Purification and properties of Gal repressor:pL-galR fusion in pKC31 plasmid vector

Abstract

The galR gene, which encodes the Gal repressor protein in Escherichia coli, has been fused to the strong pL promoter of bacteriophage lambda in plasmid pKC31. The pL promoter is kept repressed by a thermolabilie lambda repressor, CIts857, to prevent cell killing. Heat induction of the pL-galR fusion plasmid synthesizes large amounts of active Gal repressor. The protein has been purified to homogeneity in three steps. The purification is greatly aided by the reversible insolubility of active repressor in crude extract at salt concentrations of less than 200 mM. The amino-terminal amino acid sequence determined by automated Edman degradation is: N-Ala-Thr-Ile-Lys-Asp-Val-Ala-Arg-Leu-Ala-Gly-Val-Ser-Val-Ala-Thr-Val-. Comparison of this sequence with that deduced from the DNA sequence of the galR gene showed that the formyl methionine residue preceding alanine at position 1 is cleaved off. The repressor is present in solution as a dimer of a 37-kDa subunit. The protein binds to gal DNA containing wild type and not mutant operator sequences. As predicted, this sequence-specific binding is inhibited by the presence of D-galactose or D-fucose, both of which are in vivo inducers of the gal operon. Gal repressor inhibits the expresison of gal operon by binding to two spatially separated operators which flank, but do not overlap, the gal promoter segment. Experiments to study the mechanism of repressor action are discussed.