pMAF10載體：

表達PglB質(zhì)粒

Functional Expression of PglB.

In our earlier work, we showed that coexpression of the complete C. jejuni pgl locus and AcrA, a periplasmic component of a multidrug efflux pump, resulted in N-glycosylated AcrA inE. coli. Replacement of PglB by PglBmut (PglB with amino acid substitutions W458A and Y459A), abolished glycosylation (6). We have now constructed pMAF10, a plasmid that expresses an HA-tagged version of PglB, and pWA1, an analogous plasmid that expresses PglBmut. In both cases, PglB expression was controlled by an arabinose-inducible promoter. Production of PglB was verified by Western blot (data not shown). To show that HA-tagged PglB was fully functional, we introduced pMAF10 or pWA1 in E. coli cells carrying the pgl cluster expressing PglBmut, and a soluble form of a C-terminal His6-tagged AcrA. Introduction of pMAF10, but not pWA1, resulted in production of glycosylated AcrA at comparable levels to those of cells expressing the wild-type pgl locus