乙醇酸氧化酶基因在巴斯德毕赤氏酵母中的表达

将菠菜乙醇酸氧化酶基因片段克隆至表达载体pPIC3．5k。提取重组质粒，进行限制性酶切鉴定。重组质粒用Sal I酶切线性化，电导入法转化毕赤酵母(Pichia pastoris)，在缺乏组氨酸的RDB平板筛选重组子，提取酵母的染色体基因组进行PCR扩增鉴定整合情况，用甲醇诱导表达。结果表明，SDS-PAGE电泳显示表达蛋白的分子量约为39．8kD，与文献报道的乙醇酸氧化酶分子量接近。酶的活力达到了40．8IU／g湿菌体，比不含有目的片断的对照菌酶活提高了17倍，确认了导入的乙醇酸氧化酶基因片段在酵母中高效表达。

Expressing of glycolate oxidase gene in pichia pastoris

The fragment of glycolate oxidase gene was cloned into pPIC3.5K (P.pastoris expression vector). The recombinant plasmid was identified by digested with SnaB I and Not I. It was linerized by Sal I and then transferred into the yeast(Pichia pastoris). Positive clones were selected from the medium without histidine. The feature of integration was examined by PCR, using the chromosome genome of the recombinant yeast as a template. The expression of spinach glycolate oxidase gene in Pichia pastoris appeared to be induced by methanol. The molecular weight of expression protein was about 39.8 kD measured by SDS-PAGE and consisted with the date of glycolate oxidase from the literature. Under optimal conditions, the activity of glycolate oxidase enzyme was 40.8 IU/g wet cell and 17 times as high as the ghost. It approved that the recombinant yeast could express spinach glycolate oxidase efficiently.