Vectors for a 'double-tagging' assay for protein-protein interactions: localization of the CDK2-binding domain of human p21

Gene
Z X WangF J Germino

Abstract

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 clon...Continue Reading

References

Sep 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·R BernardsT P Dryja
May 1, 1983·Journal of Bacteriology·S C Cheng, P Modrich
Jan 1, 1983·Proceedings of the National Academy of Sciences of the United States of America·H A de BoerM Vasser
May 9, 1995·Proceedings of the National Academy of Sciences of the United States of America·Z X Wang, F J Germino
Nov 19, 1993·Cell·W S el-DeiryB Vogelstein
Feb 1, 1993·Proceedings of the National Academy of Sciences of the United States of America·F J GerminoS M Weissman

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