Refolding SDS-Denatured Proteins by the Addition of Amphipathic Cosolvents

Michaux, Catherine; Pomroy, Neil C.; Privé, Gilbert G.

doi:10.1016/j.jmb.2007.11.026

Cited by 67 publications

(67 citation statements)

References 53 publications

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“…Sodium dodecyl sulfate (SDS) is an anionic detergent that is widely used as protein denaturant due to its powerful dissociation and solubilization properties [53].…”

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confidence: 99%

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Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

Pan

Brown

Konermann

2010

J. Am. Soc. Mass Spectrom.

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“…Sodium dodecyl sulfate (SDS) is an anionic detergent that is widely used as protein denaturant due to its powerful dissociation and solubilization properties [53].…”

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confidence: 99%

“…However, SDS-denatured membrane proteins often retain significant residual structure, in fact, some ␣-helical elements may even be stabilized to a certain extent [45,53]. SDS-solubilized BR often serves as a starting point for folding studies [54].…”

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confidence: 99%

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

Pan

Brown

Konermann

2010

J. Am. Soc. Mass Spectrom.

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“…When expressed as IBs, solubilization and subsequent refolding of the insoluble proteins are required for production of functional and soluble proteins. Various solubilization and refolding technologies have been developed using denaturants [35,36] or detergents [37][38][39][40]. Such refolding technologies have been applied to various antibody fragments [41][42][43][44].…”

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confidence: 99%

Refolding Technology for scFv Using a New Detergent, N-Lauroyl-L-glutamate and Arginine

2012

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Monoclonal antibodies to the soluble antigens or cell surface markers hold great promise as effective human therapeutics. One of the major disadvantages is its large size, which prevents efficient penetration into the target tissues. Smaller version of antibodies, which has only antigen binding sites, is extensively investigated. It becomes increasingly apparent, however, that these smaller fragments of antibodies are rather difficult to produce, as the normally efficient mammalian secretion system does not work well for these fragments. Thus, refolding of insoluble proteins produced in Escherichia coli is a method of choice, although such refolding is mainly based on trial-and-error experiment. Here we describe a novel refolding system using a new amino acid-based detergent, N-lauroyl-L-glutamate, and arginine. This detergent appears to readily dissociate from proteins below critical micelle concentration (CMC), while remaining effective in protein solubilization above CMC. Arginine suppresses protein aggregation when the detergent concentration was reduced below CMC. The interaction of the detergent and arginine with proteins, which play an important role in protein refolding, will be discussed in great length.

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“…Recently, a simple yet effective method to recover refolded and active proteins has been developed, based on the association of an anionic detergent (the sodium dodecyl sulfate, SDS, known as denaturing agent) with an amphipathic diol solvent [3][4][5]. More precisely, it has been shown that peculiar solvents can modulate the denaturing properties of SDS and even induce the refolding from a SDS-denatured state.…”

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Characterization and optimization of a novel protein refolding methodology

Roussel

Rouse

Sansom

et al. 2013

MATEC Web of Conferences

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Controlling the refolding process of proteins is of considerable theoretical and practical interest [1,2]. Recently, a simple yet effective method to recover refolded and active proteins has been developed, based on the association of an anionic detergent (the sodium dodecyl sulfate, SDS, known as denaturing agent) with an amphipathic diol solvent [3][4][5]. More precisely, it has been shown that peculiar solvents can modulate the denaturing properties of SDS and even induce the refolding from a SDS-denatured state. Up to now, this cosolvent effect has been observed on both soluble and bacterial membrane proteins. None to say, it is crucial to have a clear picture of the physicochemical and molecular basis of these refolding processes. To this end, we have used both experimental (intrinsic fluorescence and circular dichroïsm) and theoretical (molecular dynamics [6]) approaches to help understanding such intricate phenomena including the complex interplay between protein-SDS, protein-solvent and solvent-SDS interactions.In that context, a fundamental investigation has been achieved starting from the study of the detergent/cosolvent couple and then considering the protein/detergent/cosolvent complex. In a first stage, we explore the micellization characteristics of SDS in presence (or absence) of solvents (chain length, one or several alcohols groups, position isomers, … ). Such a study gives access to the nature and strength of the interactions between the molecules, the system stability as a function of time, and the micelles formation kinetics. Original detergent/solvent pairs can be proposed in order to improve the efficiency of our refolding protocol, as well as to sharpen our understanding of its mechanism, paving the way to the full protein (especially of tricky systems as membrane proteins) characterization.

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Refolding SDS-Denatured Proteins by the Addition of Amphipathic Cosolvents

Cited by 67 publications

References 53 publications

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

Site-directed mutagenesis combined with oxidative methionine labeling for probing structural transitions of a membrane protein by mass spectrometry

Refolding Technology for scFv Using a New Detergent, N-Lauroyl-L-glutamate and Arginine

Characterization and optimization of a novel protein refolding methodology

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