc cactggaagg aacatggagc tgtcatcact 779caacaaaaaa ccgaggccct caatccacct tcaggccccg ccccatgggc ccctcaccgc 839tggttggaaa gagtgttggt gttggctggg gtgtcaataa agctgtgctt ggggtcgctg 899aaaaaaaaaa aaaaaaaaa, 918＜210〉2＜211〉239＜212〉PRT＜213〉people＜400〉2Cys Pro His Arg Leu Leu Pro Pro Leu Leu Leu Leu Leu Ala Leu Leu, 15 10 15Leu Ala Ala Ser Pro Gly Gly Ala Leu Ala Arg Cys Pro Gly Cys Gly
20 25 30Gln?Gly?Val?Gln?Ala?Gly?Cys?Pro?Gly?Gly?Cys?Val?Glu?Glu?Glu?Asp
35 40 45Gly?Gly?Ser?Pro?Ala?Glu?Gly?Cys?Ala?Glu?Ala?Glu?Gly?Cys?Leu?Arg
50 55 60Arg?Glu?Gly?Gln?Glu?Cys?Gly?Val?Tyr?Thr?Pro?Asn?Cys?Ala?Pro?Gly?65 70 75 80Leu?Gln?Cys?His?Pro?Pro?Lys?Asp?Asp?Glu?Ala?Pro?Leu?Arg?Ala?Leu
85 90 95Leu?Leu?Gly?Arg?gly?Arg?Cys?Leu?Pro?Ala?Arg?Ala?Pro?Ala?Val?Ala
100 105 110Glu?Glu?Asn?Pro?Lys?Glu?Ser?Lys?Pro?Gln?Ala?Gly?Thr?Ala?Arg?Pro
115 120 125Gln?Asp?Val?Asn?Arg?Arg?Asp?Gln?Gln?Arg?Asn?Pro?Gly?Thr?Ser?Thr
130 135 140Thr?Pro?Ser?Gln?Pro?Asn?Ser?Ala?Gly?Val?Gln?Asp?Thr?Glu?Met?Gly145 150 155 160Pro?Cys?Arg?Arg?His?Leu?Asp?Ser?Val?Leu?Gln?Gln?Leu?Gln?Thr?Glu
165 170 175Val?Tyr?Arg?Gly?Ala?Gln?Thr?Leu?Tyr?Val?Pro?Asn?Cys?Asp?His?Arg
180 185 190Gly?Phe?Tyr?Arg?Lys?Arg?Gln?Cys?Arg?Ser?Ser?Gln?Gly?Gln?Arg?Arg
195 200 205Gly?Pro?Cys?Trp?Cys?Val?Asp?Arg?Met?Gly?Lys?Ser?Leu?Pro?Gly?Ser
210 215 220Pro?Asp?Gly?Asn?Gly?Ser?Ser?Ser?Cys?Pro?Thr?Gly?Ser?Ser?Gly225 230 235<210>3<211>1196<212>DNA<213>Renilla?reniformis<220><221>CDS<222>(10)..(945)<400>3agcttaaag?atg?act?tcg?aaa?gtt?tat?gat?cca?gaa?caa?agg?aaa?cgg?atg 51
Met?Thr?Ser?Lys?Val?Tyr?Asp?Pro?Glu?Gln?Arg?Lys?Arg?Met
1 5 10ata?act?ggt?ccg?cag?tgg?tgg?gcc?aga?tgt?aaa?caa?atg?aat?gtt?ctt 99Ile?Thr?Gly?Pro?Gln?Trp?Trp?Ala?Arg?Cys?Lys?Gln?Met?Asn?Val?Leu?15 20 25 30gat?tca?ttt?att?aat?tat?tat?gat?tca?gaa?aaa?cat?gca?gaa?aat?gct 147Asp?Ser?Phe?Ile?Asn?Tyr?Tyr?Asp?Ser?Glu?Lys?His?Ala?Glu?Asn?Ala
35 40 45gtt?att?ttt?tta?cat?ggt?aac?gcg?gcc?tct?tct?tat?tta?tgg?cga?cat 195Val?Ile?Phe?Leu?His?Gly?Asn?Ala?Ala?Ser?Ser?Tyr?Leu?Trp?Arg?His
50 55 60gtt?gtg?cca?cat?att?gag?cca?gta?gcg?cgg?tgt?att?ata?cca?gat?ctt 243Val?Val?Pro?His?Ile?Glu?Pro?Val?Ala?Arg?Cys?Ile?Ile?Pro?Asp?Leu
65 70 75att?ggt?atg?ggc?aaa?tca?ggc?aaa?tct?ggt?aat?ggt?tct?tat?agg?tta 291Ile?Gly?Met?Gly?Lys?Ser?Gly?Lys?Ser?Gly?Asn?Gly?Ser?Tyr?Arg?Leu
80 85 90ctt?gat?cat?tac?aaa?tat?ctt?act?gca?tgg?ttt?gaa?ctt?ctt?aat?tta 339Leu?Asp?His?Tyr?Lys?Tyr?Leu?Thr?Ala?Trp?Phe?Glu?Leu?Leu?Ash?Leu?95 100 105 110cca?aag?aag?atc?att?ttt?gtc?ggc?cat?gat?tgg?ggt?gct?tgt?ttg?gca 387Pro?Lys?Lys?Ile?Ile?Phe?Val?Gly?His?Asp?Trp?Gly?Ala?Cys?Leu?Ala
115 120 125ttt?cat?tat?agc?tat?gag?cat?caa?gat?aag?atc?aaa?gca?ata?gtt?cac 435Phe?His?Tyr?Ser?Tyr?Glu?His?Gln?Asp?Lys?Ile?Lys?Ala?Ile?Val?His
130 135 140gct?gaa?agt?gta?gta?gat?gtg?att?gaa?tca?tgg?gat?gaa?tgg?cct?gat 483Ala?Glu?Ser?Val?Val?Asp?Val?Ile?Glu?Ser?Trp?Asp?Glu?Trp?Pro?Asp
145 150 155att?gaa?gaa?gat?att?gcg?ttg?atc?aaa?tct?gaa?gaa?gga?gaa?aaa?atg 531Ile?Glu?Glu?Asp?Ile?Ala?Leu?Ile?Lys?Ser?Glu?Glu?Gly?Glu?Lys?Met
160 165 170gtt?ttg?gag?aat?aac?ttc?ttc?gtg?gaa?acc?atg?ttg?cca?tca?aaa?atc 579Val?Leu?Glu?Asn?Asn?Phe?Phe?Val?Glu?Thr?Met?Leu?Pro?Set?Lys?Ile175 180 185 190atg?aga?aag?tta?gaa?cca?gaa?gaa?ttt?gca?gca?tat?ctt?gaa?cca?ttc 627Met?Arg?Lys?Leu?Glu?Pro?Glu?Glu?Phe?Ala?Ala?Tyr?Leu?Glu?Pro?Phe
195 200 205aaa?gag?aaa?ggt?gaa?gtt?cgt?cgt?cca?aca?tta?tca?tgg?cct?cgt?gaa 675Lys?Glu?Lys?Gly?Glu?Val?Arg?Arg?Pro?Thr?Leu?Ser?Trp?Pro?Arg?Glu
210 215 220atc?ccg?tta?gta?aaa?ggt?ggt?aaa?cct?gac?gtt?gta?caa?att?gtt?agg 723Ile?Pro?Leu?Val?Lys?Gly?Gly?Lys?Pro?Asp?Val?Val?Gln?Ile?Val?Arg
225 230 235aat?tat?aat?gct?tat?cta?cgt?gca?agt?gat?gat?tta?cca?aaa?atg?ttt 771Asn?Tyr?Asn?Ala?Tyr?Leu?Arg?Ala?Ser?Asp?Asp?Leu?Pro?Lys?Met?Phe
240 245 250att?gaa?tcg?gat?cca?gga?ttc?ttt?tcc?aat?gct?att?gtt?gaa?ggc?gcc 819Ile?Glu?Ser?Asp?Pro?Gly?Phe?Phe?Ser?Asn?Ala?Ile?Val?Glu?Gly?Ala255 260 265 270aag?aag?ttt?cct?aat?act?gaa?ttt?gtc?aaa?gta?aaa?ggt?ctt?cat?ttt 867Lys?Lys?Phe?Pro?Asn?Thr?Glu?Phe?Val?Lys?Val?Lys?Gly?Leu?His?Phe
275 280 285tcg?caa?gaa?gat?gca?cct?gat?gaa?atg?gga?aaa?tat?atc?aaa?tcg?ttc 915Ser?Gln?Glu?Asp?Ala?Pro?Asp?Glu?Met?Gly?Lys?Tyr?Ile?Lys?Ser?Phe
290 295 300gtt?gag?cga?gtt?ctc?aaa?aat?gaa?caa?taa?ttactttggt?tttttattta 965Val?Glu?Arg?Val?Leu?Lys?Asn?Glu?Gln
305 310catttttccc?gggtttaata?atataaatgt?cattttcaac?aattttattt?taactgaata?1025tttcacaggg?aacattcata?tatgttgatt?aatttagctc?gaactttact?ctgtcatatc?1085attttggaat?attacctctt?tcaatgaaac?tttataaaca?gtggttcaat?taattaatat?1145atattataat?tacatttgtt?atgtaataaa?ctcggtttta?ttataaaaaa?a 1196<210>4<211>311<212>PRT<213>Renilla?reniformis<400>4Met?Thr?Ser?Lys?Val?Tyr?Asp?Pro?Glu?Gln?Arg?Lys?Arg?Met?Ile?Thr 1 5 10 15Gly?Pro?Gln?Trp?Trp?Ala?Arg?Cys?Lys?Gln?Met?Asn?Val?Leu?Asp?Ser
20 25 30Phe?Ile?Asn?Tyr?Tyr?Asp?Ser?Glu?Lys?His?Ala?Glu?Asn?Ala?Val?Ile
35 40 45Phe?Leu?His?Gly?Asn?Ala?Ala?Ser?Ser?Tyr?Leu?Trp?Arg?His?Val?Val
50 55 60Pro?His?Ile?Glu?Pro?Val?Ala?Arg?Cys?Ile?Ile?Pro?Asp?Leu?Ile?Gly?65 70 75 80Met?Gly?Lys?Ser?Gly?Lys?Ser?Gly?Asn?Gly?Ser?Tyr?Arg?Leu?Leu?Asp
85 90 95His?Tyr?Lys?Tyr?Leu?Thr?Ala?Trp?Phe?Glu?Leu?Leu?Asn?Leu?Pro?Lys
100 105 110Lys?Ile?Ile?Phe?Val?Gly?His?Asp?Trp?Gly?Ala?Cys?Leu?Ala?Phe?His
115 120 125Tyr?Ser?Tyr?Glu?His?Gln?Asp?Lys?Ile?Lys?Ala?Ile?Val?His?Ala?Glu
130 135 140Ser?Val?Val?Asp?Val?Ile?Glu?Ser?Trp?Asp?Glu?Trp?Pro?Asp?Ile?Glu145 150 155 160Glu?Asp?Ile?Ala?Leu?Ile?Lys?Ser?Glu?Glu?Gly?Glu?Lys?Met?Val?Leu
165 170 175Glu?Asn?Asn?Phe?Phe?Val?Glu?Thr?Met?Leu?Pro?Ser?Lys?Ile?Met?Arg
180 185 190Lys?Leu?Glu?Pro?Glu?Glu?Phe?Ala?Ala?Tyr?Leu?Glu?Pro?Phe?Lys?Glu
195 200 205Lys?Gly?Glu?Val?Arg?Arg?Pro?Thr?Leu?Ser?Trp?Pro?Arg?Glu?Ile?Pro
210 215 220Leu?Val?Lys?Gly?Gly?Lys?Pro?Asp?Val?Val?Gln?Ile?Val?Arg?Asn?Tyr225 230 235 240Asn?Ala?Tyr?Leu?Arg?Ala?Ser?Asp?Asp?Leu?Pro?Lys?Met?Phe?Ile?Glu
245 250 255Ser?Asp?Pro?Gly?Phe?Phe?Ser?Asn?Ala?Ile?Val?Glu?Gly?Ala?Lys?Lys
260 265 270Phe?Pro?Asn?Thr?Glu?Phe?Val?Lys?Val?Lys?Gly?Leu?His?Phe?Ser?Gln
275 280 285Glu?Asp?Ala?Pro?Asp?Glu?Met?Gly?Lys?Tyr?Ile?Lys?Ser?Phe?Val?Glu
290 295 300Arg Val Leu Lys Asn Glu Gln305 310＜210〉5＜211〉543＜212〉DNA＜213〉people＜220〉＜221〉CDS＜222〉(1) .. (543)＜400〉5atg gga atc cca atg ggg aag tcg atg ctg gtg ctt ctc acc ttc ttg 48Met Gly Ile Pro Met Gly Lys Ser Met Leu Val Leu Leu Thr Phe Leu 15 10 15gcc ttc gcc tcg tgc tgc att gct gct tac cgc ccc agt gag acc ctg 96Ala Phe Ala Ser Cys Cys Ile Ala Ala Tyr Arg Pro Ser Glu Thr Leu
20 25 30tgc?ggc?ggg?gag?ctg?gtg?gac?acc?ctc?cag?ttc?gtc?tgt?ggg?gac?cgc 144Cys?Gly?Gly?Glu?Leu?Val?Asp?Thr?Leu?Gln?Phe?Val?Cys?Gly?Asp?Arg
35 40 45ggc?ttc?tac?ttc?agc?agg?ccc?gca?agc?cgt?gtg?agc?cgt?cgc?agc?cgt 192Gly?Phe?Tyr?Phe?Ser?Arg?Pro?Ala?Ser?Arg?Val?Ser?Arg?Arg?Ser?Arg
50 55 60ggc?atc?gtt?gag?gag?tgc?tgt?ttc?cgc?agc?tgt?gac?ctg?gcc?ctc?ctg 240Gly?Ile?Val?Glu?Glu?Cys?Cys?Phe?Arg?Ser?Cys?Asp?Leu?Ala?Leu?Leu?65 70 75 80gag?acg?tac?tgt?gct?acc?ccc?gcc?aag?tcc?gag?agg?gac?gtg?tcg?acc 288Glu?Thr?Tyr?Cys?Ala?Thr?Pro?Ala?Lys?Ser?Glu?Arg?Asp?Val?Ser?Thr
85 90 95cct?ccg?acc?gtg?ctt?ccg?gac?aac?ttc?ccc?aga?tac?ccc?gtg?ggc?aag 336Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val?Gly?Lys
100 105 110ttc?ttc?caa?tat?gac?acc?tgg?aag?cag?tcc?acc?cag?cgc?ctg?cgc?agg 384Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln?Arg?Leu?Arg?Arg
115 120 125ggc?ctg?cct?gcc?ctc?ctg?cgt?gcc?cgc?cgg?ggt?cac?gtg?ctc?gcc?aag 432Gly?Leu?Pro?Ala?Leu?Leu?Arg?Ala?Arg?Arg?Gly?His?Val?Leu?Ala?Lys
130 135 140gag?ctc?gag?gcg?ttc?agg?gag?gcc?aaa?cgt?cac?cgt?ccc?ctg?att?gct 480Glu?Leu?Glu?Ala?Phe?Arg?Glu?Ala?Lys?Arg?His?Arg?Pro?Leu?Ile?Ala145 150 155 160cta?ccc?acc?caa?gac?ccc?gcc?cac?ggg?ggc?gcc?ccc?cca?gag?atg?gcc 528Leu?Pro?Thr?Gln?Asp?Pro?Ala?His?Gly?Gly?Ala?Pro?Pro?Glu?Met?Ala
165 170 175agc?aat?cgg?aag?tga 543Ser?Asn?Arg?Lys
180＜210〉6＜211〉180＜212〉PRT＜213〉people＜400〉6Met Gly Ile Pro Met Gly Lys Ser Met Leu Val Leu Leu Thr Phe Leu, 15 10 15 Ala Phe Ala Ser Cys Cys Ile Ala Ala Tyr Arg Pro Ser Glu Thr Leu
20 25 30Cys?Gly?Gly?Glu?Leu?Val?Asp?Thr?Leu?Gln?Phe?Val?Cys?Gly?Asp?Arg
35 40 45Gly?Phe?Tyr?Phe?Ser?Arg?Pro?Ala?Ser?Arg?Val?Ser?Arg?Arg?Ser?Arg
50 55 60Gly?Ile?Val?Glu?Glu?Cys?Cys?Phe?Arg?Ser?Cys?Asp?Leu?Ala?Leu?Leu?65 70 75 80Glu?Thr?Tyr?Cys?Ala?Thr?Pro?Ala?Lys?Ser?Glu?Arg?Asp?Val?Ser?Thr
85 90 95Pro?Pro?Thr?Val?Leu?Pro?Asp?Asn?Phe?Pro?Arg?Tyr?Pro?Val?Gly?Lys
100 105 110Phe?Phe?Gln?Tyr?Asp?Thr?Trp?Lys?Gln?Ser?Thr?Gln?Arg?Leu?Arg?Arg
115 120 125Gly?Leu?Pro?Ala?Leu?Leu?Arg?Ala?Arg?Arg?Gly?His?Val?Leu?Ala?Lys
130 135 140Glu?Leu?Glu?Ala?Phe?Arg?Glu?Ala?Lys?Arg?His?Arg?Pro?Leu?Ile?Ala145 150 155 160Leu?Pro?Thr?Gln?Asp?Pro?Ala?His?Gly?Gly?Ala?Pro?Pro?Glu?Met?Ala
165 170 175Ser?Asn?Arg?Lys
180＜210〉7＜211〉717＜212〉DNA＜213〉artificial sequence＜220〉＜221〉CDS＜222〉(1) .. (717)＜220〉＜223〉artificial sequence description: humanization egfp cDNA＜400〉7atg agc aag ggc gag gaa ctg ttc act ggc gtg gtc cca att ctc gtg 48Met Sar Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val, 15 10 15gaa ctg gat ggc gat gtg aat ggg cac aaa ttt tct gtc agc gga gag 96Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Sar Val Ser Gly Glu
20 25 30ggt?gaa?ggt?gat?gcc?aca?tac?gga?aag?ctc?acc?ctg?aaa?ttc?atc?tgc 144Gly?Glu?Gly?Asp?Ala?Thr?Tyr?Gly?Lys?Leu?Thr?Leu?Lys?Phe?Ile?Cys
35 40 45acc?act?gga?aag?ctc?cct?gtg?cca?tgg?cca?aca?ctg?gtc?act?acc?ttc 192Thr?Thr?Gly?Lys?Leu?Pro?Val?Pro?Trp?Pro?Thr?Leu?Val?Thr?Thr?Phe
50 55 60tct?tat?ggc?gtg?cag?tgc?ttt?tcc?aga?tac?cca?gac?cat?atg?aag?cag 240Ser?Tyr?Gly?Val?Gln?Cys?Phe?Sar?Arg?Tyr?Pro?Asp?His?Met?Lys?Gln?65 70 75 80cat?gac?ttt?ttc?aag?agc?gcc?atg?ccc?gag?ggc?tat?gtg?cag?gag?aga 288His?Asp?Phe?Phe?Lys?Sar?Ala?Met?Pro?Glu?Gly?Tyr?Val?Gln?Glu?Arg
85 90 95acc?atc?ttt?ttc?aaa?gat?gac?ggg?aac?tac?aag?acc?cgc?gct?gaa?gtc 336Thr?Ile?Phe?Phe?Lys?Asp?Asp?Gly?Asn?Tyr?Lys?Thr?Arg?Ala?Glu?Val
100 105 110aag?ttc?gaa?ggt?gac?acc?ctg?gtg?aat?aga?atc?gag?ctg?aag?ggc?att 384Lys?Phe?Glu?Gly?Asp?Thr?Leu?Val?Asn?Arg?Ile?Glu?Leu?Lys?Gly?Ile
115 120 125gac?ttt?aag?gag?gat?gga?aac?att?ctc?ggc?cac?aag?ctg?gaa?tac?aac 432Asp?Phe?Lys?Glu?Asp?Gly?Asn?Ile?Leu?Gly?His?Lys?Leu?Glu?Tyr?Asn
130 135 140tat?aac?tcc?cac?aat?gtg?tac?atc?atg?gcc?gac?aag?caa?aag?aat?ggc 480Tyr?Asn?Sar?His?Asn?Val?Tyr?Ile?Met?Ala?Asp?Lys?Gln?Lys?Asn?Gly145 150 155 160atc?aag?gtc?aac?ttc?aag?atc?aga?cac?aac?att?gag?gat?gga?tcc?gtg 528Ile?Lys?Val?Asn?Phe?Lys?Ile?Arg?His?Asn?Ile?Glu?Asp?Gly?Ser?Val
165 170 175cag?ctg?gcc?gac?cat?tat?caa?cag?aac?act?cca?atc?ggc?gac?ggc?cct 576Gln?Leu?Ala?Asp?His?Tyr?Gln?Gln?Asn?Thr?Pro?Ile?Gly?Asp?Gly?Pro
180 185 190gtg?ctc?ctc?cca?gac?aac?cat?tac?ctg?tcc?acc?cag?tct?gcc?ctg?tct 624Val?Leu?Leu?Pro?Asp?Asn?His?Tyr?Leu?Ser?Thr?Gln?Ser?Ala?Leu?Ser
195 200 205aaa?gat?ccc?aac?gaa?aag?aga?gac?cac?atg?gtc?ctg?ctg?gag?ttt?gtg 672Lys?Asp?Pro?Asn?Glu?Lys?Arg?Asp?His?Met?Val?Leu?Leu?Glu?Phe?Val
210 215 220acc gct gct ggg atc aca cat ggc atg gac gag ctg tac aag tga 717Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys225,230 235＜210〉8＜211〉238＜212〉PRT＜213〉artificial sequence＜400〉8Met Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val, 15 10 15Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
20 25 30Gly?Glu?Gly?Asp?Ala?Thr?Tyr?Gly?Lys?Leu?Thr?Leu?Lys?Phe?Ile?Cys
35 40 45Thr?Thr?Gly?Lys?Leu?Pro?Val?Pro?Trp?Pro?Thr?Leu?Val?Thr?Thr?Phe
50 55 60Ser?Tyr?Gly?Val?Gln?Cys?Phe?Ser?Arg?Tyr?Pro?Asp?His?Met?Lys?Gln?65 70 75 80His?Asp?Phe?Phe?Lys?Ser?Ala?Met?Pro?Glu?Gly?Tyr?Val?Gln?Glu?Arg
85 90 95Thr?Ile?Phe?Phe?Lys?Asp?Asp?Gly?Asn?Tyr?Lys?Thr?Arg?Ala?Glu?Val
100 105 110Lys?Phe?Glu?Gly?Asp?Thr?Leu?Val?Asn?Arg?Ile?Glu?Leu?Lys?Gly?Ile
115 120 125Asp?Phe?Lys?Glu?Asp?Gly?Asn?Ile?Leu?Gly?His?Lys?Leu?Glu?Tyr?Asn
130 135 140Tyr?Asn?Ser?His?Asn?Val?Tyr?Ile?Met?Ala?Asp?Lys?Gln?Lys?Asn?Gly145 150 155 160Ile?Lys?Val?Asn?Phe?Lys?Ile?Arg?His?Asn?Ile?Glu?Asp?Gly?Ser?Val
165 170 175Gln?Leu?Ala?Asp?His?Tyr?Gln?Gln?Asn?Thr?Pro?Ile?Gly?Asp?Gly?Pro
180 185 190Val?Leu?Leu?Pro?Asp?Asn?His?Tyr?Leu?Ser?Thr?Gln?Ser?Ala?Leu?Ser
195 200 205Lys?Asp?Pro?Asn?Glu?Lys?Arg?Asp?His?Met?Val?Leu?Leu?Glu?Phe?Val
210 215 220Thr Ala Ala Gly Ile Thr His Gly Met Asp Glu Leu Tyr Lys225 230 235＜210〉9＜211〉333＜212〉DNA＜213〉people＜220〉＜221〉CDS＜222〉(1) .. (333)＜400〉9atg gcc ctg tgg atg cgc ctc ctg ccc ctg ctg gcg ctg ctg gcc ctc 48Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu 15 10 15tgg gga cct gac cca gcc gca gcc ttt gtg aac caa cac ctg tgc ggc 96Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly
20 25 30tca?cac?ctg?gtg?gaa?gct?ctc?tac?cta?gtg?tgc?ggg?gaa?cga?ggc?ttc 144Ser?His?Leu?Val?Glu?Ala?Leu?Tyr?Leu?Val?Cys?Gly?Glu?Arg?Gly?Phe
35 40 45ttc?tac?aca?ccc?aag?acc?cgc?cgg?gag?gca?gag?gac?ctg?cag?gtg?ggg 192Phe?Tyr?Thr?Pro?Lys?Thr?Arg?Arg?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
50 55 60cag?gtg?gag?ctg?ggc?ggg?ggc?cct?ggt?gca?ggc?agc?ctg?cag?ccc?ttg 240Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?65 70 75 80gcc?ctg?gag?ggg?tcc?ctg?cag?aag?cgt?ggc?att?gtg?gaa?caa?tgc?tgt 288Ala?Leu?Glu?Gly?Ser?Leu?Gln?Lys?Arg?Gly?Ile?Val?Glu?Gln?Cys?Cys
85 90 95acc?agc?atc?tgc?tcc?ctc?tac?cag?ctg?gag?aac?tac?tgc?aac?tag 333Thr?Ser?Ile?Cys?Ser?Leu?Tyr?Gln?Leu?Glu?Asn?Tyr?Cys?Asn
100 105 110＜210〉10＜211〉110＜212〉PRT＜213〉people＜400〉10Met Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu, 15 10 15Trp Gly Pro Asp Pro Ala Ala Ala Phe Val Asn Gln His Leu Cys Gly
20 25 30Ser?His?Leu?Val?Glu?Ala?Leu?Tyr?Leu?Val?Cys?Gly?Glu?Arg?Gly?Phe
35 40 45Phe?Tyr?Thr?Pro?Lys?Thr?Arg?Arg?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
50 55 60Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?65 70 75 80Ala?Leu?Glu?Gly?Ser?Leu?Gln?Lys?Arg?Gly?Ile?Val?Glu?Gln?Cys?Cys
85 90 95Thr?Ser?Ile?Cys?Ser?Leu?Tyr?Gln?Leu?Glu?Asn?Tyr?Cys?Asn
100 105 110
Claims (32)
1. measure in the viable cell whether interactional method of first kind of protein and second kind of protein for one kind, method comprises:
A) in cell, provide with first kind of protein of donor luciferase compound and with second kind of protein of acceptor fluorescence group compound;
B) placement first kind of protein of compound and second kind of protein of compound make it close mutually in cell; With
C) detect any fluorescence of rolling into a ball from acceptor fluorescence;
When first kind of protein when second kind of protein, the donor luciferase can pass to acceptor fluorescence group with luminous resonance energy; And
The luminous resonance energy transmission of donor luciferase causes acceptor fluorescence group to send fluorescence, shows that then interaction has taken place for first kind of protein and second kind of protein.
2. the method for claim 1, wherein provide with first kind of protein of donor luciferase compound with acceptor fluorescence and roll into a ball second kind of protein of compound, comprise genetically engineered DNA and change genetically engineered DNA over to viable cell, this cause cell produce with first kind of protein of donor luciferase compound and with second kind of protein of acceptor fluorescence group's compound.
3. the process of claim 1 wherein provide with first kind of proteinic cell of donor luciferase compound be mammalian cell.
4. the process of claim 1 wherein provide with acceptor fluorescence group second kind of proteinic cell of compound be mammalian cell.
5. the process of claim 1 wherein that the donor luciferase that is provided is the Renilla luciferase.
6. the process of claim 1 wherein that the acceptor fluorescence group that is provided is egfp.
7. the process of claim 1 wherein that the acceptor fluorescence group that is provided is the jellyfish egfp.
8. the process of claim 1 wherein and adopt any fluorescence of spectrofluorimetry detection from the donor luciferase.
9. measure in the viable cell whether interactional method of first kind of molecule and second kind of molecule for one kind, method comprises:
A) in cell, provide with first kind of molecule of donor luciferase compound and with second kind of molecule of acceptor fluorescence group compound;
B) placement first kind of molecule of compound and second kind of molecule of compound make it close mutually in cell; With
C) detect any fluorescence of rolling into a ball from acceptor fluorescence;
When the close second kind of branch period of the day from 11 p.m. to 1 a.m of first kind of molecule, the donor luciferase can pass to acceptor fluorescence group with luminous resonance energy; And
The luminous resonance energy transmission of donor luciferase causes acceptor fluorescence group to send fluorescence, shows that then interaction has taken place for first kind of molecule and second kind of molecule.
10. the method for claim 9, wherein first kind of molecule is first kind of protein, and second kind of molecule is second kind of protein; And
Wherein provide with first kind of protein of donor luciferase compound with acceptor fluorescence and roll into a ball second kind of protein of compound, comprise genetically engineered DNA and change genetically engineered DNA over to viable cell, this cause cell produce with first kind of protein of donor luciferase compound and with second kind of protein of acceptor fluorescence group's compound.
11. the method for claim 10, wherein provide with first kind of proteinic cell of donor luciferase compound be mammalian cell.
12. the method for claim 10, wherein providing and having rolled into a ball second kind of proteinic cell of compound with acceptor fluorescence is mammalian cell.
13. the method for claim 9, the donor luciferase that is wherein provided is the Renilla luciferase.
14. the method for claim 9, the acceptor fluorescence that is wherein provided group is egfp.
15. the method for claim 9, the acceptor fluorescence that is wherein provided group is the jellyfish egfp.
16. the method for claim 9 is wherein with any fluorescence of spectrofluorimetry detection from the donor luciferase.
17. measure whether interactional method of first kind of protein and second kind of protein for one kind, method comprises:
A) provide with first kind of protein of donor luciferase compound and with second kind of protein of acceptor fluorescence group compound;
B) first kind of protein of compound and second kind of protein of compound is close mutually; With
C) detect any fluorescence of rolling into a ball from acceptor fluorescence;
When first kind of protein when second kind of protein, the donor luciferase can pass to acceptor fluorescence group with luminous resonance energy; And
The luminous resonance energy transmission of donor luciferase causes acceptor fluorescence group to send fluorescence, shows that then interaction has taken place for first kind of protein and second kind of protein.
18. the method for claim 17, wherein provide with first kind of protein of donor luciferase compound with acceptor fluorescence and roll into a ball second kind of protein of compound, comprise genetically engineered DNA and genetically engineered DNA imported viable cell, this cause cell produce with first kind of protein of donor luciferase compound and with second kind of protein of acceptor fluorescence group's compound.
19. the method for claim 18, wherein provide with first kind of proteinic cell of donor luciferase compound be mammalian cell.
20. the method for claim 18, wherein providing and having rolled into a ball second kind of proteinic cell of compound with acceptor fluorescence is mammalian cell.
21. the method for claim 17, the donor luciferase that is wherein provided is the Renilla luciferase.
22. the method for claim 17, the acceptor fluorescence that is wherein provided group is egfp.
23. the method for claim 17, the acceptor fluorescence that is wherein provided group is the jellyfish egfp.
24. the method for claim 17 is wherein used any fluorescence of spectrofluorimetry detection from the donor luciferase.
25. measure whether interactional method of first kind of molecule and second kind of molecule for one kind, method comprises:
A) provide with first kind of molecule of donor luciferase compound and with second kind of molecule of acceptor fluorescence group compound;
B) first kind of molecule of compound and second kind of molecule is close mutually; With
C) detect any fluorescence of rolling into a ball from acceptor fluorescence;
When first kind of molecule and second kind of molecule near the time, the donor luciferase can pass to acceptor fluorescence group with luminous resonance energy; With
The luminous resonance energy transmission of donor luciferase causes acceptor fluorescence group to send fluorescence, shows that then interaction has taken place for first kind of molecule and second kind of molecule.
26. the method for claim 25, wherein first kind of molecule is first kind of protein and second kind of molecule is second kind of protein; And
Wherein provide with first kind of protein of donor luciferase compound with acceptor fluorescence and roll into a ball second kind of protein of compound, comprise genetically engineered DNA and change genetically engineered DNA over to viable cell, this cause cell produce with first kind of protein of donor luciferase compound and with second kind of protein of acceptor fluorescence group's compound.
27. the method for claim 26, wherein provide with first kind of proteinic cell of donor luciferase compound be mammalian cell.
28. the method for claim 26, wherein providing and having rolled into a ball second kind of proteinic cell of compound with acceptor fluorescence is mammalian cell.
29. the method for claim 25, the donor luciferase that is wherein provided is the Renilla luciferase.
30. the method for claim 25, the acceptor fluorescence that is wherein provided group is egfp.
31. the method for claim 25, the acceptor fluorescence that is wherein provided group is the jellyfish egfp.
32. the method for claim 25 wherein adopts the fluorescence of spectrofluorimetry detection from the donor luciferase.
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 (2)
| Publication Number | Publication Date |
|---|---|
| CN1323353A true CN1323353A (en) | 2001-11-21 |
| CN1160470C CN1160470C (en) | 2004-08-04 |
Family
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB99811958XA Expired - Fee Related CN1160470C (en) | 1998-09-03 | 1999-09-02 | Method for studying protein interactions i(invivo) |
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| 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 (5)
| 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 |
| CN102798717A (en) * | 2012-06-15 | 2012-11-28 | 杭州师范大学 | Method for detecting activity of O6-methylguanine-DNA (Deoxyribose Necleic Acid) methyltransferase |
| CN103616502A (en) * | 2013-09-12 | 2014-03-05 | 西北农林科技大学 | Method for detection of protein-protein interaction through a BRET technology based on bacterial Luciferase |
| CN110794129A (en) * | 2018-08-01 | 2020-02-14 | 清华大学 | Method for detecting interaction between biological molecules and regulating factor thereof in cell and used reagent |
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| AU2003280420A1 (en) | 2002-06-26 | 2004-01-19 | Yale University | Modulators and modulation of the interacton between rgm and neogenin |
| EP2053409A1 (en) | 2003-11-20 | 2009-04-29 | F. Hoffmann-La Roche Ag | Specific markers for metabolic syndrome |
| ES2542501T3 (en) | 2005-09-30 | 2015-08-06 | Abbvie Deutschland Gmbh & Co Kg | Protein binding domains of the protein family of repulsive targeting molecules (RGM) and functional fragments thereof, as well as their use |
| US8962803B2 (en) | 2008-02-29 | 2015-02-24 | AbbVie Deutschland GmbH & Co. KG | Antibodies against the RGM A protein and uses thereof |
| CA2748500C (en) | 2009-01-29 | 2017-10-17 | Commonwealth Scientific And Industrial Research Organisation | Measuring g protein coupled receptor activation |
| BR112012013734A2 (en) | 2009-12-08 | 2017-01-10 | Abbott Gmbh & Co Kg | monoclonal antibodies against the rgm a protein for use in the treatment of retinal nerve fiber layer degeneration. |
| 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 |
| EP2561068A1 (en) | 2010-04-19 | 2013-02-27 | Medizinische Universität Innsbruck | Tmem195 encodes for tetrahydrobiopterin-dependent alkylglycerol monooxygenase activity |
| MY194587A (en) | 2012-01-27 | 2022-12-05 | Abbvie Inc | Composition and method for the diagnosis and treatment of diseases associated with neurite degeneration |
| US10415960B2 (en) | 2015-04-06 | 2019-09-17 | Worldvu Satellites Limited | Elevation angle estimating system and method for user terminal placement |
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Family Cites Families (11)
| 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 |
| ES8607563A1 (en) * | 1983-10-13 | 1986-06-01 | Univ Georgia Res Found | Bioluminescent immunoassays. |
| US5683888A (en) * | 1989-07-22 | 1997-11-04 | University Of Wales College Of Medicine | Modified bioluminescent proteins and their use |
| US5292658A (en) * | 1989-12-29 | 1994-03-08 | University Of Georgia Research Foundation, Inc. Boyd Graduate Studies Research Center | Cloning and expressions of Renilla luciferase |
| AT401526B (en) * | 1993-02-10 | 1996-09-25 | Scheirer Winfried | REAGENT SOLUTION TO STABILIZE LUMINESCENCE IN LUCIFERASE MEASUREMENT |
| US5491084A (en) * | 1993-09-10 | 1996-02-13 | The Trustees Of Columbia University In The City Of New York | Uses of green-fluorescent protein |
| US5605793A (en) * | 1994-02-17 | 1997-02-25 | Affymax Technologies N.V. | Methods for in vitro 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 |
| WO1999066324A2 (en) * | 1998-06-16 | 1999-12-23 | Biosignal Packard Inc. | A bioluminescence resonance energy transfer (bret) system and its use |
-
1999
- 1999-09-02 EP EP99945460A patent/EP1109931A4/en not_active Withdrawn
- 1999-09-02 JP JP2000569011A patent/JP2002524087A/en active Pending
- 1999-09-02 WO PCT/US1999/020207 patent/WO2000014271A1/en not_active Application Discontinuation
- 1999-09-02 AU AU58056/99A patent/AU752675B2/en not_active Ceased
- 1999-09-02 CN CNB99811958XA patent/CN1160470C/en not_active Expired - Fee Related
- 1999-09-02 CA CA002341314A patent/CA2341314A1/en not_active Abandoned
Cited By (8)
| 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 |
| CN102798717A (en) * | 2012-06-15 | 2012-11-28 | 杭州师范大学 | Method for detecting activity of O6-methylguanine-DNA (Deoxyribose Necleic Acid) methyltransferase |
| 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 |
| CN103616502A (en) * | 2013-09-12 | 2014-03-05 | 西北农林科技大学 | Method for detection of protein-protein interaction through a BRET technology based on bacterial Luciferase |
| 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 |
| CN110794129A (en) * | 2018-08-01 | 2020-02-14 | 清华大学 | Method for detecting interaction between biological molecules and regulating factor thereof in cell and used reagent |
| 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| AU752675B2 (en) | 2002-09-26 |
| JP2002524087A (en) | 2002-08-06 |
| EP1109931A4 (en) | 2004-12-15 |
| EP1109931A1 (en) | 2001-06-27 |
| AU5805699A (en) | 2000-03-27 |
| WO2000014271A1 (en) | 2000-03-16 |
| CA2341314A1 (en) | 2000-03-16 |
| CN1160470C (en) | 2004-08-04 |
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