Immobilized metal-affinity chromatography protein-recovery screening is predictive of crystallographic structure success

被引：83

作者：

Choi, Ryan ^{[1
,2
]}

Kelley, Angela ^{[1
,2
]}

Leibly, David ^{[1
,2
]}

Hewitt, Stephen Nakazawa ^{[1
,2
]}

Napuli, Alberto ^{[1
,2
]}

Van Voorhis, Wesley ^{[1
,2
]}

机构：

[1] SSGCID, Seattle, WA 98125 USA

[2] Univ Washington, Dept Med, Div Allergy & Infect Dis, Sch Med, Seattle, WA 98195 USA

来源：

ACTA CRYSTALLOGRAPHICA SECTION F-STRUCTURAL BIOLOGY COMMUNICATIONS | 2011年 / 67卷

基金：

美国国家卫生研究院;

关键词：

HETEROLOGOUS EXPRESSION; PLASMODIUM-FALCIPARUM; ESCHERICHIA-COLI; SOLUBILITY; CLONING;

D O I：

10.1107/S1744309111017374

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

The recombinant expression of soluble proteins in Escherichia coli continues to be a major bottleneck in structural genomics. The establishment of reliable protocols for the performance of small-scale expression and solubility testing is an essential component of structural genomic pipelines. The SSGCID Protein Production Group at the University of Washington (UW-PPG) has developed a high-throughput screening (HTS) protocol for the measurement of protein recovery from immobilized metal-affinity chromatography (IMAC) which predicts successful purification of hexahistidine-tagged proteins. The protocol is based on manual transfer of samples using multichannel pipettors and 96-well plates and does not depend on the use of robotic platforms. This protocol has been applied to evaluate the expression and solubility of more than 4000 proteins expressed in E. coli. The UW-PPG also screens large-scale preparations for recovery from IMAC prior to purification. Analysis of these results show that our low-cost non-automated approach is a reliable method for the HTS demands typical of large structural genomic projects. This paper provides a detailed description of these protocols and statistical analysis of the SSGCID screening results. The results demonstrate that screening for proteins that yield high recovery after IMAC, both after small-scale and large-scale expression, improves the selection of proteins that can be successfully purified and will yield a crystal structure.

引用

页码：998 / 1005

页数：8

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