title: Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis.
abstract: Brucella melitensis is a gram-negative bacteria known to cause brucellosis and to produce severe infections in humans. Whilst brucella's outer membrane proteins have been extensively studied due to their potential role as antigens or virulence factors, their function is still poorly understood at the structural level, as the 3D structure of Brucella β-barrel membrane proteins are still unknown. In this context, the B. melitensis trimeric Omp2a porin has been overexpressed and refolded in n-dodecyl-β-d-maltopyranoside. We here show that this refolding process is insensitive to urea but is temperature- and ionic strength-dependent. Reassembled species were characterized by fluorescence, size-exclusion chromatography and circular dichroism. A refolding mechanism is proposed, suggesting that Omp2a first refolds under a monomeric form and then self-associates into a trimeric state. This first complete in vitro refolding of a membrane protein from B. melitensis shall eventually lead to functional and 3D structure determination.
authors.affiliation: Unité de Chimie Physique Théorique et Structurale, University of Namur, FUNDP, 61 rue de Bruxelles, 5000 Namur, Belgium.
authors.forename: G
authors.initials: G
authors.lastname: Roussel
authors.forename: A
authors.initials: A
authors.lastname: Matagne
authors.forename: X
authors.initials: X
authors.lastname: De Bolle
authors.forename: E A
authors.initials: EA
authors.lastname: Perpète
authors.forename: C
authors.initials: C
authors.lastname: Michaux
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D017347:Brucella melitensis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D008320:Maltose / Q000031:analogs & derivatives / Q000737:chemistry; D008969:Molecular Sequence Data; D018272:Porins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; C096735:Omp2a protein, Brucella; D018272:Porins; D011994:Recombinant Proteins; C411362:dodecyl maltopyranoside; D008320:Maltose