来自毕赤酵母的人Csk同源激酶的谷胱甘肽S-转移酶（GST）融合激酶结构域的表达有助于轻松纯化。

Expression of GST-fused kinase domain of human Csk homologous kinase from Pichia pastoris facilitates easy purification.

作者信息

Murthy T V S

机构信息

Division of Experimental Medicine, Harvard Institutes of Medicine, 4-Blackfan Circle, Boston, MA 02115, USA.

出版信息

Biotechnol Lett. 2004 Mar;26(5):443-9. doi: 10.1023/b:bile.0000018265.25289.6d.

DOI:10.1023/b:bile.0000018265.25289.6d

PMID:15104145

Abstract

Human Csk Homologous Kinase (CHK), a protein of 527 amino acid residues, is involved in suppression of breast tumors. The kinase domain of CHK (amino acid residues 228 to 485) expressed with C-terminal 6HIS fusion in Pichia pastoris is heavily glycosylated. Expression of the C-terminal 6HIS fused kinase domain of CHK, with an N-terminal glutathione S-transferase fusion, in Pichia pastoris alleviated the hyperglycosylation. The expressed protein was purified by affinity chromatography to 1 mg l(-1) culture and remained active. A simple plate assay to identify colonies of P. pastoris expressing the recombinant protein is also presented.

摘要

人Csk同源激酶（CHK）是一种由527个氨基酸残基组成的蛋白质，参与乳腺肿瘤的抑制过程。在毕赤酵母中以C端6HIS融合形式表达的CHK激酶结构域（氨基酸残基228至485）高度糖基化。在毕赤酵母中表达带有N端谷胱甘肽S-转移酶融合的CHK的C端6HIS融合激酶结构域，可减轻高糖基化现象。通过亲和层析将表达的蛋白纯化至1 mg l(-1)培养物，且该蛋白仍保持活性。本文还介绍了一种用于鉴定表达重组蛋白的毕赤酵母菌落的简单平板检测方法。