Thermo-inducible expression of a recombinant fusion protein by Escherichia coli lac repressor mutants.

The lac I gene of Escherichia coli encodes the lactose repressor. We isolated temperature-sensitive mutants of the lac I gene by in vitro mutagenesis with hydroxylamine. The new mutation sites were determined, and replacement of a single amino acid had respectively occurred at amino acid positions 241 (Ala-->Thr), 265 (Gly-->Asp) and 300 (Ser-->Asn). These mutation sites were located in the core region of the lac repressor protein. Temperature-dependent expression of beta-galactosidase was observed in the strains having these mutant lac I genes. By using these temperature-sensitive lac I genes, we developed a thermo-inducible expression system for a foreign gene under the control of the lac promoter. A recombinant fusion protein, consisting of a derivative of E. coli beta-galactosidase and the human calcitonin precursor peptide, was efficiently produced by using this system.