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Applied Microbiology and Biotechnology

, Volume 99, Issue 1, pp 327–336 | Cite as

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

  • Suma SreenivasEmail author
  • Sateesh M. Krishnaiah
  • Nagaraja Govindappa
  • Yogesh Basavaraju
  • Komal Kanojia
  • Niveditha Mallikarjun
  • Jayaprakash Natarajan
  • Amarnath Chatterjee
  • Kedarnath N. Sastry
Applied genetics and molecular biotechnology

Abstract

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 over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression 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.

Keywords

Two-chain Glargine Kex2 protease Over-expression Pichia pastoris 

Notes

Acknowledgments

We thank Molecular Biology group of R&D for their support and suggestions. We thank Biocon Research Limited for supporting this work.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Suma Sreenivas
    • 1
    Email author
  • Sateesh M. Krishnaiah
    • 2
  • Nagaraja Govindappa
    • 1
  • Yogesh Basavaraju
    • 1
  • Komal Kanojia
    • 1
  • Niveditha Mallikarjun
    • 1
  • Jayaprakash Natarajan
    • 1
  • Amarnath Chatterjee
    • 1
  • Kedarnath N. Sastry
    • 1
  1. 1.Biocon Research LimitedBangaloreIndia
  2. 2.Molecular Diagnostics Laboratory, Dept. of Microbiology & BiotechnologyBangalore UniversityBangaloreIndia

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