Purification and DNA binding properties of theblalgene product, repressor for the β-lactamase gene,blaP, ofBacillus licheniformis

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Abstract
The location of the repressor gene, blal, for the β-lactamase gene blaP of Bacillus licheniformis 749, on the 5' side of blaP, was confirmed by sequencing the bla region of the constitutive mutant 749/C. An amber stop codon, likely to result in a nonfunctional truncated repressor, was found at codon 32 of the 128 codon blal open reading frame (ORF) located 5' to blaP. In order to study the DNA binding activity of the repressor, the structural gene for blal, from strain 749, with its ribosome binding site was expressed using a two plasmid T7 RNA polymerase/promotor system (S. Tabor and C. C. Richardson. Proc. Natl. Acad. Sci. 82, 1074–1078 (1985). Heat induction of this system in Escherichia coli K38 resulted in the production of Blal as 5–10% of the soluble cell protein. Repressor protein was then purified by ammonium sulfate fractionation and cation exchange chromatography. The sequence of the N-terminal 28 amino acid residues was determined and was as predicted from the DNA. Binding of Blal to DNA was detected by the slower migration of protein DNA complexs during polyacrylamide gel electrophoresis. Blal was shown to selectively bind DNA fragments carrying the promoter regions of blal and blaP.