Stability analysis of a Pichia pastoris recombinant clone expressing human insulin precursor

Abstract

Abstract Current recombinant human insulin production utilizes two major expression systems, Escherichia coli and Saccharomyces cerevisiae-based expression systems. Methylotropic yeast, Pichia pastoris, has appeared as a promising alternate yeast recombinant host for insulin precursor (IP) expression due to its ability to produce high titers, efficient secretion, and growth to very high cell densities. Similar to the S. cerevisiae system, P. pastoris secreted soluble IP into the culture supernatant. In the previous study, we have established P. pastoris recombinant clones harboring synthetic insulin precursor (IP) expression cassette integrated into their genomes through homologous recombination. It is essential to verify that the expression cassettes of the IP encoding gene remain stably integrated with the genome during such prolonged methanol induction. Therefore, we aimed to analyze the stability of one recombinant clone (CL-4) expressing the human insulin precursor by verifying the stable integration of the IP expression cassette into the genome by PCR, and the IP protein expression after prolonged methanol induction over 70 generations. We found that the expression cassette was stably integrated into the genome of the CL-4 recombinant clone and the IP expression was sustained after 72 generations of cultivations in the culture and induction media without antibiotic selection.