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Recombinant Glargine Insulin Production Process Using Escherichia coli.

Recombinant Glargine Insulin Production Process Using Escherichia coli.
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Hwang HG, Kim KJ, Lee SH, Kim CK, Min CK, Yun JM, Lee SU, Son YJ,


Hwang HG, Kim KJ, Lee SH, Kim CK, Min CK, Yun JM, Lee SU, Son YJ, (click to view)

Hwang HG, Kim KJ, Lee SH, Kim CK, Min CK, Yun JM, Lee SU, Son YJ,

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Journal of microbiology and biotechnology 26(10) 1781-1789 doi 10.4014/jmb.1602.02053
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 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.

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