人类超氧化物歧化酶在酿酒酵母系统中的高效表达

High Level Expression of Human Cu-Zn Superoxide Dismutase in Saccharomyces cerevisiae

超氧化物歧化酶 (SOD)是一种在生物界广泛存在的抗氧化酶 ,在抗衰老、抗肿瘤、抗免疫疾病和电磁辐射上都起着重要的作用 .对一从人类胎肝cDNA文库中筛到的SOD基因片段进行重组、克隆 ,并得到带有启动子PADH2 GAPDH和终止子TADH1的高效稳定的酿酒酵母表达载体 pHC11 hSOD .转化酿酒酵母DCO4后获得工程菌DCO4 /pHC11 hSOD .表达产物经破壁后SDS PAGE电泳检测为 2 0ku的条带 ;酶活性测定结果表明发酵液有 4 0万U/L的hSOD活性 .此外 ,还研究了工程菌的发酵条件和hSOD产物的纯化方法 .纯化产物在SDS PAGE上和Superdex分子筛检测显示 ,所得产物的纯度已达 95

Superoxide dismutase (SOD) is found ubiquitously in various organisms. It plays an important role in anti ageing, tumor, immune diseases and radiation processes. A recombinant SOD gene fragment screened from a cDNA library of human fetal liver was cloned under the control of a hybrid promoter P (ADH2-GAPDH) and a terminator T (ADH1)into plasmid pHC11, which was highly stable in Saccharomyces cerevisiae , to form an expression vector pHC11-hSOD. After transformation of this expression vector into a Saccharomyces cerevisiae strain DCO4, an SOD expressing yeast strain DCO4/pHC11hSOD was obtained. A multistage procedure including yeast fermentation and cell lysis was designed to collect the protein product. Eletrophoresis analysis of the product showed a new band of 20 ku on the gel. Biochemical analysis proved that the lysate supernatant exhibited a high SOD activity as 400 000 U/L. Explorations were made to reach a high-density fermentation and to purify the product. The single chain hSOD wes already purified to reach a purity level of over 95%, as judged by SOD-PAGE and molecular sieve.