Enhancement of recombinant monomeric insulin production in methylotrophic yeasts by increasing copy number of gene

Abstract

Currently, methylotrophic yeast expression system has been developed and widely used for recombinant proteins production. In this research, Pichia pastoris strains, X-33, GS115, KM71H, and Hansenula polymorpha NRRL2214, 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, respectively, were successfully constructed in pPICZαA expression vector and transformed into the hosts. 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 recombinant P. pastoris KM71H 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. Effect of host strains on the MIP expression level was comparison of MIP concentration between recombinant strains which harboring 1 copy of MIP gene which cultured in MMH for 72 hours, P. pastoris KM71H (MutS phenotype) 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 and the expression level was unrelated with gene copy number.