title: Towards a universal method for protein refolding: the trimeric beta barrel membrane Omp2a as a test case.
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.
authors.affiliation: Unité de Chimie Physique Théorique et Structurale-UCPTS, University of Namur-FUNDP, 61 Rue de Bruxelles, Namur 5000, Belgium.
authors.forename: Guillaume
authors.initials: G
authors.lastname: Roussel
authors.forename: Eric A
authors.initials: EA
authors.lastname: Perpète
authors.forename: André
authors.initials: A
authors.lastname: Matagne
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Tinti
authors.forename: Catherine
authors.initials: C
authors.lastname: Michaux
mesh_terms: D000438:Alcohols / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods; D008956:Models, Chemical; D009994:Osmolar Concentration; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012965:Sodium Chloride / Q000737:chemistry
chemical_list: D000438:Alcohols; D001426:Bacterial Proteins; C096735:Omp2a protein, Brucella; D018272:Porins; D011994:Recombinant Proteins; D012965:Sodium Chloride