An expression vector tailored for large-scale, high-throughput purification of recombinant proteins

被引：133

作者：

Donnelly, MI ^{[1
]}

Zhou, M ^{[1
]}

Millard, CS ^{[1
]}

Clancy, S ^{[1
]}

Stols, L ^{[1
]}

Eschenfeldt, WH ^{[1
]}

Collart, FR ^{[1
]}

Joachimiak, A ^{[1
]}

机构：

[1] Argonne Natl Lab, Biosci Div, Argonne, IL 60439 USA

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2006年 / 47卷 / 02期

关键词：

high-throughput; structural genomics; maltose-binding protein; TVMV protease; ligation-independent cloning;

D O I：

10.1016/j.pep.2005.12.011

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Production of milligram quantities of numerous proteins for structural and functional studies requires an efficient purification pipeline. We found that the dual tag, his(6)-tag-maltose-binding protein (MBP), intended to facilitate purification and enhance proteins' solubility, disrupted such a pipeline, requiring additional screening and purification steps. Not all proteins rendered soluble by fusion to MBP remained soluble after its proteolytic removal, and in those cases where the protein remained soluble, standard purification protocols failed to remove completely the stoichiometric amount of his(6)-tagged MBP generated by proteolysis. Both liabilities were alleviated by construction of a vector that produces fusion proteins in which MBP, the his(6)-tag and the target protein are separated by highly specific protease cleavage sites in the configuration MBP-site-his(6)-site-protein. In vivo cleavage at the first site by co-expressed protease generated untagged MBP and his(6)-tagged target protein. Proteins not truly rendered soluble by transient association with MBP precipitated, and untagged MBP was easily separated from the his-tagged target protein by conventional protocols. The second protease cleavage site allowed removal of the his(6)-tag. (c) 2006 Elsevier Inc. All rights reserved.

引用

页码：446 / 454

页数：9

共 26 条

[1] High throughput protein production for functional proteomics [J].