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.
Institute for Biochemistry, University of Freiburg, FRG.
M L
ML
Schmitz
P A
PA
Baeuerle
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000096:biosynthesis*; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000502:physiology; D005822:Genetic Vectors; D006639:Histidine; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D006639:Histidine