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Mechanism of Gemini Disulfide Detergent Mediated Oxidative Refolding of Lysozyme in a New Artificial Chaperone System

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Abstract

Gemini surfactants are a new class of surfactants that consist of two hydrophilic head groups and two hydrophobic tails separated by a spacer group. As the properties of geminis are different to their monomeric counterparts, a large number of applications have been investigated. Here we report on the use of a new class of gemini detergents containing a disulfide bond in the spacer (Det-SS-Det) for protein refolding. Using lysozyme as a model protein we could demonstrate that the disulfide gemini detergents allow oxidative refolding of the protein in the absence of any external redox system in an “artificial chaperone system”. Refolding kinetics using gemini disulfide detergents differing in their hydrophobicity were analysed to determine the folding and aggregation rate constants. The results point to an important role of the transiently formed mixed disulfides between the protein and the detergent (Prot-SS-Det) in the oxidative refolding process of lysozyme.

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Abbreviations

ACN:

Acetonitrile

ALP:

Alkaline phosphatase

β-CD:

β-cyclodextrin

BSA:

Bovine serum albumin

CD:

Cyclodextrin

CMC:

Critical micelle concentration

CTAB:

Cetyltrimethylammonium bromide

C9SS:

Nonyl-dimethyl-(2-sulfanylethyl)azanium-disulfide

C10SS:

Decyl-dimethyl-(2-sulfanylethyl)azanium-disulfide

C11SS:

Undecyl-dimethyl-(2-sulfanylethyl)azanium-disulfide

C12SS:

Dodecyl-dimethyl-(2-sulfanylethyl)azanium-disulfide

C13SS:

Tridecyl-dimethyl-(2-sulfanylethyl)azanium-disulfide

C14SS:

Tetradecyl-dimethyl-(2-sulfanylethyl)azanium-disulfide

C15SS:

Pentadecyl-dimethyl-(2-sulfanylethyl)azanium-disulfide

C16SS:

Hexadecyl-dimethyl-(2-sulfanylethyl)azanium-disulfide

DTAB:

Dodecyltrimethylammonium bromide

DTTox:

Oxidized dithiothreitol

DTTred:

Reduced dithiothreitol

E. coli:

Escherichia coli

EDTA:

Ethylenediaminetetraacetic acid

GdmCl:

Guanidine hydrochloride

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

HPLC:

High pressure liquid chromatography

IB:

Inclusion body

m-β-CD:

Methyl-β-cyclodextrin

RNAse A:

Ribonuclease A

RP-HPLC:

Reversed phase high pressure liquid chromatography

RT:

Room temperature

TCEP:

Tris (2-carboxyethyl) phosphine hydrochloride

TFA:

Trifluoroacetic acid

Tris:

Tris (hydroxymethyl)-aminomethan

TTAB:

Tetradecyltrimethylammonium bromide

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Acknowledgments

This work was supported by the Landesstiftung Baden-Württemberg.

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Authors and Affiliations

  1. Institute for Biochemistry, University of Applied Sciences, Mannheim, Germany

    Marc Potempa & Christian Frech

  2. Institute of Molecular Biology and Cell Culture Technology, University of Applied Sciences, Mannheim, Germany

    Mathias Hafner

Authors
  1. Marc Potempa
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  2. Mathias Hafner
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  3. Christian Frech
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Correspondence to Christian Frech.

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Potempa, M., Hafner, M. & Frech, C. Mechanism of Gemini Disulfide Detergent Mediated Oxidative Refolding of Lysozyme in a New Artificial Chaperone System. Protein J 29, 457–465 (2010). https://doi.org/10.1007/s10930-010-9279-8

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  • DOI: https://doi.org/10.1007/s10930-010-9279-8

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