双亚基丹酚酸酯酶基因的表达载体构建及其生物转化
Vector construction and biotransformation of double subunits salvianolic acid- esterase
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摘要: 为了实现丹酚酸酯酶的外源性表达,利用了大肠杆菌和毕赤酵母两种表达系统,原核大肠杆菌采用p ET 28a和p ET 32a构建表达载体,并对重组大肠杆菌进行分别诱导表达和共表达;真核毕赤酵母采用p PIC9K构建共表达载体,对重组毕赤酵母进行诱导表达。研究结果表明,两种体系均能诱导表达出丹酚酸酯酶,蛋白在大肠杆菌系统中得到了高效表达,但未显示出该酶活力。毕赤酵母系统可使蛋白分泌性表达,表达产物具有一定的丹酚酸酯酶活力,但表达量不高。说明大肠杆菌系统体系更为适合大量表达丹酚酸酯酶,由此提供了一种该酶的外源表达方法。Abstract: In order to achieve the salvianolic acid- esterase express in vitro,the salvianolic acid- esterase were heterologous expressed by E.coli and Pichia pasioris respectively.The E.coil system constructed expression vector with p ET 28 a and p ET 32 a and carried out induced expression and coexpression of the recombinant E.coil. The Pichia pastoris system constructed coexpression vector with p PIC9 K and carried out induced expression of the recombinant Pichia pastoris. The research found that the salvianolic acid- esterase were induced in E.coli and Pichia pasioris.The protein was highly expressed in E.coli system,but it did not exhibit the enzyme activity. The salvianolic acid- esterase were expressed in Pichia pasioris,and the expression product had salvianolic acid-esterase activity.But the quantity of expression was not high.It showed that the E.coli expression system was more suitable for the salvianolic acid- esterase and provided a exogenous expression method of salvianolic acid-esterase.