His-tag对胆固醇氧化酶基因在大肠杆菌中表达的影响

为考察组氨酸标签(His-tag)对Brevibacterium sp.DGCDC-82中胆固醇氧化酶基因(ChoAb)在大肠杆菌中表达的影响,将PCR扩增后得到的结构基因与pET28a(+)连接,构建重组质粒pETChoAb(不带His-tag),pETChoAbn(His-tag位于N端)和pETChoAbc(His-tag位于C端)并在大肠杆菌中进行表达.对重组酶进行酶活检测,结果表明His-tag位于ChoAb的C端和N端,COD单位体积酶活由未带标签时的1.72 U/mL分别提高到4.03 U/mL和11.36 U/mL.利用软件Quantity One对SDS-PAGE电泳条带进行灰度分析,结果显示与不带His-tag的COD相比,His-tag位于ChoAb的C端和N端,COD表达量由8.8％增加到16.4％与72.3％.同时菌体浓度分别提高了1.2倍和3.2倍.作为纯化标签,该研究结果对His-tag用于诊断用酶COD的分离纯化可以提供一定的理论指导.

Effects of His-tag on cholesterol oxidase expression in Escherichia coli

Effects of the His-tag on the expression of cholesterol oxidase （COD） in Escherichia coll were studied. Three different recombinant COD in Escherichia coli were expressed： the untagged COD, and two His-tag COD, identical with the untagged COD except carrying the additional C or N terminal His-tag. Detection of recombinase activity showed that the activity of the untagged COD was 1.72 U/mL, while the activities of the latter two were improved to 4.03 U/mL and 11.36 U/mL, respectively. As well as the express lever, compared to cells expressing the untagged COD, the expression of the modified recombinase with C or N terminal His-tag rose from 8. 8% to 16. 4% and 72. 3%. While the corresponding cell concentration also increased by 1.2 times and 3.2 times. The results of this study showed clearly that both growth properties and COD expression levels were improved for E. coli cells expressing the His-tag COD.