AU5805699A

AU5805699A - Method for studying protein interactions (in vivo)

Info

Publication number: AU5805699A
Application number: AU58056/99A
Authority: AU
Inventors: Aladar A. Szalay; Yubao Wang; Gefu Wang-Pruski
Original assignee: Loma Linda University
Current assignee: Loma Linda University
Priority date: 1998-09-03
Filing date: 1999-09-02
Publication date: 2000-03-27
Anticipated expiration: 2019-09-02
Also published as: WO2000014271A1; JP2002524087A; CN1160470C; AU752675B2; CN1323353A; EP1109931A4; EP1109931A1; CA2341314A1

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

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.