title: Large-Scale Refolding and Enzyme Reaction of Human Preproinsulin for Production of Human Insulin.
abstract: Human insulin is composed of 21 amino acids of an A-chain and 30 amino acids of a B-chain. This is the protein hormone that has the role of blood sugar control. When the recombinant human proinsulin is expressed in Escherichia coli, a serious problem is the formation of an inclusion body. Therefore, the inclusion body must be denatured and refolded under chaotropic agents and suitable reductants. In this study, H27R-proinsulin was refolded from the denatured form with β-mercaptoethanol and urea. The refolding reaction was completed after 15 h at 15°C, whereas the reaction at 25°C was faster than that at 15°C. The refolding yield at 15°C was 17% higher than that at 25°C. The refolding reaction could be carried out at a high protein concentration (2 g/l) using direct refolding without sulfonation. The most economical and optimal refolding condition for human preproinsulin was 1.5 g/l protein, 10 mM glycine buffer containing 0.6 M urea, pH 10.6, and 0.3 mM β-mercaptoethanol at 15°C for 16 h. The maximum refolding yield was 74.8% at 15°C with 1.5 g/l protein. Moreover, the refolded preproinsulin could be converted into normal mature insulin with two enzymes. The average amount of human insulin was 138.2 g from 200 L of fermentation broth after enzyme reaction with H27R-proinsulin. The direct refolding process for H27R-proinsulin was successfully set up without sulfonation. The step yields for refolding and enzyme reaction were comparatively high. Therefore, our refolding process for production of recombinant insulin may be beneficial to the large-scale production of other biologically active proteins.
authors.affiliation: Division of Animal Resources and Life Science, Sangji University, Wonju 220-702, Republic of Korea.
authors.forename: Chang-Kyu
authors.initials: CK
authors.lastname: Kim
authors.affiliation: Division of Animal Resources and Life Science, Sangji University, Wonju 220-702, Republic of Korea.
authors.forename: Seung-Bae
authors.initials: SB
authors.lastname: Lee
authors.affiliation: Department of Pharmacy, Sunchon National University, Jeonnam, Suncheon 540-742, Republic of Korea.
authors.forename: Young-Jin
authors.initials: YJ
authors.lastname: Son
mesh_terms: D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D011498:Protein Precursors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013678:Technology, Pharmaceutical / Q000379:methods; D013696:Temperature; D013997:Time Factors
chemical_list: D007328:Insulin; D011498:Protein Precursors; D011994:Recombinant Proteins; C013737:preproinsulin
keywords: enzyme reaction; pilot scale; preproinsulin; refolding