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 plasmids (pTrc.BCCP and pTrc.EZZ::BCCP) which encode different 'tags' for the capture of a target protein of interest on a filter coated with either 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 Ptac promoter, while the second produces fusions to an IgG-binding domain (EZZ). The gene encoding a protein of interest can be inserted into these plasmids and thereby direct the production of a fusion protein which is biotinylated in vivo and can bind to avidin, or a fusion protein which can bind to IgG. The third is a positive-selection, phase lambda expression vector (lambdaFJG2) which permits the construction of lacZ::cDNA fusion proteins which retain beta-galactosidase activity. The insertion of an active ecoRVR gene between the cloning sites (EcoRI and HindII or NotI) permits the positive selection of inserts. The C-terminal two-thirds of the mouse retinoblastoma-encoding gene (containing the E1A-binding pocket) was cloned into pTrc.BCCP and pTrc.EZZ::BCCP, while the 13S E1a gene was cloned into lambdaFJG2. We show that the interaction between these two proteins can be detected using the 'double-tagging' filter assay, and that this assay has high sensitivity and specificity for detecting this interaction. Finally, we have used these vectors to localize the CDK2-binding domain of the cyclin-dependent kinase inhibitor, p21. These results closely correspond to those obtained using the yeast two-hybrid assay, as well as in vitro binding assays.
Wang
Z X
ZX
Cancer Institute of New Jersey, University of Medicine and Dentistry of NJ, New Brunswick, NJ 08901, USA.
Bhargava
A
A
Sarkar
R
R
Germino
F J
FJ
D017500:Adenovirus E1A Proteins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D001681:Biological Assay / Q000379:methods; D042846:CDC2-CDC28 Kinases; D003001:Cloning, Molecular; D051357:Cyclin-Dependent Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21; D018844:Cyclin-Dependent Kinases / Q000235:genetics / Q000378:metabolism*; D016213:Cyclins / Q000378:metabolism*; D004247:DNA; D004791:Enzyme Inhibitors / Q000378:metabolism*; D004926:Escherichia coli; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism
D017500:Adenovirus E1A Proteins; C493956:CDKN1A protein, human; C493957:Cdkn1a protein, mouse; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D016160:Retinoblastoma Protein; D004247:DNA; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; C495893:Cdk2 protein, mouse; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases