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

Guillaume Roussel, Eric A. Perpète, André Matagne, Emmanuel Tinti, Catherine Michaux

Research output: Contribution to journalArticle

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 4mg/mL) and ionic strength (up to 1M 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.

Original languageEnglish
Pages (from-to)417-423
Number of pages7
JournalBiotechnology and Bioengineering
Volume110
Issue number2
DOIs
Publication statusPublished - 1 Feb 2013

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Protein Refolding
Sodium dodecyl sulfate
Proteins
Membranes
Enzymes
Carbonic anhydrase
Sodium Dodecyl Sulfate
Detergents
Dichroism
Carbonic Anhydrase II
Ionic strength
Porins
Alcohols
Muramidase
Circular Dichroism
Protein Binding
Osmolar Concentration
Salts
Membrane Proteins
Temperature

Keywords

  • 2-methyl-2,4-pentanediol
  • Circular dichroism
  • Protein
  • Refolding
  • Sodium dodecyl sulfate

Cite this

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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 4mg/mL) and ionic strength (up to 1M 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.",
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Towards a universal method for protein refolding : The trimeric beta barrel membrane Omp2a as a test case. / Roussel, Guillaume; Perpète, Eric A.; Matagne, André; Tinti, Emmanuel; Michaux, Catherine.

In: Biotechnology and Bioengineering, Vol. 110, No. 2, 01.02.2013, p. 417-423.

Research output: Contribution to journalArticle

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AU - Michaux, Catherine

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