title: Functional refolding of the Campylobacter jejuni MOMP (major outer membrane protein) porin by GroEL from the same species.
abstract: Functional and structural studies of outer membrane proteins from Gram-negative bacteria are frequently carried out using refolded proteins. Recombinant proteins are produced in Escherichia coli as inclusion bodies and then tediously refolded by dilution in buffered detergent solutions. In the present work, we obtained the refolding of MOMP (major outer membrane protein) from Campylobacter assisted by the molecular chaperone GroEL. Refolded MOMP recovered its native pore-forming activity when reconstituted in planar lipid bilayers. Both proteins were purified from the Campylobacter jejuni strain 85H. The purity of GroEL was assessed by silver staining and MS. Its native ultrastructure was observed by the use of transmission electron microscopy. Denaturation of MOMP was performed in urea at 65 degrees C followed by dialysis against 100 mM acetic acid, and was assessed by CD analysis. MOMP refolding reached a maximum efficiency in the presence of GroEL (at a MOMP/GroEL molar ratio of 9:1) and ATP. Under these conditions, 95% of denatured MOMP was refolded after a 15 min incubation. This approach represents an alternative method in studies of membrane protein refolding.
authors.affiliation: EA 2197, IFR 48, Faculté de Médecine, 27 boulevard Jean Moulin, 13385 Marseille cedex 05, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Goulhen
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Dé
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Pagès
authors.forename: Jean-Michel
authors.initials: JM
authors.lastname: Bolla
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018834:Chaperonin 60 / Q000378:metabolism*; D007700:Kinetics; D018272:Porins / Q000737:chemistry* / Q000378:metabolism; D011489:Protein Denaturation; D017510:Protein Folding
chemical_list: D001426:Bacterial Proteins; D018834:Chaperonin 60; D018272:Porins; C095080:major outer membrane protein, Campylobacter jejuni