Heterogenous expression and characterization of cag4 in Helicobacter pylori

Abstract:

Abstract:［Objective］To construct prokaryotic fusion gene expression vector pET-28a- cag pathogenicity island protein 4(cag4)，express the recombinant fusion protein cag4 and analyze the enzyme activity of recombinant protein.［Methods］We cloned cag4 gene, this encoded lytic transglycosylase in CagPAI of Helicobacter pylori NCTC11637 via PCR method. After TA cloning and restriction enzyme digested confirmed, we constructed prokaryotic expression plasmid pET-28a- cag4 and transformed the plasmid into E.coli BL21 (DE3) for heterogenesis expression after it is sequenced. We performed SDS-PAGE and Western blot. We purified and collected the recombinant protein by using Ni2＋-NTA columns under denaturation condition.We renatured the recombinant protein via dialysis. Besides, we isolated peptidoglycan from Micrococcus lysodeikticus by SDS boiling method.We detected the enzyme activity of recombinant protein by zymogram analysis and the effect of pH values by turbidimetric analysis.［Results］The recombinant protein has the enzyme activity of lytic transglycosylase.The enzyme activity, which defined as the rate of degraded (DA?min-1mg-1), was higher at pH 6.0 group than other groups (pH 5.0 and 7.0).［Conclusion］The cag4 of Helicobacter pylori NCTC11637 has the enzyme activity of lytic transglycosylase.

Key words:Keywords: Helicobacter pylori; Ⅳ type secretion system; cag4 protein; Heterogenous expression; activity analysis