摘要
为了研究His_6-tag标签位置对鹰嘴豆孢克鲁维酵母CBS4857外切菊粉酶(Kluyveromyces cicerisporus CBS4857 exo-inulinase,KcINU1)酶活性的影响,实验首先根据Swiss-Model构建的KcINU1三维模型预测了组氨酸标签在KcINU1的位置,然后将编码His_6-tag的基因通过PCR方法分别引入到kcINU1的N端和C端,并将重组的kcINU1基因克隆到毕赤酵母表达载体pPICZαA,重组质粒进一步经电转化法整合到毕赤酵母宿主菌X-33中,最后用甲醇诱导进行分泌表达,镍柱亲和层析法纯化目的蛋白,DNS去进行外切菊粉酶的活性测定。结果显示,实验中成功表达了具有活性的、His_6-tag标签位置不同的重组蛋白,并且N-His_6-KcINU1糖基化程度比KcINU1-His-C高,后者的比活是前者的82.2%,表明N端携带His_6-tag标签不影响蛋白的糖基化修饰,可以保持较高的酶活。该研究为获得高活性且便于分离纯化的重组KcINU1奠定了基础,对糖苷水解酶32家族标签位置的选择具有指导意义。
The purpose of this research is to investigate the effects of different positions of His_6-tag in the protein sequence of exo-inulinase of Kluyveromyces cicerisporus CBS4857 （KclNU1） on tile enzyme activity. Firstly, the 3D structure of KcINUl was modeled by the Swiss-Model Server to predict the position of His6- tag in the protein sequence of KcINU1. Secondly, tile recombinant kclNU1 genes with sequences encoding N-His6-tag and C-His6-tag were amplified by PCR, and were cloned into pPICZaA vector respectively. The recombinant plasmids were further transformed into Pichia pastoris X-33 by electrotransformation. Finally, the recombinant KcINUIs were successfully expressed tinder the methanol induction and purified by His- tamine affinity. The exo-inulinase activity of recombinant KcINU1 was determined by DNS. The results showed that the recombinant proteins, which contained different positions of His6-tag and had exo-inulinase activity, were successfully constructed. The glycosylation efficiency of N-His6-KcINU1 was higher than that of KcINU1-His6-C, and the ratio of specific activity of KcINU1-His6-C to N-His6-KcINU1 was 82.2%. It indicated that N terminal with His6-tag in KcINU1 did not affect the glycosylation efficiency of the protein and eould maintain a higher enzyme activity as well. This finding may lay the foundation for easily obtaining and purifying the recombinant KcINU1 with high activity and have the guiding significanee for selection of label position of glycoside hydrolase family 32.
出处
《生命科学研究》
CAS
CSCD
2016年第3期218-223,277,共7页
Life Science Research
基金
国家高技术研究发展规划(863计划)项目(2011AA10A205
2012AA021205)
