Expression and Characterization of Helicobacter pylori Adhesin Protein Linked to Cholera Toxin A2/B Subunits in Escherichia coli
2000
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 8 M Urea and 0.1 M 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 orderly-assembled 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 168 kDa as estimated by size exclusion chromatography. The Adhesin/CTXA2B protein may be used as a candidate antigen for oral immunization against H. pylori.
Keywords:
- Correction
- Source
- Cite
- Save
- Machine Reading By IdeaReader
0
References
1
Citations
NaN
KQI