Purification of integral outer-membrane protein OmpC, a surface antigen from Salmonella typhi for structure-function studies: a method applicable to enterobacterial major outer-membrane protein.
Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDS-PAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure-function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins.
Arockiasamy
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A
Bioinformatics Centre, School of Biotechnology, Madurai Kamaraj University, India.
Krishnaswamy
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S
D002462:Cell Membrane / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004755:Enterobacteriaceae / Q000276:immunology; D004926:Escherichia coli / Q000737:chemistry / Q000276:immunology; D008070:Lipopolysaccharides / Q000378:metabolism; D018272:Porins / Q000737:chemistry* / Q000302:isolation & purification*; D020033:Protein Isoforms; D012485:Salmonella typhi / Q000737:chemistry / Q000276:immunology*; D013329:Structure-Activity Relationship
D008070:Lipopolysaccharides; C113724:OmpC protein; D018272:Porins; D020033:Protein Isoforms