酿酒酵母CICC1747醛糖还原酶基因的克隆及表达

Cloning and expression of the S.cerevisiae CICC1747 aldose reductase gene

采用PCR技术以酿酒酵母CICC1747基因组DNA为模板扩增得到醛糖还原酶基因GRE3,插入到pET-15b载体的NdeⅠ和BamHⅠ酶切位点之间,构建了酿酒酵母醛糖还原酶原核表达载体pET-15b-GRE3。将该载体转化到大肠杆菌菌株Rosetta(DE3)中,重组菌株用IPTG诱导表达,采用紫外分光光度法测定醛糖还原酶活力,并对其表达条件进行初步优化。SDS-PAGE电泳结果显示在分子量约37 kD处有明显的特异性蛋白质条带。发酵液的比酶活最高为54.94 mU/mg,与酿酒酵母野生菌株相比提高了近10倍。

With S. cerevisiae CICC1747 genomic DNA as the template, GRE3 gene fragment was amplified by PCR reaction and ligated to the Nde Ⅰ/BamH Ⅰ sites of pET-15b vector as the expression vector pET-15b- GRE3. E. coil Rosetta （DE3） competent cells were transformed with the recombinant plasmid. The aldose reductase activity of recombinant strain was determined with spectrum assay after IPTG induction. Under the optimal expression condition, the experiment results showed protein band about 37 kD as the molecular weight by the SDS-PAGE and the specific activity was 54.94 mU/mg, which was enhanced about ten-fold as that of the initial strain.