Abstract：Abstract: The purpose of this research is to investigate the effects of different positions of His6-tag in the protein sequence of exo-inulinase of Kluyveromyces cicerisporus CBS4857 (KcINU1) on the enzyme activity. Firstly, the 3D structure of KcINU1 was modeled by the Swiss-Model Server to predict the position of His6-tag in the protein sequence of KcINU1. Secondly, the recombinant kcINU1 genes with sequences encoding N-His6-tag and C-His6-tag were amplified by PCR, and were cloned into pPICZαA vector respectively. The recombinant plasmids were further transformed into Pichia pastoris X-33 by electrotransformation. Finally, the recombinant KcINU1s were successfully expressed under the methanol induction and purified by Histamine 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 could 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 significance for selection of label position of glycoside hydrolase family 32.
引用本文:
马君燕, 谭海东, 王文霞, 杜昱光, 尹 恒. 组氨酸标签位置对重组鹰嘴豆孢克鲁维酵母外切菊粉酶活性的影响[J]. 生命科学研究, 2016, 20(3): 218-223. MA Jun-Yan, TAN Hai-Dong, WANG Wen-Xia, DU Yu-Guang, YIN Heng. The Effects of His6-tag Position in Kluyveromyces cicerisporus Exo-inulinase on Its Activity. Life Science Research, 2016, 20(3): 218-223.
2016,Vol. 20
