在大肠杆菌中作为谷胱甘肽S-转移酶融合蛋白表达的人α-L-岩藻糖苷酶前体的纯化。

Purification of human alpha-L-fucosidase precursor expressed in Escherichia coli as a glutathione S-transferase fusion protein.

作者信息

de Carlos Alejandro, Montenegro Dolores, Alonso-Rodríguez Ana, Páez de la Cadena María, Rodríguez-Berrocal Francisco Javier, Martínez-Zorzano Vicenta Soledad

机构信息

Department of Biochemistry, Genetics and Immunology, Faculty of Sciences, University of Vigo, 36200, Vigo, Spain.

出版信息

J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Mar 25;786(1-2):7-15. doi: 10.1016/s1570-0232(02)00721-3.

DOI:10.1016/s1570-0232(02)00721-3

PMID:12650997

Abstract

Alpha-L-fucosidase (FUC) is a glycosidase involved in the degradation of fucose-containing glycoconjugates. A cDNA representing the complete sequence of human FUC was inserted into the prokaryotic expression vector pGEX-2T. High levels of the glutathione S-transferase (GST) fusion protein were detected in Escherichia coli cells after induction with isopropyl thio-beta-D-galactopyranoside. The GST-FUC protein was mostly found as inclusion bodies and attempts to optimise its expression as a soluble form were unsuccessful. Nevertheless, the recombinant protein was purified by affinity chromatography on glutathione-sepharose and its fucosidase activity was characterised. After thrombin cleavage of the GST tag, the FUC precursor protein was purified by electro-elution.

摘要

α-L-岩藻糖苷酶（FUC）是一种参与含岩藻糖糖缀合物降解的糖苷酶。将代表人类FUC完整序列的cDNA插入原核表达载体pGEX-2T中。用异丙基硫代-β-D-半乳糖苷诱导后，在大肠杆菌细胞中检测到高水平的谷胱甘肽S-转移酶（GST）融合蛋白。GST-FUC蛋白大多以包涵体形式存在，将其表达优化为可溶形式的尝试未成功。尽管如此，重组蛋白通过谷胱甘肽-琼脂糖亲和层析进行了纯化，并对其岩藻糖苷酶活性进行了表征。在凝血酶切割GST标签后，通过电洗脱纯化了FUC前体蛋白。