The wzy/rfc gene, encoding the O-antigen polymerase, of Salmonella enterica serovar Typhimurium has been previously cloned and sequenced. In the present work, the wzy transcriptional startpoint was initially identified by primer extension. Next, wzy promoter strength in Escherichia coli K-12 was measured, and was found to be greater than that of the induced lac promoter. To define the Wzy translational startpoint, DNA including the wzy promoter and the putative first five residues of the Wzy protein was fused to the Nterminus of glutathione-S-transferase, and the fusion protein purified by affinity chromatography. N-terminal amino acid sequencing yielded the Wzy translational startpoint. Next, the Wzy protein was C-terminally tagged with the FLAG peptide, and immunoblotting of an S. typhimurium strain expressing a low-copy wzy-FLAG gene (five copies per cell) localized the intact Wzy protein in the cytoplasmic membrane of S. typhimurium cells. The Wzy protein was not well-expressed from a multi-copy wzy-FLAG M plasmid in S. typhimurium , or in E. coli K-12.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.