Yabuta M, Onai-Miura S, Ohsuye K
Suntory Bio-Pharma Tech Center, Gunma, Japan.
J Biotechnol. 1995 Feb 21;39(1):67-73. doi: 10.1016/0168-1656(94)00144-2.
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.
大肠杆菌的lac I基因编码乳糖阻遏物。我们通过用羟胺进行体外诱变分离出了lac I基因的温度敏感突变体。确定了新的突变位点，并且在氨基酸位置241（丙氨酸→苏氨酸）、265（甘氨酸→天冬氨酸）和300（丝氨酸→天冬酰胺）分别发生了单个氨基酸的替换。这些突变位点位于lac阻遏蛋白的核心区域。在具有这些突变lac I基因的菌株中观察到了β-半乳糖苷酶的温度依赖性表达。通过使用这些温度敏感的lac I基因，我们开发了一种在lac启动子控制下用于外源基因的热诱导表达系统。利用该系统高效生产了一种由大肠杆菌β-半乳糖苷酶衍生物和人降钙素前体肽组成的重组融合蛋白。
