2 research outputs found
Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris
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 overexpression
of endogenous Kex2 proprotein convertase,
taking advantage of dibasic amino acid sequence (ArgArg)
at the end of B-chain of Glargine. KEX2 gene overexpression
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 material