title: Production and solid-phase refolding of human glucagon-like peptide-1 using recombinant Escherichia coli.
abstract: Human glucagon-like peptide-1 (GLP-1), an incretin hormone with pharmaceutical potential in treating type 2 diabetes mellitus is known to be rapidly degraded when expressed in Escherichia coli. For the efficient production of the intact GLP-1 using a recombinant E. coli system, a fusion protein of GLP-1 was designed to be composed of the 6-lysine tag, ubiquitin and GLP-1 (K6UbGLP-1) in a row. A fed-batch fermentation of recombinant E. coli BL21(DE3)/pAPK6UbGLP-1 was carried out in a pH-stat feeding strategy and resulted in 11.3 g/L of K6UbGLP-1 as a form of inclusion body. Solid-phase refolding of K6UbGLP-1 inclusion body using a cation exchanger of the SP Sepharose FF led to a refolding yield over 90% even at 5.2 mg protein/mL resin. On-column cleavage of the refolded K6UbGLP-1 with ubiquitin-specific protease 1 gave an authentic form of GLP-1. Instrumental analyses using mass spectrometry and reverse-phase HPLC showed that the recombinant GLP-1 released from K6UbGLP-1 was identical to the standard GLP-1.
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: So-Yeon
authors.initials: SY
authors.lastname: Shin
authors.forename: Yong-Cheol
authors.initials: YC
authors.lastname: Park
authors.forename: Chul-Soo
authors.initials: CS
authors.lastname: Shin
authors.forename: Jin-Ho
authors.initials: JH
authors.lastname: Seo
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D056148:Chromatography, Reverse-Phase; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D052216:Glucagon-Like Peptide 1 / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D002479:Inclusion Bodies; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics
chemical_list: D011993:Recombinant Fusion Proteins; D052216:Glucagon-Like Peptide 1