caa a22 4500 605497877 CHVBK 20210128100543.0 cr unu---uuuuu 210128e20150101xx s 000 0 eng 10.1007/s00253-014-6052-5 doi (NATIONALLICENCE)springer-10.1007/s00253-014-6052-5 Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris [Elektronische Daten] [Suma Sreenivas, Sateesh Krishnaiah, Nagaraja Govindappa, Yogesh Basavaraju, Komal Kanojia, Niveditha Mallikarjun, Jayaprakash Natarajan, Amarnath Chatterjee, Kedarnath Sastry] 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. Springer-Verlag Berlin Heidelberg, 2014 Two-chain Glargine nationallicence Kex2 protease nationallicence Over-expression nationallicence Pichia pastoris nationallicence Sreenivas Suma Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut Krishnaiah Sateesh Molecular Diagnostics Laboratory, Dept. of Microbiology & Biotechnology, Bangalore University, JnanaBharathi Campus, 560056, Bangalore, Karnataka, India aut Govindappa Nagaraja Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut Basavaraju Yogesh Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut Kanojia Komal Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut Mallikarjun Niveditha Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut Natarajan Jayaprakash Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut Chatterjee Amarnath Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut Sastry Kedarnath Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/1(2015-01-01), 327-336 0175-7598 99:1<327 2015 99 253 https://doi.org/10.1007/s00253-014-6052-5 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-014-6052-5 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Sreenivas Suma Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut NATIONALLICENCE 700 1- Krishnaiah Sateesh Molecular Diagnostics Laboratory, Dept. of Microbiology & Biotechnology, Bangalore University, JnanaBharathi Campus, 560056, Bangalore, Karnataka, India aut NATIONALLICENCE 700 1- Govindappa Nagaraja Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut NATIONALLICENCE 700 1- Basavaraju Yogesh Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut NATIONALLICENCE 700 1- Kanojia Komal Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut NATIONALLICENCE 700 1- Mallikarjun Niveditha Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut NATIONALLICENCE 700 1- Natarajan Jayaprakash Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut NATIONALLICENCE 700 1- Chatterjee Amarnath Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut NATIONALLICENCE 700 1- Sastry Kedarnath Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, 560099, Bangalore, Karnataka, India aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/1(2015-01-01), 327-336 0175-7598 99:1<327 2015 99 253