A VECTOR, PHISGAL, ALLOWING BACTERIAL PRODUCTION OF PROTEINS FUSED TOA HEXAHISTIDINE-TAGGED GAL4 DNA-BINDING DOMAIN

A vector pHisGal, was constructed that allows isopropyl-beta-D-thiogal actopyranoside (IPTG)-inducible expression of the DNA-binding domain o f the yeast transcription factor Ga14 in Escherichia coli. Protein seq uences to be tested for transcription regulatory potential in conjunct ion with the Ga14 DNA-binding domain can be inserted in-frame into a m ultiple cloning site at the C terminus of Ga14. A hexahistidine sequen ce fused to the N terminus of Ga14 allows efficient purification of th e bacterially produced protein by affinity chromatography on nickel ni trilotriacetic acid (Ni-NTA) columns. Purified Ga14 fusion proteins pr oduced in E. coli showed DNA-binding activity in electrophoretic mobil ity shift assays and were highly active in cell-free transcription ass ays from higher eukaryotic cells.