Objective: The aim of this study was to further increase the yield of insulin precursor by Pichia pastoris.
Methods: For this, we transformed the expression vector pPICZalpha-IP into P. pastoris X-33 using electroporation and screened two mutant strains B4 and S6 on the YPD plate containing 100 microg/mL zeocin. Both could overexpress human insulin precursor. Taking B4 and S6 as start strains, we repeatedly transformed SacI linearizing pPICZalpha-IP into P. pastoris X-33 by electroporation, then screened a new mutant strain 2B4 (with 7 copies) on the 1000 microg/mL zeocin YPD plate.
Results: After cultivation, the human insulin precursor yield of 2B4 strain was 2.7 fold higher than that of B7. Meanwhile, the cell growth was not inhibited. The target gene transcription level of 2B4 was 2 fold higher than that of B7 by real-time quantification PCR.
Conclusion: The strategy of combining resistance screening and repeated electroporation was efficient to increase the copy number of target gene, so as to facilitate higher transcription level and enhance objective recombinant protein yield.