Enhancement of recombinant monomeric insulin production in pichia pastoris strains by increasing copy number of gene

Abstract

Currently, Pichia pastoris expression system has been developed and widely used for recombinant proteins production. In this research, P. pastoris 3 strains (X-33, GS115 and KM71H) and Hansenula polymorpha (NRRL2214), which are methylotrophic yeasts, were used as hosts for recombinant monomeric insulin production. Recombinant plasmids, TP1, TP2, TP4, which have 1, 2 and 4 copy(s) of monomeric insulin precursor (MIP) gene, were successfully constructed in pPICZalphaA expression vector with the size of 3,709 bp, 5,501 bp and 9,085 bp, respectively. The recombinant yeasts which harboring TP1, TP2, TP4 plasmids which integrated into their genome were cultured in two steps; the first step in complex medium for cell production and the second step in minimal methanol histidine (MMH) medium for inducing the expression of MIP. A simple and specific dot-blotting technique was chose to monitor the expression level while indirect competitive ELISA was used to quantitatively determine the MIP concentration. By dot-blot analysis, the MIP expression of recombinants P. pastoris KM71H (MutS phenotype) could be detected since 24 hours in an induction phase while those of other recombinants were detected at 48 or 72 hours. By indirect competitive ELISA, results showed that the MIP expression progressively increased together with the time of induction. Comparison of the MIP expression between yeast strains which harbored 1 copy of MIP gene in culture at 72 hours, P. pastoris KM71H has the highest MIP concentration (4.19±0.96 mg.L-1), following by P. pastoris GS115 (2.69±0.48 mg.L-1), P. pastoris X-33 (0.93±0.08 mg.L-1) and H. polymorpha (0.04±0.01 mg.L-1). In view of gene copy number, we found that recombinant yeast strains which differ in gene copy number have different expression level of the MIP.