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Open Access Highly Accessed Methodology article

Use of anionic denaturing detergents to purify insoluble proteins after overexpression

Benjamin Schlager*, Anna Straessle and Ernst Hafen

Author Affiliations

Institute for Molecular Systems Biology, ETH Zurich, Wolfgang Pauli-Strasse 16, Zurich, 8093, Switzerland

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BMC Biotechnology 2012, 12:95  doi:10.1186/1472-6750-12-95

Published: 11 December 2012

Abstract

Many proteins form insoluble protein aggregates, called “inclusion bodies”, when overexpressed in E. coli. This is the biggest obstacle in biotechnology. Ever since the reversible denaturation of proteins by chaotropic agents such as urea or guanidinium hydrochloride had been shown, these compounds were predominantly used to dissolve inclusion bodies. Other denaturants exist but have received much less attention in protein purification. While the anionic, denaturing detergent sodiumdodecylsulphate (SDS) is used extensively in analytical SDS-PAGE, it has rarely been used in preparative purification.

Here we present a simple and versatile method to purify insoluble, hexahistidine-tagged proteins under denaturing conditions. It is based on dissolution of overexpressing bacterial cells in a buffer containing sodiumdodecylsulfate (SDS) and whole-lysate denaturation of proteins. The excess of detergent is removed by cooling and centrifugation prior to affinity purification. Host- and overexpressed proteins do not co-precipitate with SDS and the residual concentration of detergent is compatible with affinity purification on Ni/NTA resin. We show that SDS can be replaced with another ionic detergent, Sarkosyl, during purification. Key advantages over denaturing purification in urea or guanidinium are speed, ease of use, low cost of denaturant and the compatibility of buffers with automated FPLC.

Ionic, denaturing detergents are useful in breaking the solubility barrier, a major obstacle in biotechnology. The method we present yields detergent-denatured protein. Methods to refold proteins from a detergent denatured state are known and therefore we propose that the procedure presented herein will be of general application in biotechnology.

Keywords:
Inclusion Bodies; Sodiumdodecylsulphate (SDS); N-lauroylsarcosine sodium salt (Sarkosyl); Immobilized Metal Ion Affinity Chromatography (IMAC)