In vitro assay for protein-protein interaction: carboxyl-terminal 40 residues of simian virus 40 structural protein VP3 contain a determinant for interaction with VP1.
Intermolecular interactions between polypeptide chains play essential roles in the functioning of proteins. We describe here an in vitro assay system for identifying and characterizing such interactions. Such interactions are difficult to study in vivo. We have translated synthetic, nonmethyl-capped RNAs in a cell-free protein-synthesizing system. The translation products were allowed to interact posttranslationally to form protein-protein complexes. The chemical nature of the protein interaction(s) was determined by coimmunoprecipitation of associating proteins, sedimentation through sucrose gradients, followed by NaDodSO4/polyacrylamide gel electrophoresis or by nonreducing NaDodSO4/polyacrylamide gel electrophoresis. The system has been utilized to show the self-assembly of monomeric VP1, the major structural protein of simian virus 40, into disulfide-linked pentamers and to show the noncovalent interaction of another structural protein, VP3, with VP1 at low monomer concentrations. Additionally, we show that the carboxyl-terminal 40 amino acids of VP3 are essential and sufficient for its interaction with VP1 in vitro. The in vitro assay system described here provides a method for identifying the domains involved in, and the molecular nature of, protein-protein interactions, which play an important role in such biological phenomena as replication, transcription, translation, transport, ligand binding, and assembly.
Gharakhanian
E
E
Department of Biology, University of California, Los Angeles 90024-1606.
Takahashi
J
J
Clever
J
J
Kasamatsu
H
H
D000596:Amino Acids / Q000032:analysis; D011108:Polymers; D014176:Protein Biosynthesis; D013539:Simian virus 40; D014760:Viral Fusion Proteins / Q000378:metabolism; D014764:Viral Proteins; D015678:Viral Structural Proteins; D001616:beta-Galactosidase / Q000378:metabolism
D000596:Amino Acids; D011108:Polymers; D014760:Viral Fusion Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; D001616:beta-Galactosidase