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microbiology, Vol 145, 89-98, Copyright © 1999 by Society for General Microbiology
ARTICLES |
FL Thies, A Weishaupt, H Karch, HP Hartung and G Giegerich
Department of Neurology, Julius-Maximilians-University, Wurzburg, Germany.
The groESL bicistronic operon from the enteric pathogen Campylobacter jejuni was cloned and sequenced. It consists of two ORFs encoding proteins with molecular masses of 9.5 and 57.9 kDa, which showed a high degree of homology to other bacterial GroES and GroEL proteins. Northern blot analysis suggested that the groESL operon is transcribed as a bicistronic mRNA, and its steady-state level was markedly increased after temperature upshift. By primer extension assay, one potential transcription start point preceding the groESL genes could be demonstrated, and a putative promoter region compatible with both Escherichia coli and C. jejuni sigma70 consensus sequences was identified. A conserved inverted repeat, which is believed to be involved in the regulation of the groESL genes, was found between the - 10 promoter box and the groES translation start site. The complete coding region of groEL was fused with pET-22b(+) and expressed in E. coli as a His6-tagged recombinant protein (rCjHsp60-His). After purification, the protein was recognized by an anti-HSP60 monoclonal antibody. ELISA and Western immunoblotting experiments showed that IgG and IgA antibody responses against rCjHsp60-His were not significantly increased in sera from 24 patients with sporadic Campylobacter infection when compared to sera from 16 healthy controls.
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