Screening for in vivo protein-protein interactions.
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.
Germino
F J
FJ
Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06510-8005.
Wang
Z X
ZX
Weissman
S M
SM
D000103:Acetyl-CoA Carboxylase; D000818:Animals; D001360:Avidin / Q000378:metabolism; D010582:Bacteriophage lambda / Q000235:genetics; D001710:Biotin / Q000378:metabolism; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D054889:Fatty Acid Synthase, Type II; D007124:Immunoenzyme Techniques; D016350:Leucine Zippers; D051379:Mice; D015335:Molecular Probes; D015513:Oncogene Proteins / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification*; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D002352:Carrier Proteins; D015335:Molecular Probes; D015513:Oncogene Proteins; D011506:Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D001360:Avidin; D001710:Biotin; D001616:beta-Galactosidase; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein