Towards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case

Authors

  • Guillaume Roussel,

    1. Unité de Chimie Physique Théorique et Structurale (UCPTS), University of Namur (FUNDP), 61 rue de Bruxelles, Namur 5000, Belgium; telephone: +32-81-72-45-57; fax: +32-81-72-45-46
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  • Eric A. Perpète,

    1. Unité de Chimie Physique Théorique et Structurale (UCPTS), University of Namur (FUNDP), 61 rue de Bruxelles, Namur 5000, Belgium; telephone: +32-81-72-45-57; fax: +32-81-72-45-46
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  • André Matagne,

    1. Laboratory of Enzymology and Protein Folding, Centre for Protein Engineering, Institut de Chimie B6, University of Liège, Liège, Belgium
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  • Emmanuel Tinti,

    1. Unité de Chimie Physique Théorique et Structurale (UCPTS), University of Namur (FUNDP), 61 rue de Bruxelles, Namur 5000, Belgium; telephone: +32-81-72-45-57; fax: +32-81-72-45-46
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  • Catherine Michaux

    Corresponding author
    1. Unité de Chimie Physique Théorique et Structurale (UCPTS), University of Namur (FUNDP), 61 rue de Bruxelles, Namur 5000, Belgium; telephone: +32-81-72-45-57; fax: +32-81-72-45-46
    • Unité de Chimie Physique Théorique et Structurale (UCPTS), University of Namur (FUNDP), 61 rue de Bruxelles, Namur 5000, Belgium; telephone: +32-81-72-45-57; fax: +32-81-72-45-46.
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Abstract

It has recently been reported that 2-methyl-2,4-pentanediol (MPD) can modulate the protein-binding properties of sodium dodecyl sulfate (SDS), turning it into a non-denaturing detergent. Indeed both alpha (the lysozyme) and beta (the carbonic anhydrase II) soluble enzymes, as well as a beta membrane protein (PagP) have been successfully refolded into their native form by using this amphiphatic alcohol. In order to support the universal character of our MPD-based technique, we have extended its transferability to the Omp2a trimeric membrane porin. The far-UV circular dichroism signature of Omp2a refolded with our original procedure is identical to that obtained by classical techniques, clearly indicating a proper refolding. Moreover, we show that the optimal SDS/MPD ratio for refolding Omp2a is similar to what has been observed for other types of proteins. While the protocol allows refolding at higher protein concentration (up to 4 mg/mL) and ionic strength (up to 1 M NaCl) than other refolding methods, it is also more efficient at basic pH values and medium temperature (20–40°C). Finally, the key role of the cosolvent was highlighted by a thorough study of the efficiency of MPD analogues, and a high variability was observed, as they can be able or unable to induce refolding at low or high salt concentrations. Biotechnol. Bioeng. 2013; 110: 417–423. © 2012 Wiley Periodicals, Inc.

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