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KMID : 0545120000100010056
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Journal of Microbiology and Biotechnology
2000 Volume.10 No. 1 p.56 ~ p.62
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Expression and Characterization of Helicobacter pylori Adhesin Protein Linked to Cholera Toxin A2/B Subunits in Escherichia coli
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Kim, Byung Ho
Shin, Sung Seup/Yoo, Young Hyo/Pyo, Suhk Neung
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Abstract
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The hpa gene genetically linked to the ctxa2b gene was cloned into the pTED expression vector, and the constructed pTEDhpa/ctxa2b was transformed into Escherichia coli. The fusion protein, the adhesin fused to the cholera toxin subunit A2B (CTXA2B) subunit, was expressed to high levels as inclusion bodies in E. coli. The expressed protein was partially purified by washing the inclusion bodies with working solution containing 8M Urea and 0.1M DTT. Refolding of denatured fusion protein was carried out in the presence of glutathione redox buffer. The refolded fusion protein was purified by size exclusion chromatography. The expressed fusion protein was verified by SDS-PAGE, western blotting with antibodies to both antigenic components of adhesin and cholera toxin subunit B (CTXB), and its N-terminal amino acid sequence was analyzed. The orderlyassembled fusion protein was confirmed by modified G_M1- ganglioside ELISA with Abs to adhesin. The results indicate that the purified fusion protein is an Adhesin/CTXA2B protein containing the H. pylori adhesin and G_m1-ganglioside binding activity of CTXB and the expressed fusion protein in E. coli could be easily purified by the refolding process. Its molecular weight was 168kDa as estimated by size exclusion chromatography. The Adhesin/CTXA2B protein may be used as a candidate antigen for oral immunization against H. pylori.
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