A vector, pHisGal, allowing bacterial production of proteins fused to a hexahistidine-tagged Gal4 DNA-binding domain.

A vector, pHisGal, was constructted that allows isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible expression of the DNA-binding domain of the yeast transcription factor Gal4 in Escherichia coli. Protein sequences to be tested for transcription regulatory potential in conjunction with the Gal4 DNA-binding domain can be inserted in-frame into a multiple cloning site at the C terminus of Gal4. A hexahistidine sequence fused to the N terminus of Gal4 allows efficient purification of the bacterially produced protein by affinity chromatography on nickel nitrilotriacetic acid (Ni-NTA) columns. Purified Gal4 fusion proteins produced in E. coli showed DNA-binding activity in electrophoretic nobility shift assays and were highly active in cell-free transcription assays from higher eukaryotic cells.