title: Detection of soluble expression and in vivo interactions of the inner membrane protein OppC using green fluorescent protein.
abstract: In this study, the in vivo interaction system of oligopeptide permease (Opp) proteins was analyzed, and a high expression system of inner membrane protein OppC was constructed by flexible usage of the green fluorescent protein (GFP). The Escherichia coli OppC gene, which encodes a transmembrane component of oligopeptide transporter, was cloned into different vectors. Recombinant plasmids were transformed into different E. coli strains, and the expression conditions were optimized. The effect of plasmids and expression strains on OppC production was evaluated by in-gel and western blot analyses. OppC produced by the pWaldo-GFPe vector, harboring the GFP reporter gene, transformed into E. coli C43(DE3) provided sufficient functional protein for biochemical and biophysical studies. In vivo protein-protein interactions were detected among oligopeptide permease proteins using a GFP fragment reassembly protocol. The substrate binding protein OppA showed no interaction with the other components, while the ATP-binding component OppD did not interact with OppF. OppD and OppF interacted with the transmembrane components OppB and OppC. OppB also showed direct interaction with OppC. In vivo OppC functionality was determined by constructing an OppC gene deletion strain. OppC was shown to be essential for peptide uptake, and non-essential for cell viability. These results could help in elucidating the oligopeptide transport mechanism in bacteria.
authors.affiliation: Department of Microbiology, College of Resource and Environmental Sciences, Sichuan Agricultural University, Chengdu, China.|College of Life Sciences, Sichuan Key Laboratory of Molecular Biology and Biotechnology, Sichuan University, Chengdu, China.
authors.forename: Q J
authors.initials: QJ
authors.lastname: Xiang
authors.affiliation: Department of Gynecology, Central Hospital of Xuzhou, Affiliated Hospital of Southeast University, Xuzhou, China.
authors.forename: J F
authors.initials: JF
authors.lastname: Zhai
authors.affiliation: Department of Gynecology, Central Hospital of Xuzhou, Affiliated Hospital of Southeast University, Xuzhou, China.
authors.forename: M
authors.initials: M
authors.lastname: Zhang
authors.affiliation: Department of Gynecology, Central Hospital of Xuzhou, Affiliated Hospital of Southeast University, Xuzhou, China.
authors.forename: B
authors.initials: B
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D002470:Cell Survival / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011485:Protein Binding
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C043426:inner membrane protein, E coli; D049452:Green Fluorescent Proteins; C037230:oligopeptide permease, Bacteria