Open AccessTwo-hybrid reporter vectors for gap repair cloning
Author(s) -
Jennifer I. Semple,
Graham Prime,
Lise J. Wallis,
Christopher M. Sanderson,
David Markie
Publication year - 2005
Publication title -
biotechniques/biotechniques
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.617
H-Index - 131
eISSN - 1940-9818
pISSN - 0736-6205
DOI - 10.2144/05386rr03
Subject(s) - insert (composites) , cloning (programming) , biology , multiple cloning site , plasmid , computational biology , genetics , shuttle vector , vector (molecular biology) , cloning vector , genomic library , reporter gene , gene , yeast , recombinant dna , peptide sequence , computer science , gene expression , mechanical engineering , engineering , programming language
Yeast two-hybrid analysis is a valuable approach to the discovery and characterization of protein interactions. We have developed vectors that can indicate the presence of an insert when used in two-hybrid bait and prey construction by gap repair cloning. The strategy uses a recombination cloning site flanked by sequences encoding the GAL4 activation and binding domains. After gap repair cloning in standard hosts carrying an ADE2 reporter gene, disruption of GAL4 by an insert can be identified by the development of red colony color, while empty vector plasmids produce white colonies. Function in yeast two-hybrid applications was initially validated using known interacting proteins in pair-wise analyses, and subsequently, the bait vectors were used in library screens with the mouse Mad212 and human Mccd1 proteins, identifying a number of putative new interactions for these proteins. These vectors should facilitate high-throughput yeast two-hybrid screens in which large numbers of bait and prey constructs may be required.