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.
Yabuta
M
M
Suntory Bio-Pharma Tech Center, Gunma, Japan.
Onai-Miura
S
S
Ohsuye
K
K
D000595:Amino Acid Sequence; D001431:Bacteriological Techniques; D001483:Base Sequence; D001709:Biotechnology; D002116:Calcitonin / Q000096:biosynthesis / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D007763:Lac Operon; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011498:Protein Precursors / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics*; D012097:Repressor Proteins / Q000235:genetics*; D013696:Temperature; D001616:beta-Galactosidase / Q000096:biosynthesis / Q000235:genetics
D017931:DNA Primers; D011498:Protein Precursors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D002116:Calcitonin; D001616:beta-Galactosidase