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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.

SUBMITTER: Goulhen F

PROVIDER: S-EPMC1224022 | biostudies-other | 2004 Mar

REPOSITORIES: biostudies-other

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Project description:Gram-negative bacteria constitutively release outer membrane vesicles (OMVs) during cell growth that play significant roles in bacterial survival, virulence and pathogenesis. In this study, comprehensive proteomic analysis of OMVs from a human gastrointestinal pathogen Campylobacter jejuni NCTC11168 was performed using high-resolution mass spectrometry. The OMVs of C. jejuni NCTC11168 were isolated from culture supernatants then characterized using electron microscopy and dynamic light scattering revealing spherical OMVs of an average diameter of 50nm. We then identified 134 vesicular proteins using high-resolution LTQ-Orbitrap mass spectrometry. Subsequent functional analysis of the genes revealed the relationships of the vesicular proteins. Furthermore, known N-glycoproteins were identified from the list of the vesicular proteome, implying the potential role of the OMVs as a delivery means for biologically relevant bacterial glycoproteins. These results enabled us to elucidate the overall proteome profile of pathogenic bacterium C. jejuni and to speculate on the function of OMVs in bacterial infections and communication.Biological significanceThis work demonstrates the importance of understanding vesicular proteomes from a human pathogen Campylobacter jejuni. From the secreted outer membrane vesicles (OMVs) of C. jejuni NCTC11168, we found a variety of virulence factors and essential proteins for bacterial survival. Bioinformatics analysis of these proteins predicted functional enrichment and localization. The most highly enriched were redox enzymes, which are considered to be essential for survival in oxygen-limiting environments and are predicted to be on the twin-arginine translocation (Tat) pathway suggesting a role for this pathway in the biogenesis of OMVs. This study additionally implicates a biological role for N-linked glycoproteins in OMVs. These approaches allow for a better understanding of the physiology of this important human pathogen.

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Functional refolding of the Campylobacter jejuni MOMP (major outer membrane protein) porin by GroEL from the same species.

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