A general path for large-scale solubilization of cellular proteins: From membrane receptors to multiprotein complexes

被引：16

作者：

Pullara, Filippo ^{[1
,6
]}

Guerrero-Santoro, Jennifer ^{[1
,4
]}

Calero, Monica ^{[1
]}

Zhang, Qiangmin ^{[1
]}

Peng, Ye ^{[3
]}

Spahr, Henrik ^{[5
]}

Kornberg, Guy L. ^{[5
]}

Cusimano, Antonella ^{[2
,6
]}

Stevenson, Hilary P. ^{[3
]}

Santamaria-Suarez, Hugo ^{[1
]}

Reynolds, Shelley L. ^{[1
]}

Brown, Ian S. ^{[1
]}

Monga, Satdarshan P. S. ^{[2
]}

Van Houten, Bennett ^{[3
]}

Rapic-Otrin, Vesna ^{[4
]}

Calero, Guillermo ^{[1
]}

Levine, Arthur S. ^{[4
]}

机构：

[1] Univ Pittsburgh, Sch Med, Dept Biol Struct, Pittsburgh, PA 15260 USA

[2] Univ Pittsburgh, Sch Med, Dept Pathol, Pittsburgh, PA 15260 USA

[3] Univ Pittsburgh, Sch Med, Dept Pharmacol & Chem Biol, Pittsburgh, PA 15260 USA

[4] Univ Pittsburgh, Sch Med, Dept Microbiol & Mol Genet, Pittsburgh, PA 15260 USA

[5] Stanford Univ, Dept Biol Struct, Stanford, CA 94305 USA

[6] Ri MED Fdn, Palermo, Italy

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2013年 / 87卷 / 02期

基金：

美国国家卫生研究院;

关键词：

Protein purification; Detergents; Multiprotein complexes; Membrane receptors; NUCLEOTIDE EXCISION-REPAIR; LIGHT-SCATTERING; MINIMAL SET; DNA; TRANSCRIPTION; PURIFICATION; EXPRESSION; TFIIH; AGGREGATION; BINDING;

D O I：

10.1016/j.pep.2012.10.007

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

Expression of recombinant proteins in bacterial or eukaryotic systems often results in aggregation rendering them unavailable for biochemical or structural studies. Protein aggregation is a costly problem for biomedical research. It forces research laboratories and the biomedical industry to search for alternative, more soluble, non-human proteins and limits the number of potential "druggable" targets. In this study we present a highly reproducible protocol that introduces the systematic use of an extensive number of detergents to solubilize aggregated proteins expressed in bacterial and eukaryotic systems. We validate the usefulness of this protocol by solubilizing traditionally difficult human protein targets to milligram quantities and confirm their biological activity. We use this method to solubilize monomeric or multimeric components of multi-protein complexes and demonstrate its efficacy to reconstitute large cellular machines. This protocol works equally well on cytosolic, nuclear and membrane proteins and can be easily adapted to a high throughput format. (C) 2012 Elsevier Inc. All rights reserved.

引用

页码：111 / 119

页数：9

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