Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris


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Suma Sreenivas, . and Sateesh, M. and Krishnaiah, . and Nagaraja Govindappa, . and Yogesh Basavaraju, . and Komal Kanojia, . and Niveditha Mallikarjun, . and Jayaprakash Natarajan, . and Amarnath Chatterjee, . and Kedarnath N. Sastry, . (2015) Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris. Applied Microbiology and Biotechnology, 99 (1). pp. 327-336. ISSN 1432-0614

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Official URL: https://doi.org/10.1007/s00253-014-6052-5


Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving overexpression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (ArgArg) at the end of B-chain of Glargine. KEX2 gene overexpression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials

Item Type: Article
Uncontrolled Keywords: Two-chain GlargineKex2 proteaseOver-expressionPichia pastoris
Subjects: Faculty of Science > Environmental and Biological Sciences > Microbiology & Biotechnology
Divisions: Jnana Bharathi / Central College Campus > Department of Microbiology and Biotechnology
Depositing User: Mr H.G. Srikanth
Date Deposited: 30 Aug 2016 05:54
Last Modified: 30 Aug 2016 05:54
URI: http://eprints-bangaloreuniversity.in/id/eprint/4946

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