[논문]Recombinant Glargine Insulin Production Process Using Escherichia coli

Hwang, Hae-Gwang; Kim, Kwang-Jin; Lee, Se-Hoon; Kim, Chang-Kyu; Min, Cheol-Ki; Yun, Jung-Mi; Lee, Su Ui; Son, Young-Jin

doi:10.4014/jmb.1602.02053

Recombinant Glargine Insulin Production Process Using Escherichia coli 원문보기

Journal of microbiology and biotechnology, v.26 no.10, 2016년, pp.1781 - 1789

Hwang, Hae-Gwang (Department of Pharmacy, Sunchon National University) , Kim, Kwang-Jin (Department of Pharmacy, Sunchon National University) , Lee, Se-Hoon (Department of Pharmacy, Sunchon National University) , Kim, Chang-Kyu (Division of Animal Resources and Life Science, Sangji University) , Min, Cheol-Ki (Department of Integrated Biotechnology, Sogang University) , Yun, Jung-Mi (Department of Food and Nutrition, Chonnam National University) , Lee, Su Ui (Natural Medicine Research Center, Korea Research Institute of Bioscience and Biotechnology) , Son, Young-Jin (Department of Pharmacy, Sunchon National University)

Abstract ▼ AI-Helper

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.

주제어

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제안 방법

The purpose of this study was to establish a production process for high-purity recombinant human glargine insulin and a method to block Arg (B31)-insulin formation. We expressed a large amount of prepeptide fusion glargine insulin in E.

성능/효과

Using deacylation, the natural glargine insulin was recovered from modified glargine insulin after the enzyme reaction. Because of citraconylation, the formation of Arg (B31)-insulin was reduced and the enzymatic conversion yield of glargine insulin increased by 3.2-fold compared with that without citraconylation.
Even though the retention times of the purified recombinant glargine insulin and Lantus (as a standard) were not consistent, we considered that the final purified glargine insulin was equal to the standard because the chromatogram of the mixture showed one peak (Fig
It is considered that the cleavage substrate 1 for trypsin, peptide 1 – Arg – Arg - peptide 2 was more sensitive than the cleavage substrate 2 for trypsin, peptide 1 – Arg – Glu - peptide 2. However, the modification of prepeptide fusion glargine with citraconylation changed the substrate sensitivity, so formation of the impurity was decreased dramatically. The adequate amount of addition of citraconic anhydride was 4.
We performed SDS-PAGE to determine the time course of expression during cell culture; a sample was collected at each time point after raising the temperature. The SDS-PAGE analysis showed that the expression of prepeptide fusion glargine was significantly increased after raising the temperature to 37℃ (Fig. 2B).
The purity of the final glargine insulin product was 98.11% (included 1.96% desamido insulin) based on the HPLC chromatogram from a Protein & Peptide C4 analytical column (Fig
5 for insulin glargine. The recovery of insulin glargine was 92.3% and the content of the impurity was a minimum at pH 4.5.
That meant the prepeptide fusion glargine was more hydrophobic than the prepeptide fusion insulin [37]. Therefore prepeptide fusion glargine had different characteristics for refolding and protease cleavage compared with human insulin. Citraconylation is the adoption of one or more citraconyl groups into a substance by the acylation reaction.
Arg (B31)-insulin is very similar to glargine insulin in physicochemical characteristics, so it is hard to separate during the purification process. Therefore, blocking Arg (B31)-insulin formation during the enzymatic modification is requisite in order to develop a cost-effective process for glargine insulin production. To minimize the amount of Arg (B31)-insulin, we performed citraconylation and deacylation during trypsin cleavage of recombinant prepeptide fusion glargine.

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