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. 1993 Feb 1;90(3):933–937. doi: 10.1073/pnas.90.3.933

Screening for in vivo protein-protein interactions.

F J Germino 1, Z X Wang 1, S M Weissman 1
PMCID: PMC45784  PMID: 8430108

Abstract

We describe an in vivo approach for the isolation of proteins interacting with a protein of interest. The protein of interest is "tagged" with a portion of the biotin carboxylase carrier protein (BCCP), encoded on a specially constructed plasmid, so that it becomes biotinylated in vivo. The "query" proteins (e.g., those in a cDNA library) are tagged by fusing them to the 3' end of the lacZ gene on a lambda vector in such a way that the beta-galactosidase activity is not disrupted. These phage are transfected into cells containing the plasmid encoding the BCCP-tagged protein. The infection lyses the cells and exposes the protein complexes. The BCCP-tagged protein and any associated protein(s) are "captured" by using avidin, streptavidin, or anti-biotin antibody-coated filters. The detection of bound protein is accomplished by directly assaying for beta-galactosidase activity on the filters. Positive plaques can be plaque-purified for DNA sequencing. We have tested this approach by using c-Fos and c-Jun as our model system. We show that avidin, streptavidin, or polyclonal anti-biotin (but not a monoclonal anti-biotin) antibody is capable of specifically capturing in vivo biotinylated beta-galactosidase and c-Jun and that this capture is dependent upon the presence of both avidin and the BCCP moiety. Further, complexes containing c-Jun and c-Fos can also be isolated in this manner, and the isolation of this complex is dependent on the presence of c-Fos, c-Jun, avidin, and the BCCP moiety. We discuss the possible uses and limitations of this technique for isolating proteins that interact with a known protein.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Blanar M. A., Rutter W. J. Interaction cloning: identification of a helix-loop-helix zipper protein that interacts with c-Fos. Science. 1992 May 15;256(5059):1014–1018. doi: 10.1126/science.1589769. [DOI] [PubMed] [Google Scholar]
  2. Chien C. T., Bartel P. L., Sternglanz R., Fields S. The two-hybrid system: a method to identify and clone genes for proteins that interact with a protein of interest. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9578–9582. doi: 10.1073/pnas.88.21.9578. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Cicchetti P., Mayer B. J., Thiel G., Baltimore D. Identification of a protein that binds to the SH3 region of Abl and is similar to Bcr and GAP-rho. Science. 1992 Aug 7;257(5071):803–806. doi: 10.1126/science.1379745. [DOI] [PubMed] [Google Scholar]
  4. Cronan J. E., Jr Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins. J Biol Chem. 1990 Jun 25;265(18):10327–10333. [PubMed] [Google Scholar]
  5. Defeo-Jones D., Huang P. S., Jones R. E., Haskell K. M., Vuocolo G. A., Hanobik M. G., Huber H. E., Oliff A. Cloning of cDNAs for cellular proteins that bind to the retinoblastoma gene product. Nature. 1991 Jul 18;352(6332):251–254. doi: 10.1038/352251a0. [DOI] [PubMed] [Google Scholar]
  6. Fearon E. R., Finkel T., Gillison M. L., Kennedy S. P., Casella J. F., Tomaselli G. F., Morrow J. S., Van Dang C. Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):7958–7962. doi: 10.1073/pnas.89.17.7958. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fields S., Song O. A novel genetic system to detect protein-protein interactions. Nature. 1989 Jul 20;340(6230):245–246. doi: 10.1038/340245a0. [DOI] [PubMed] [Google Scholar]
  8. Helin K., Lees J. A., Vidal M., Dyson N., Harlow E., Fattaey A. A cDNA encoding a pRB-binding protein with properties of the transcription factor E2F. Cell. 1992 Jul 24;70(2):337–350. doi: 10.1016/0092-8674(92)90107-n. [DOI] [PubMed] [Google Scholar]
  9. Kaelin W. G., Jr, Krek W., Sellers W. R., DeCaprio J. A., Ajchenbaum F., Fuchs C. S., Chittenden T., Li Y., Farnham P. J., Blanar M. A. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. doi: 10.1016/0092-8674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  10. Li S. J., Cronan J. E., Jr The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. J Biol Chem. 1992 Jan 15;267(2):855–863. [PubMed] [Google Scholar]
  11. Macgregor P. F., Abate C., Curran T. Direct cloning of leucine zipper proteins: Jun binds cooperatively to the CRE with CRE-BP1. Oncogene. 1990 Apr;5(4):451–458. [PubMed] [Google Scholar]
  12. Maina C. V., Riggs P. D., Grandea A. G., 3rd, Slatko B. E., Moran L. S., Tagliamonte J. A., McReynolds L. A., Guan C. D. An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein. Gene. 1988 Dec 30;74(2):365–373. doi: 10.1016/0378-1119(88)90170-9. [DOI] [PubMed] [Google Scholar]
  13. Milner J., Medcalf E. A., Cook A. C. Tumor suppressor p53: analysis of wild-type and mutant p53 complexes. Mol Cell Biol. 1991 Jan;11(1):12–19. doi: 10.1128/mcb.11.1.12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nelbock P., Dillon P. J., Perkins A., Rosen C. A. A cDNA for a protein that interacts with the human immunodeficiency virus Tat transactivator. Science. 1990 Jun 29;248(4963):1650–1653. doi: 10.1126/science.2194290. [DOI] [PubMed] [Google Scholar]
  15. Rüther U., Koenen M., Sippel A. E., Müller-Hill B. Exon cloning: immunoenzymatic identification of exons of the chicken lysozyme gene. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6852–6855. doi: 10.1073/pnas.79.22.6852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rüther U., Müller-Hill B. Easy identification of cDNA clones. EMBO J. 1983;2(10):1791–1794. doi: 10.1002/j.1460-2075.1983.tb01659.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Scholtissek S., Grosse F. A plasmid vector system for the expression of a triprotein consisting of beta-galactosidase, a collagenase recognition site and a foreign gene product. Gene. 1988;62(1):55–64. doi: 10.1016/0378-1119(88)90579-3. [DOI] [PubMed] [Google Scholar]
  18. Sikela J. M., Hahn W. E. Screening an expression library with a ligand probe: isolation and sequence of a cDNA corresponding to a brain calmodulin-binding protein. Proc Natl Acad Sci U S A. 1987 May;84(9):3038–3042. doi: 10.1073/pnas.84.9.3038. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Steers E., Jr, Cuatrecasas P., Pollard H. B. The purification of beta-galactosidase from Escherichia coli by affinity chromatography. J Biol Chem. 1971 Jan 10;246(1):196–200. [PubMed] [Google Scholar]
  20. Vasavada H. A., Ganguly S., Germino F. J., Wang Z. X., Weissman S. M. A contingent replication assay for the detection of protein-protein interactions in animal cells. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10686–10690. doi: 10.1073/pnas.88.23.10686. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Weiss A. J., McElhinney S. A., Blankstein L. A. The catalysis of protein and nucleic acid coupling to an affinity membrane substrate. Biotechniques. 1989 Oct;7(9):1012–1016. [PubMed] [Google Scholar]
  22. Whyte P., Buchkovich K. J., Horowitz J. M., Friend S. H., Raybuck M., Weinberg R. A., Harlow E. Association between an oncogene and an anti-oncogene: the adenovirus E1A proteins bind to the retinoblastoma gene product. Nature. 1988 Jul 14;334(6178):124–129. doi: 10.1038/334124a0. [DOI] [PubMed] [Google Scholar]
  23. Yang X., Hubbard E. J., Carlson M. A protein kinase substrate identified by the two-hybrid system. Science. 1992 Jul 31;257(5070):680–682. doi: 10.1126/science.1496382. [DOI] [PubMed] [Google Scholar]
  24. Yu V. C., Delsert C., Andersen B., Holloway J. M., Devary O. V., När A. M., Kim S. Y., Boutin J. M., Glass C. K., Rosenfeld M. G. RXR beta: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements. Cell. 1991 Dec 20;67(6):1251–1266. doi: 10.1016/0092-8674(91)90301-e. [DOI] [PubMed] [Google Scholar]

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