Use of anionic denaturing detergents to purify insoluble proteins after overexpression

Abstract

BackgroundMany 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.ResultsHere 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.ConclusionIonic, 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.

Purification and structural characterization of Mce4A from Mycobacterium tuberculosis

“…Expression and purification of Mce4A was done successfully with the help of earlier published protocol [6,24] with slight modifications which yields sufficient amount of the protein. The purity of the purified Mce4A was checked through SDS-PAGE and western blotting by using anti-His tag monoclonal antibody.…”

“…The cultures were grown for next three hours and the cells were harvested by centrifugation at 5000 rpm for 20 minutes, supernatant was discarded and the pellets were collected. Purification was performed by published protocol with slight modification[19]. These pellets were dissolved in cell lysis buffer (cell lysis buffer contained8 mM Na 2 HPO 4 , 286 mM NaCl, 1.4 mM KH 2 PO 4 , 2.6 mM KCl and 1% SDS (w/v) at pH 7.4) for 15 minutes to make cell lysate homogeniesed.…”

Phosphorylation of spore coat proteins by a family of atypical protein kinases

“…For B. subtilis CotB, 1% SDS was added to the lysis buffer to aid in solubilization of the protein (61). The cells were lysed by sonication, followed by chilling to precipitate the SDS.…”

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“…SDS-PAGE (Figure 1), western blot and protease assay have showed that seven out of twelve recombinant proteases were insoluble form, whilst another five enzymes were not expressed. The major problem in protein expression would be the formation of "inclusion bodies" that occur when the heterologous protein overexpressed [30]. The insoluble protein could be formed by the association of partially folded protein or misfolded protein [31].…”