title: Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris.
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.
authors.affiliation: Biocon Research Limited, Plot No.2&3, Phase IV, Bommasandra-Jigani Link Road, Bangalore, 560099, Karnataka, India, suma.sreenivas@biocon.com.
authors.forename: Suma
authors.initials: S
authors.lastname: Sreenivas
authors.forename: Sateesh M
authors.initials: SM
authors.lastname: Krishnaiah
authors.forename: Nagaraja
authors.initials: N
authors.lastname: Govindappa
authors.forename: Yogesh
authors.initials: Y
authors.lastname: Basavaraju
authors.forename: Komal
authors.initials: K
authors.lastname: Kanojia
authors.forename: Niveditha
authors.initials: N
authors.lastname: Mallikarjun
authors.forename: Jayaprakash
authors.initials: J
authors.lastname: Natarajan
authors.forename: Amarnath
authors.initials: A
authors.lastname: Chatterjee
authors.forename: Kedarnath N
authors.initials: KN
authors.lastname: Sastry
mesh_terms: D001709:Biotechnology / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D015870:Gene Expression; D000069036:Insulin Glargine; D049528:Insulin, Long-Acting / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013678:Technology, Pharmaceutical / Q000379:methods*
chemical_list: D049528:Insulin, Long-Acting; D011994:Recombinant Proteins; D000069036:Insulin Glargine; D010447:Peptide Hydrolases