Abstract

Diabetes is one of the diseases, which have recently gained significant attention. In order to produce the recombinant insulin for treating diabetics, we have cloned and expressed a miniproinsulin in Pichia pastoris. The DNA fragment containing a gene encoding the fusion protein of miniproinsulin-6xhis was cloned into the integrative plasmid pPICZαA resulting in pPICZαA/h-mpi to construct the strain P. pastoris GS115::h-mpi expressing the recombinant MPI as secreted protein into culture. Phenotype and genotype of P. pastoris GS115::h-mpi were confirmed by PCR and restriction pattern. MPI was expressed as a fusion protein with 6xhis tag by supplementing methanol at a final concentration of 0.5% in BMMY culture medium for 96 hours after inducing.