title: Recombinant Glargine Insulin Production Process UsingEscherichia coli.
abstract: Glargine insulin is a long-acting insulin analog that helps blood glucose maintenance in patients with diabetes. We constructed the pPT-GI vector to express prepeptide glargine insulin when transformed intoEscherichia coliJM109. The transformedE. colicells were cultured by fed-batch fermentation. The final dry cell mass was 18 g/l. The prepeptide glargine insulin was 38.52% of the total protein. It was expressed as an inclusion body and then refolded to recover the biological activity. To convert the prepeptide into glargine insulin, citraconylation and trypsin cleavage were performed. Using citraconylation, the yield of enzymatic conversion for glargine insulin increased by 3.2-fold compared with that without citraconylation. After the enzyme reaction, active glargine insulin was purified by two types of chromatography (ion-exchange chromatography and reverse-phase chromatography). We obtained recombinant human glargine insulin at 98.11% purity and verified that it is equal to the standard of human glargine insulin, based on High-performance liquid chromatography analysis and Matrix-assisted laser desorption/ionization Time-of-Flight Mass Spectrometry. We thus established a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. This established process for recombinant human glargine insulin may be a model process for the production of other human insulin analogs.
authors.affiliation: Department of Pharmacy, Sunchon National University, Suncheon 57922, Republic of Korea.
authors.forename: Hae-Gwang
authors.initials: HG
authors.lastname: Hwang
authors.affiliation: Department of Pharmacy, Sunchon National University, Suncheon 57922, Republic of Korea.|Research Institute of Life and Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea.
authors.forename: Kwang-Jin
authors.initials: KJ
authors.lastname: Kim
authors.affiliation: Department of Pharmacy, Sunchon National University, Suncheon 57922, Republic of Korea.
authors.forename: Se-Hoon
authors.initials: SH
authors.lastname: Lee
authors.affiliation: Division of Animal Resources and Life Science, Sangji University, Wonju 26339, Republic of Korea.
authors.forename: Chang-Kyu
authors.initials: CK
authors.lastname: Kim
authors.affiliation: Department of Integrated Biotechnology, Sogang University, Seoul 04107, Republic of Korea.
authors.forename: Cheol-Ki
authors.initials: CK
authors.lastname: Min
authors.affiliation: Department of Food and Nutrition, Chonnam National University, Gwangju 61186, Republic of Korea.
authors.forename: Jung-Mi
authors.initials: JM
authors.lastname: Yun
authors.affiliation: Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology, Cheongju 56212, Republic of Korea.
authors.forename: Su Ui
authors.initials: SU
authors.lastname: Lee
authors.affiliation: Department of Pharmacy, Sunchon National University, Suncheon 57922, Republic of Korea.|Research Institute of Life and Pharmaceutical Sciences, Sunchon National University, Suncheon 57922, Republic of Korea.|Suncheon Research Center for Natural Medicines, Suncheon 57922, Republic of Korea.
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Son
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005285:Fermentation; D006801:Humans; D007328:Insulin / Q000235:genetics*; D000069036:Insulin Glargine / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D058849:Protein Refolding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D007328:Insulin; D011994:Recombinant Proteins; D000069036:Insulin Glargine
keywords: Arg (B31)-insulin; Diabetes; citraconylation; fed-batch fermentation; glargine