AU5805699A - Method for studying protein interactions (in vivo) - Google Patents
Method for studying protein interactions (in vivo) Download PDFInfo
- Publication number
- AU5805699A AU5805699A AU58056/99A AU5805699A AU5805699A AU 5805699 A AU5805699 A AU 5805699A AU 58056/99 A AU58056/99 A AU 58056/99A AU 5805699 A AU5805699 A AU 5805699A AU 5805699 A AU5805699 A AU 5805699A
- Authority
- AU
- Australia
- Prior art keywords
- protein
- complexed
- acceptor fluorophore
- luciferase
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000000034 method Methods 0.000 title claims description 59
- 238000001727 in vivo Methods 0.000 title description 4
- 230000006916 protein interaction Effects 0.000 title description 2
- 102000004169 proteins and genes Human genes 0.000 claims description 112
- 108090000623 proteins and genes Proteins 0.000 claims description 112
- 210000004027 cell Anatomy 0.000 claims description 67
- 108060001084 Luciferase Proteins 0.000 claims description 58
- 239000005089 Luciferase Substances 0.000 claims description 58
- 239000005090 green fluorescent protein Substances 0.000 claims description 43
- 108010052090 Renilla Luciferases Proteins 0.000 claims description 20
- 238000002165 resonance energy transfer Methods 0.000 claims description 17
- 238000004020 luminiscence type Methods 0.000 claims description 16
- 210000004962 mammalian cell Anatomy 0.000 claims description 14
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 11
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 10
- 241000243290 Aequorea Species 0.000 claims description 7
- 241000242739 Renilla Species 0.000 claims description 4
- 108091006047 fluorescent proteins Proteins 0.000 claims 1
- 102000034287 fluorescent proteins Human genes 0.000 claims 1
- 239000002299 complementary DNA Substances 0.000 description 34
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 19
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 18
- 102000048143 Insulin-Like Growth Factor II Human genes 0.000 description 18
- 102000004883 Insulin-like growth factor-binding protein 6 Human genes 0.000 description 16
- 108090001014 Insulin-like growth factor-binding protein 6 Proteins 0.000 description 16
- 230000003993 interaction Effects 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 13
- 102000004877 Insulin Human genes 0.000 description 9
- 108090001061 Insulin Proteins 0.000 description 9
- 229940125396 insulin Drugs 0.000 description 9
- YHIPILPTUVMWQT-UHFFFAOYSA-N Oplophorus luciferin Chemical compound C1=CC(O)=CC=C1CC(C(N1C=C(N2)C=3C=CC(O)=CC=3)=O)=NC1=C2CC1=CC=CC=C1 YHIPILPTUVMWQT-UHFFFAOYSA-N 0.000 description 8
- 102000037865 fusion proteins Human genes 0.000 description 8
- 108020001507 fusion proteins Proteins 0.000 description 8
- 238000001514 detection method Methods 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 239000000284 extract Substances 0.000 description 6
- 230000000536 complexating effect Effects 0.000 description 5
- 238000001506 fluorescence spectroscopy Methods 0.000 description 5
- 238000001890 transfection Methods 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 3
- 239000001506 calcium phosphate Substances 0.000 description 3
- 229910000389 calcium phosphate Inorganic materials 0.000 description 3
- 235000011010 calcium phosphates Nutrition 0.000 description 3
- 230000004927 fusion Effects 0.000 description 3
- 102000028416 insulin-like growth factor binding Human genes 0.000 description 3
- 108091022911 insulin-like growth factor binding Proteins 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 108010023799 preproinsulin-like growth factor II Proteins 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 3
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000000295 emission spectrum Methods 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000000695 excitation spectrum Methods 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000000386 microscopy Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K tripotassium phosphate Chemical compound [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- KZDCMKVLEYCGQX-UDPGNSCCSA-N 2-(diethylamino)ethyl 4-aminobenzoate;(2s,5r,6r)-3,3-dimethyl-7-oxo-6-[(2-phenylacetyl)amino]-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid;hydrate Chemical group O.CCN(CC)CCOC(=O)C1=CC=C(N)C=C1.N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 KZDCMKVLEYCGQX-UDPGNSCCSA-N 0.000 description 1
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=C[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-KKGHZKTASA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 101100018361 Homo sapiens IGFBP6 gene Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- -1 SEQ ID NO:5 Substances 0.000 description 1
- 108091006629 SLC13A2 Proteins 0.000 description 1
- 102000040739 Secretory proteins Human genes 0.000 description 1
- 108091058545 Secretory proteins Proteins 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960003942 amphotericin b Drugs 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000003169 complementation method Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000005281 excited state Effects 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000013537 high throughput screening Methods 0.000 description 1
- 238000010191 image analysis Methods 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 238000005895 oxidative decarboxylation reaction Methods 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 229910000160 potassium phosphate Inorganic materials 0.000 description 1
- 235000011009 potassium phosphates Nutrition 0.000 description 1
- 238000000159 protein binding assay Methods 0.000 description 1
- 230000004850 protein–protein interaction Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 238000001086 yeast two-hybrid system Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Computational Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Enzymes And Modification Thereof (AREA)
- Peptides Or Proteins (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
WO 00/14271 PCT/US99/20207 1 METHOD FOR STUDYING PROTEIN INTERACTIONS IN VIVO BACKGROUND The study of interactions between proteins in living cells is often necessary to understand the proteins' functions and their mechanisms of action. These interactions are currently studied using immuno-precipitation, the yeast two hybrid method, and -gal complementation method. ) However, these methods are associated with several disadvantages. For example, these methods are associated with false positives. Second, they do not permit the determination of quantitative information regarding the interactions. Further, they do not allow for in vivo real time monitoring of the interactions. Therefore, it would be advantageous to have another method of studying 5 interactions between proteins in vivo, which does not have these disadvantages. Further preferably, the method could be used with a wide variety of proteins and in a wide variety of living cells. Also preferably, the method could be used to determine the interactions between molecules other than proteins. SUMMARY ) According to one embodiment of the present invention, there is provided a WO 00/14271 PCT/US99/20207 2 method for determining whether a first protein interacts with a second protein within a living cell. The method comprises providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore within the cell. The donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore 5 when the first protein is in proximity to the second protein. Then, the complexed first protein and the complexed second protein are allowed to come into proximity to each other within the cell. Next, any fluorescence from the acceptor fluorophore is detected. Fluorescence of the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase to acceptor fluorophore the indicates that the first protein 10 has interacted with the second protein. In a preferred embodiment, providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore comprises genetically engineering DNA and transferring the genetically engineered DNA to the living cell causing the cell to produce the first protein complexed to a donor luciferase and the second protein 15 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 20 Aequorea green fluorescent protein. In a particularly preferred embodiment, the detection of acceptor fluorophore fluorescence is performed using spectrofluorometery. DESCRIPTION The present invention includes a method for determining whether a first 25 protein interacts with a second protein in a living cell using luminescent resonance energy transfer (LRET). Luminescence resonance energy transfer results from the transfer of excited state energy from a donor luciferase to an acceptor fluorophore. In order for LRET to occur, there must be an overlap between the emission spectrum of the donor luciferase and the excitation spectrum of the acceptor fluorophore. 30 The efficiency of luminescence resonance energy transfer is dependent on the distance separating the donor luciferase and the acceptor fluorophore, among other variables.
WO 00/14271 PCT/US99/20207 3 Generally, significant energy transfers occur only where the donor luciferase and acceptor fluorophore are less than about 80 A of each other. This short distance is considerably less than the distance needed between for optical resolution between two entities using conventional microscopy. Therefore, detecting luminescence resonance energy transfer 5 between a donor luciferase and an acceptor fluorophore indicates that the donor luciferase and acceptor fluorophore have come within the distance needed for LRET to occur, that is less than about 80 A of each other. The present invention utilizes luminescence resonance energy transfer to determine whether an interaction takes place between a first protein and a second protein in a 10 living cell. This is accomplished by complexing a first protein to the donor luciferase and complexing the second protein to the acceptor fluorophore and placing the complexed first protein and the complexed second protein in the cell under conditions suitable for an interaction between the first protein and the second protein to take place. If the first protein interacts with the second protein, the donor luciferase will come close enough to the acceptor 15 fluorophore for luminescence resonance energy transfer to take place and the acceptor fluorophore will fluoresce. Detection of fluorescence from the acceptor fluorophore will, thereby, indicate that the first protein has interacted with the second protein. Advantageously, this method allows for the detection of interaction between the first protein and the second protein even though the interaction cannot be detected by optical methods 20 such as conventional microscopy. There are several advantages of using luminescent resonance energy transfer to detect the interaction between two proteins according to the present invention. First, the specific labeling of the proteins in living cells can be achieved through genetic engineering methods where the introduction of fluorescent dyes into living cells is very difficult. 25 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 30 and the first protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein. Similarly, "complexing a second protein to WO 00/14271 PCT/US99/20207 4 the acceptor fluorophore" refers to joining the acceptor fluorophore to the second protein in a manner that the acceptor fluorophore and the second protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein. Such complexing can be done, for example, by genetically engineering the cell to produce a 5 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 10 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 15 accurate quantitative determination of light production. 'Humanized' GFP is a 27 kDa protein fluorophore that has an excitation maximum at 480 nm. It has a single amino acid difference from wild-type Aequorea green fluorescent protein. 'Humanized' GFP was chosen as the acceptor fluorophore because its excitation spectrum overlaps with the emission spectra of Renilla luciferase. Additionally, 20 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, 25 insulin-like growth factor binding protein 6 (IGFBP 6) and insulin-like growth factor II (IGF II) were selected as the first protein and second protein. IGFBP 6 is a protein known to have a marked binding affinity for IGF-II. The Renilla luciferase cDNA was fused to IGFBP 6 cDNA and 'humanized' GFP cDNA was fused to IGF-II cDNA. Living cells were transfected with the fused cDNAs 30 and the fusion proteins were expressed. Cell extracts were produced and mixed. The substrate for the Renilla luciferase moiety of the fused Renilla luciferase-IGFPB 6 protein WO 00/14271 PCT/US99/20207 5 was added. Finally, fluorescence from the 'humanized' GFP moiety of the fused 'humanized' GFP-IGF-II protein was detected. Demonstration one method according to the present invention will now be described in greater detail. A) The Cloning of Fused IGFBP-6 cDNA to Renilla Luciferase cDNA; Fused IGF-II 5 cDNA to 'humanized' GFP cDNA; and Fused Insulin cDNA to 'humanized' GFP cDNA: First, three fused cDNAs were produced: 1) fused IGFBP-6 cDNA and Renilla luciferase cDNA; 2) fused IGF-II cDNA and 'humanized' GFP cDNA; and 3) fused insulin cDNA and 'humanized' GFP cDNA. IGFBP-6 cDNA, SEQ ID NO: 1, GenBank accession number M69054, encoded IGFBP-6, SEQ ID NO:2, which was used as the first protein. 10 Renilla luciferase cDNA, SEQ ID NO:3, GenBank accession number M63501, encoded Renilla luciferase, SEQ ID NO:4, which was used as the donor luciferase. IGF-II cDNA, SEQ ID NO:5, encoded IGF-II, SEQ ID NO:6, which was used as the second protein. 'Humanized' GFP cDNA, SEQ ID NO:7, GenBank accession numberU50963, encoded 'humanized' GFP, SEQ ID NO:8,which was used as the acceptor fluorophore. Insulin 15 cDNA, SEQ ID NO:9, accession number AH002844, encoded insulin, SEQ ID NO:10. Insulin, fused to 'humanized' GFP, was used as a control protein because insulin is homologous to IGF-II, but it does not bind to IGFBP-6. The IGFBP-6 cDNA, SEQ ID NO:1, IGF-II cDNA, SEQ ID NO:5, and insulin cDNA, SEQ ID NO:9, were modified using PCR as follows. 20 First, the cDNA of prepro-IGF-II carried on an EcoRI fragment was cloned into pBluescript KS (+) II vector. The insert was sequenced using T7 and T3 primers and confirmed to contain the known cDNA sequence of prepro-IGF-II. The 5' end of the IGF-II precursor was connected to the T7 promoter in the pBluescript KS (+) II vector. An IGF-II 3' primer was designed to generate a Notice of Allowance restriction site, to remove the D 25 and E domains of prepro-IGF-II, and to maintain the Notice of Allowance fragment of the 'humanized' GFP in frame with the open reading frame of IGF-II. Next, the IGF-II fragment was amplified with PCR using the T7 promoter primer and the IGF-II 3' primer. The PCR-amplified IGF-II fragment was digested by EcoRI and Not I and cloned into pCDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, US) 30 producing pCDNA-IGF-II. Then, the Notice of Allowance fragment of the 'humanized' GFP was inserted into the Not I site of pCDNA-IGF-II producing pC-IGF-II-GFP.
WO 00/14271 PCT/US99/20207 6 The cDNA for precursor of insulin, which contained a signal peptide the B, C and A domains, was modified in a manner corresponding to the IGF-II fragment, above. The 'humanized' GFP cDNA was then linked to the 3' end of the modified insulin cDNA to produce pC-INS-GFP. 5 Finally, IGFBP 6 cDNA was amplified by PCR from a plasmid named Rat-tagged human IGFBP6. The stop codon of IGFBP 6 was removed and the open reading frame of IGFBP 6 was in frame with Renilla luciferase cDNA from pCEP4-RUC (Mayerhofer R, Langridge WHR, Cormier MG and Szalay AA. Expression of recombinant Renilla luciferase in transgenic plants results in high levels of light emission. The Plant 10 Journal 1995;7;1031-8). The linking of the Renilla luciferase cDNA to the 3' end of modified IGFBP 6 cDNA produced pC-IGFBP 6-RUC. The sequences of the insert DNA fragments from all the constructs were verified by DNA sequencing analysis. Qiagen Maxi Plasmid Kit (Qiagen, Inc., Valencia, CA) was used for the purification of plasmid DNA. 15 B) Transient Transfection of Mammalian Cells With pC-IGF-II-GFP, pC-INS-GFP and pC-IGFBP 6-RUC Using the Calcium Phosphate Precipitation Method: Next, mammalian cells were transfected with the cloned fusion DNAs. First, COS-7 cells (African green monkey kidney cell, American Type Culture Collection CRL 1651) were grown at 37 C in Dulbecco's Modified Eagle Medium (DMEM) with 20 L-Glutamine supplemented with 10% fetal bovine serum and antibiotic antimycotic solution containing a final concentration of penicillin 100 unit/ml, streptomycin 100 mg/ml and amphotericin B 250 ng/ml (Sigma-Aldrich Co., St. Louis, MO, US) in 5% CO 2 . Groups of lx106 of these cells were plated the day before transfection and were approximately 50% to 60% confluent at the time of transfection. 25 Forty mg of each plasmid fusion DNA were precipitated and resuspended into Dulbecco's Phosphate Buffered Saline Solution and the plasmid fusion DNAs was introduced into mammalian cells using the standard calcium phosphate precipitation method. Transfection efficiency was estimated by fluorescence microscopy after 24 hours. The number of green fluorescent cells per plate were comparable in plates of pC-IGF-II-GFP 30 DNA transfected cells, pC-INS-GFP DNA transfected cells and cells transfected with a plasmid DNA containing GFP only, which was used as a positive control.
WO 00/14271 PCT/US99/20207 7 C) Confirmation of Expression of Fusion Proteins: Twenty-four hours after DNA transfection using DNA calcium phosphate precipitation method, individual plasmid DNA transfected COS-7 cells were visualized using fluorescence microscopy by detection of GFP fluorescence. pC-IGF-II-GFP and 5 pC-INS-GFP transfected cells showed similar fluorescence patterns typical of secretory protein translocated through ER to Golgi. The pC-IGFBP 6-RUC transfected cells did not fluoresce. However, the pC-IGFBP 6-RUC transfected cells did show luminescence using a low light imaging system after the addition of coelenterazine. Further, the presence of fusion proteins IGF-II-GFP and IGFBP 6-RUC, 10 having the expected molecular weights of about 36 kDa and 56 kDa, respectively, were detected using immunoblot analysis. This confirmed the presence of both fusion proteins in the transiently transfected cells. D) Detection of Protein Interactions by Spectrofluorometry: Having confirmed the presence of the expected fusion proteins IGF-II-GFP 15 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 20 NaC1, 1 mM EDTA and 0.1 M potassium phosphate at a pH 7.5. The harvested cells were sonicated 3 times for 10 seconds with an interval of 10 seconds using a Fisher Model 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, US) to produce cell extracts. Next, the cell extracts containing IGF-II-GFP and IGFBP 6-RUC were mixed and 0.1 Ag of coelenterazine was immediately added. Spectrofluorometry was performed 25 using a SPEX FluoroMax® (Instruments S.A., Inc., Edison, NJ). The spectrum showed a single emission peak at 471 nm, which corresponds to the known emission of Renilla luciferase. Following the first spectrofluorometry, the mixtures were kept at room temperature for 30 minutes and the spectra were traced again after fresh coelenterazine was 30 added. The trace at 30 minutes showed two peaks with emission maximum at 471 nm and 503 nm. The spectrofluorometry of the cell extracts was carried out at a longer time, but the WO 00/14271 PCT/US99/20207 8 spectral pattern did not change over time. Control cell extract mixtures from cells transfected with pC-INS-GFP and pC-IGFBP 6-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 5 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 10 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, 15 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 20 these methods by spectrofluorometry by low light image analysis in cells, colonies and tissues. Additionally, high through put screening of colonies can be accomplished using the present methods combined with cell sorting and low light video analysis of micro titre dishes or multiple array detection. Therefore, the spirit and scope of the appended claims should not be limited to the description of preferred embodiments contained herein.
Claims (32)
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 5 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 10 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 15 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 20 to a donor luciferase is a mammalian cell.
4. The method of claim 1, where the cell provided with the second protein complexed to a acceptor fluorophore is a mammalian cell.
5. The method of claim 1, where the donor luciferase provided is Renilla luciferase.
6. The method of claim 1, where the acceptor fluorophore provided is a green 25 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. 30
9. A method for determining whether a first molecule interacts with a second molecule within a living cell, the method comprising: WO 00/14271 PCT/US99/20207 10 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 5 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 10 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 15 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. 20
12. The method of claim 10, where the cell provided with the second protein complexed to a acceptor fluorophore is a mammalian cell.
13. The method of claim 9, where the donor luciferase provided is Renilla luciferase.
14. The method of claim 9, where the acceptor fluorophore provided is a green fluorescent protein. 25
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, 30 the method comprising: a) providing the first protein complexed to a donor luciferase and the second protein WO 00/14271 PCT/US99/20207 11 complexed to an acceptor fluorophore; b) placing the complexed first protein and the complexed second protein in proximity to each other; and c) detecting any fluorescence from the acceptor fluorophore; 5 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. 10
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. 15
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 20 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. 25
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 30 molecule complexed to an acceptor fluorophore; b) placing the complexed first molecule and the complexed second molecule in WO 00/14271 PCT/US99/20207 12 proximity to each other; and c) detecting any fluorescence from the acceptor fluorophore; where the donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore when the first molecule is in proximity to the second molecule; and 5 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 10 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. 15
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 20 luciferase.
30. The method of claim 25, where the acceptor fluorophore provided is a green fluorescent protein.
31. The method of claim 25, where the acceptor fluorophore provided is a Aequorea green fluorescent protein. 25
32. The method of claim 25, where detecting any fluorescence from the donor luciferase is performed using spectrofluorometery.
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US9906898P | 1998-09-03 | 1998-09-03 | |
| US60/099068 | 1998-09-03 | ||
| US13583599P | 1999-05-24 | 1999-05-24 | |
| US60/135835 | 1999-05-24 | ||
| PCT/US1999/020207 WO2000014271A1 (en) | 1998-09-03 | 1999-09-02 | METHOD FOR STUDYING PROTEIN INTERACTIONS $i(IN VIVO) |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU5805699A true AU5805699A (en) | 2000-03-27 |
| AU752675B2 AU752675B2 (en) | 2002-09-26 |
Family
ID=26795497
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU58056/99A Ceased AU752675B2 (en) | 1998-09-03 | 1999-09-02 | Method for studying protein interactions (in vivo) |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP1109931A4 (en) |
| JP (1) | JP2002524087A (en) |
| CN (1) | CN1160470C (en) |
| AU (1) | AU752675B2 (en) |
| CA (1) | CA2341314A1 (en) |
| WO (1) | WO2000014271A1 (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| JP2009510002A (en) | 2005-09-30 | 2009-03-12 | アボット ゲゼルシャフト ミット ベシュレンクテル ハフツング ウント コンパニー コマンディトゲゼルシャフト | Binding domains of proteins of the repulsion-inducing molecule (RGM) protein family, and functional fragments thereof, and uses thereof |
| US8962803B2 (en) | 2008-02-29 | 2015-02-24 | AbbVie Deutschland GmbH & Co. KG | Antibodies against the RGM A protein and uses thereof |
| SG172321A1 (en) | 2009-01-29 | 2011-07-28 | Commw Scient Ind Res Org | Measuring g protein coupled receptor activation |
| CN101620233B (en) * | 2009-05-27 | 2012-10-31 | 华中科技大学 | A method for detection of protein interaction |
| CN102656190A (en) | 2009-12-08 | 2012-09-05 | 雅培股份有限两合公司 | 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 |
| WO2011131626A1 (en) | 2010-04-19 | 2011-10-27 | Medizinische Universität Innsbruck | Tmem195 encodes for tetrahydrobiopterin-dependent alkylglycerol monooxygenase activity |
| SG10201600316SA (en) | 2012-01-27 | 2016-02-26 | Abbvie Deutschland | Composition and method for diagnosis and treatment of diseases associated with neurite degeneration |
| CN102798717B (en) * | 2012-06-15 | 2014-11-26 | 杭州师范大学 | A kind of detection method of O6-methylguanine-DNA methyltransferase activity |
| 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 |
| US10415960B2 (en) | 2015-04-06 | 2019-09-17 | Worldvu Satellites Limited | Elevation angle estimating system and method for user terminal placement |
| US11579149B2 (en) | 2017-11-01 | 2023-02-14 | Queen's University At Kingston | Hippo pathway bioluminescent biosensor |
| CN110794129B (en) * | 2018-08-01 | 2020-12-01 | 清华大学 | Methods and reagents for intracellular detection of interactions between biomolecules and their regulatory factors |
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 |
| DK487784A (en) * | 1983-10-13 | 1985-04-14 | Univ Georgia | IMMUNOASSAY |
| 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 |
| CA2335305C (en) * | 1998-06-16 | 2006-05-23 | Biosignal Packard Inc. | A bioluminescence resonance energy transfer (bret) system and its use |
-
1999
- 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
- 1999-09-02 JP JP2000569011A patent/JP2002524087A/en active Pending
- 1999-09-02 EP EP99945460A patent/EP1109931A4/en not_active Withdrawn
- 1999-09-02 WO PCT/US1999/020207 patent/WO2000014271A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP1109931A4 (en) | 2004-12-15 |
| EP1109931A1 (en) | 2001-06-27 |
| JP2002524087A (en) | 2002-08-06 |
| CN1160470C (en) | 2004-08-04 |
| AU752675B2 (en) | 2002-09-26 |
| CA2341314A1 (en) | 2000-03-16 |
| WO2000014271A1 (en) | 2000-03-16 |
| CN1323353A (en) | 2001-11-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU5805699A (en) | Method for studying protein interactions (in vivo) | |
| CN102344494B (en) | Nicotinamide adenine dinucleotide gene coding fluorescent probe as well as preparation method and application thereof | |
| WO2019174633A1 (en) | Fluorescent probe for branched chain amino acids and use thereof | |
| JP2007508841A5 (en) | ||
| Wang et al. | A study of protein-protein interactions in living cells using luminescence resonance energy transfer (LRET) from Renilla luciferase to Aequorea GFP | |
| US8552151B2 (en) | Mutant blue fluorescent protein and method of using the same for fluorescence energy transfer and blue fluorescent fish | |
| CN108519484A (en) | Imaging and tracking protein interactions in living cells using bimolecular fluorescence complementation with self-attaching tags | |
| CN104403003B (en) | NADH fluorescence probe of gene code and its preparation method and application | |
| JP6051438B2 (en) | Calcium sensor protein using red fluorescent protein | |
| EP2687847B1 (en) | Probe for analyzing biological tissue and method for utilizing same | |
| WO2021253126A1 (en) | Cleavable linkers for protein translation reporting | |
| CN112813049B (en) | Fusion protein for live cell RNA marking and application | |
| CN104277120B (en) | NAD gene code fluorescence probe and its preparation method and application | |
| KR20200137596A (en) | Composition for diagnosis acute kidney transplant rejection reaction and diagnostic kit containing the same | |
| CN118360332B (en) | Report carrier for target gene 3' UTR splicing and application thereof | |
| CN102181465B (en) | Fluorescent clone screening vector and preparation and application thereof | |
| Zlobovskaya et al. | Infrared fluorescent protein iRFP as an acceptor for resonance excitation energy transfer | |
| EP1840213A1 (en) | Target physiological function inactivator using photosensitizer-labeled fluorescent protein | |
| WO2016038750A1 (en) | Split-type recombinant luciferase, and analysis method using same | |
| CN114836470A (en) | Vector for expressing combined fluorescent protein fragment and application thereof | |
| van der Linden et al. | Exploration of mScarlet for development of a red lifetime sensor for calcium imaging | |
| CN117165613A (en) | Expression vector and construction method and application thereof | |
| CN119291199A (en) | Nucleic acid probe for detecting VEGFA-VEGFR interaction on single living cells and preparation and use methods thereof | |
| WO2014016455A1 (en) | Variants of the calcium-sensitive fusion protein tdtomato-aequorin | |
| NZ614600B2 (en) | Probe for analyzing biological tissue and method for utilizing same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FGA | Letters patent sealed or granted (standard patent) |