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

摘要

Glargine is an analog of Insulin currently beingudproduced by recombinant DNA technology using twouddifferent hosts namely Escherichia coli and Pichiaudpastoris. Production from E. coli involves the steps ofudextraction of inclusion bodies by cell lysis, refolding,udproteolytic cleavage and purification. In P. pastoris, audsingle-chain precursor with appropriate disulfide bondingudis secreted to the medium. Downstream processing currentlyudinvolves use of trypsin which converts the precursorudinto two-chain final product. The use of trypsin in theudprocess generates additional impurities due to presence ofudLys and Arg residues in the Glargine molecule. In thisudstudy, we describe an alternate approach involving overexpressionudof endogenous Kex2 proprotein convertase,udtaking advantage of dibasic amino acid sequence (ArgArg)udat the end of B-chain of Glargine. KEX2 gene overexpressionudin Pichia was accomplished by using promotersudof varying strengths to ensure production of greaterudlevels of fully functional two-chain Glargine product,udconfirmed by HPLC and mass analysis. In conclusion,udthis new production process involving Kex2 proteaseudover-expression improves the downstream process efficiency,udreduces the levels of impurities generated anduddecreases the use of raw materials