Purification, refolding and characterization of the trimeric Omp2a outer membrane porin from Brucella melitensis

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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.

Original languageEnglish
Pages (from-to)198-204
Number of pages7
JournalProtein Expression and Purification
Volume83
Issue number2
DOIs
Publication statusPublished - Jun 2012

Keywords

  • Biophysics
  • Brucella melitensis
  • Outer membrane protein
  • Porin
  • Refolding
  • Trimer

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