title: Production of disulfide bond-rich peptides by fusion expression using small transmembrane proteins of Escherichia coli.
abstract: Recombinant expression in Escherichia coli allows the simple, economical, and effective production of bioactive peptides. On the other hand, the production of native peptides, particularly those rich in disulfide bonds, is a major problem. Previous studies have reported that the use of carrier proteins for fusion expression can result in good peptide yields, but few are folded correctly. In this study, two transmembrane small proteins in E. coli, YoaJ and YkgR, which both orientate with their N-termini in cytoplasm and their C-termini in periplasm, were used for fusion expression. The recombinant production of two peptides, asteropsin A (ASPA) and β-defensin (BD), was induced in the periplasm of E. coli using a selected carrier protein. Both peptides were expressed at high levels, at yields of approximately 5-10 mg/L of culture. Mass spectrometry showed that the resulting peptide had the same molecular weight as their natural forms. After purification, single peaks were observed by reversed phase high-performance liquid chromatography (RP-HPLC), demonstrating the absence of isoforms. Furthermore, cytoplasmically expressed fusion proteins with a carrier at their C-termini did not contain disulfide bonds. This study provides new carrier proteins for fusion expression of disulfide bond-rich peptides in E. coli.
authors.affiliation: Department of Chemistry and Chemistry Institute of Functional Materials, Pusan National University, Busan, 609-735, Korea.
authors.forename: Ziwei
authors.initials: Z
authors.lastname: Chang
authors.forename: Ming
authors.initials: M
authors.lastname: Lu
authors.forename: Yunqi
authors.initials: Y
authors.lastname: Ma
authors.forename: Dong-Geon
authors.initials: DG
authors.lastname: Kwag
authors.forename: Seo-Hyun
authors.initials: SH
authors.lastname: Kim
authors.forename: Ji-Min
authors.initials: JM
authors.lastname: Park
authors.forename: Bo-Hye
authors.initials: BH
authors.lastname: Nam
authors.forename: Young-Ok
authors.initials: YO
authors.lastname: Kim
authors.forename: Cheul-Min
authors.initials: CM
authors.lastname: An
authors.forename: Huayue
authors.initials: H
authors.lastname: Li
authors.forename: Jee H
authors.initials: JH
authors.lastname: Jung
authors.forename: Jang-Su
authors.initials: JS
authors.lastname: Park
mesh_terms: D004220:Disulfides / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D019897:Periplasm / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D023083:beta-Defensins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D004220:Disulfides; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C581719:asteropsin A; D023083:beta-Defensins