Article

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

  1. Guillaume Roussel1,
  2. Eric A. Perpète1,
  3. André Matagne2,
  4. Emmanuel Tinti1,
  5. Catherine Michaux1,*

Article first published online: 18 SEP 2012

DOI: 10.1002/bit.24722

Issue

Biotechnology and Bioengineering

Biotechnology and Bioengineering

Volume 110, Issue 2, pages 417–423, February 2013

Additional Information

How to Cite

Roussel, G., Perpète, E. A., Matagne, A., Tinti, E. and Michaux, C. (2013), Towards a universal method for protein refolding: The trimeric beta barrel membrane Omp2a as a test case. Biotechnol. Bioeng., 110: 417–423. doi: 10.1002/bit.24722

Author Information

  1. 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

  2. 2

    Laboratory of Enzymology and Protein Folding, Centre for Protein Engineering, Institut de Chimie B6, University of Liège, Liège, Belgium

*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.

Publication History

  1. Issue published online: 20 DEC 2012
  2. Article first published online: 18 SEP 2012
  3. Accepted manuscript online: 4 SEP 2012 11:06AM EST
  4. Manuscript Accepted: 20 AUG 2012
  5. Manuscript Revised: 9 AUG 2012
  6. Manuscript Received: 1 JUN 2012

Funded by

  • Belgian National Fund for Scientific Research
  • Fonds de la Recherche Fondamentale et Collective. Grant Numbers: 2.4550.05, 2.4530.09
  • Federal Office for Scientific Technical and Cultural Affaires

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Keywords:

  • refolding;
  • protein;
  • circular dichroism;
  • 2-methyl-2,4-pentanediol;
  • sodium dodecyl sulfate

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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