title: Electrostatic interaction-induced inclusion body formation of glucagon-like peptide-1 fused with ubiquitin and cationic tag.
abstract: In an attempt to produce glucagon-like peptide-1 (GLP-1) using recombinant Escherichia coli, ubiquitin (Ub) as a fusion partner was fused to GLP-1 with the 6-lysine tag (K6) for simple purification. Despite the high solubility of ubiquitin, the fusion protein K6UbGLP-1 was expressed mainly as insoluble inclusion bodies in E. coli. In order to elucidate this phenomenon, various N- and C-terminal truncates and GLP-1 mutants of K6UbGLP-1 were constructed and analyzed for their characteristics by various biochemical and biophysical methods. The experiment results obtained in this study clearly demonstrated that the insoluble aggregation of K6UbGLP-1 was attributed to the electrostatic interaction between the N-terminal 6-lysine tag and the C-terminal GLP-1 before the completion of folding which might be one of the reasons for protein misfolding frequently observed in many foreign proteins introduced with charged amino acid residues such as the His tag and the protease recognition sites. The application of a cation exchanger for neutralizing the positive charge of the 6-lysine tag in solid-phase refolding of K6UbGLP-1 successfully suppressed the electrostatic interaction-driven aggregation even at a high protein concentration, resulting in properly folded K6UbGLP-1 for GLP-1 production.
authors.affiliation: Department of Biomedical Science, Youngdong University, Chungbuk 370-701, Republic of Korea.
authors.forename: Sung-Gun
authors.initials: SG
authors.lastname: Kim
authors.forename: Won-Ki
authors.initials: WK
authors.lastname: Min
authors.forename: Yong-Taek
authors.initials: YT
authors.lastname: Rho
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Seo
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003956:Dialysis; D004926:Escherichia coli; D052216:Glucagon-Like Peptide 1 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D002479:Inclusion Bodies / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011107:Polylysine / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D012995:Solubility; D055672:Static Electricity; D025801:Ubiquitin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D011107:Polylysine; D052216:Glucagon-Like Peptide 1