TAILIEUCHUNG - Báo cáo Y học: Conformational analysis of opacity proteins from Neisseria meningitidis

Opacity-associated (Opa) proteins are outer membrane proteins which play a critical role in the adhesion of patho-genicNeisseriaspp. to epithelial and endothelial cells and polymorphonuclear neutrophils. The adherence is mainly mediated by the CD66-epitope-containing members of the carcinoembryonic-antigen family of human cell-adhesion molecules (CEACAM). For the analysis of the specific in-teractions of individual Opa proteins with their receptors, pure protein is needed in its native conformation. . | Eur. J. Biochem. 269 5215-5223 2002 FEBS 2002 doi Conformational analysis of opacity proteins from Neisseria meningitidis Marien I. de Jonae1 2 Martine P. Bos3. Hendrik J Hamstra1. Wim Jiskoot4. Peter van Ulsen4 Jan Tommassen4 Loek van Alphen1 and Peter van der Ley1 1 Laboratory of Vaccine Research National Institute of Public Health and the Environment RIVM Bilthoven the Netherlands department of Medical Microbiology University of Amsterdam AMC Amsterdam the Netherlands 3Department of Molecular Microbiology and department of Pharmaceutics Utrecht University Utrecht the Netherlands Opacity-associated Opa proteins are outer membrane proteins which play a critical role in the adhesion of pathogenic Neisseria spp. to epithelial and endothelial cells and polymorphonuclear neutrophils. The adherence is mainly mediated by the CD66-epitope-containing members of the carcinoembryonic-antigen family of human cell-adhesion molecules CEACAM . For the analysis of the specific interactions of individual Opa proteins with their receptors pure protein is needed in its native conformation. In this study we describe the isolation and structural analysis of opacity proteins OpaJ129 and OpaB128 derived from Neisseria meningitidis strain H44 76. When the Opa proteins were produced with the phoE signal sequence in Escherichia coli they were localized at the cell surface and the recombinant bacteria were found to specifically interact with CEACAM1. For refolding and purification the proteins were overproduced without their signal sequences in E. coli resulting in its cytoplasmic accumulation in the form of inclusion bodies. After solubilization of the inclusion bodies in urea the proteins could be folded efficiently in vitro under alkaline conditions by dilution in ethanolamine and the detergent n-dodecyl-N N-dimethyl-1-ammonio-3-propane-sulfonate SB12 . The structure of the refolded and purified proteins determined by circular dichroism indicated a high

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