VECTORS FOR A DOUBLE-TAGGING ASSAY FOR PROTEIN-PROTEIN INTERACTIONS -LOCALIZATION OF THE CDK2-BINDING DOMAIN OF HUMAN P21

We report the construction of three new vectors which can be used for the 'double-tagging' assay previously reported [Germino et al., Proc. Natl, Acad. Sci. USA 90 (1993) 933-937]. The vectors include two plasm ids (pTrc.BCCP and pTrc.EZZ::BCCP) which encode different 'tags' for t he capture of a target protein of interest on a filter coated with eit her avidin or IgG, respectively. The first plasmid (pTrc.BCCP) encodes the C terminus of the biotin carboxylase carrier protein (BCCP) under the control of the P-tac promoter, while the second produces fusions to an IgG-binding domain (EZZ). The gene encoding a protein of interes t can be inserted into these plasmids and thereby direct the productio n of a fusion protein which is biotinylated in vivo and can bind to av idin, or a fusion protein which can bind to IgG. The third is a positi ve-selection, phage lambda expression vector (lambda FJG2) which permi ts the construction of lacZ::cDNA fusion proteins which retain beta-ga lactosidase activity. The insertion of an active ecoRVR gene between t he cloning sites (EcoRI and HindIII or NotI) permits the positive sele ction of inserts. The C-terminal two-thirds of the mouse retinoblastom a-encoding gene (containing the E1A-binding pocket) was cloned into pT rc.BCCP and pTrc.EZZ::BCCP, while the 13S E1a gene was cloned into lam bda FJG2. We show that the interaction between these two proteins can be detected using the 'double-tagging' filter assay, and that this ass ay has high sensitivity and specificity for detecting this interaction , Finally, we have used these vectors to localize the CDK2-binding dom ain of the cyclin-dependent kinase inhibitor, p21. These results close ly correspond to those obtained using the yeast two-hybrid assay, as w ell as in vitro binding assays.