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

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• 作者：Suma Sreenivas (1)
  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)
  
• 关键词：Two ; chain Glargine ; Kex2 protease ; Over ; expression ; Pichia pastoris
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2015
• 出版时间：January 2015
• 年：2015
• 卷：99
• 期：1
• 页码：327-336
• 全文大小：907 KB
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  12. Olsen JV, Ong SE, Mann M (2004) Trypsin cleaves exclusively C-terminal to arginine and lysine residues. Mol Cell Proteomics 3(6):608鈥?14 mcp.T400003-MCP200" target="_blank" title="It opens in new window">CrossRef
  13. Poon K, King AB (2010) Glargine and detemir: safety and efficacy profiles of the long-acting basal insulin analogs. Drug Healthc Patient Saf 2:213鈥?23
  14. Seeboth PG, Hein J (1991) In-vitro processing of yeast alpha factor leader fusion proteins using a soluble yscF (Kex2) variant. Appl Microbiol Biotechnol 35:771鈥?76 CrossRef
  15. Seidah NG, Gaspar L, Mion P, Marcinkiewicz M, Mbikay M, Chrttien M (1990) cDNA sequence of two distinct pituitary proteins homologous to kex2 and furin gene products: tissue specific mRNAs encoding candidates for prohormone processing proteinases. DNA Cell Biol 9:415鈥?24 CrossRef
  16. Seidah NG, Marcinkiewicz M, Benjannet S, Gaspar L, Beaubien G, Mattei MG, Lazure C, Mbikay M, Chrdtien M (1991) Cloning and primary sequence of a mouse candidate prohormone convertase PC1 homologous to PC2, Furin and kex2: distinct chromosomal localization and messenger RNA distribution in brain and pituitary compared to PC2. Mol Endocrinol 5:111鈥?22 mend-5-1-111" target="_blank" title="It opens in new window">CrossRef
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  22. Yang S, Kuang Y, Li H, Liu Y, Hui X, Li P, Jiang Z, Zhou Y, Wang Y, Xu A, Li S, Liu P, Wu D (2013) Enhanced production of recombinant secretory proteins in / Pichia pastoris by optimizing Kex2 P1鈥?site. PLoS ONE 8(9):e75347. doi:10.1371/journal.pone.0075347 CrossRef
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  24. Zimmerman RE, Stokell DJ, Akers MP (2012) Glargine proinsulin and methods of producing glargine insulin analogs therefrom. US 20120214965 A1
  
• 作者单位：Suma Sreenivas (1)
  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. Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, Bangalore, 560099, Karnataka, India
  2. Molecular Diagnostics Laboratory, Dept. of Microbiology & Biotechnology, Bangalore University, JnanaBharathi Campus, Bangalore, 560056, Karnataka, India
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Biotechnology
  Microbiology
  Microbial Genetics and Genomics
  
• 出版者：Springer Berlin / Heidelberg
• ISSN：1432-0614

文摘

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.