• Journal of Microbiology and Biotechnology
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• v.26 no.10
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• pp.1781-1789
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• 2016

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 into Escherichia coli JM109. The transformed E. coli cells 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.

• Journal of Environmental Health Sciences
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• v.30 no.4
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• pp.314-319
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• 2004

Glycinin of more than $97\%$ purity was modified using maleic anhydride. Glycinin samples of $0\%,\;65\%,\;and\;95\%$ lysine residue modifications were used to determine the changes in turbidimetric characteristics of the protein due to maleylation. The solubility behavior of the protein as a function of pH was changed with maleylation. The isoelectric point of $65\%\;and\;95\%$ modified glycinin shifted to pH 4.0 and pH 3.5-4.0, respectively, as compared to pH 4.6 for native glycinin. Maleylated glycinins exhibited increased solubility at pH above 4.6. Turbidity of native glycinin decreased substantially by the addition of NaCl, but the stabilizing effect of NaCl decreased when the protein was chemically modified. The effect of NaCl on $65\%$ modified glycinin was intermediate between native glycinin and $95\%$ modified sample. Thermal aggregation of native glycinin was completed within 5 min of heating at $80^{\circ}C$. Maleylation contributed significantly to the thermostability of the protein at pH of 7.0 and 9.0, exhibiting little turbidity. Addition of NaCl suppressed thermal aggregation of native glycinin, but turbidity actually increased for the samples of $65\%\;and\;95\%$ modification.