Transformation and characterization of human insulin precursor gene in Pichia pastoris X-33

The case of diabetes increases significantly and has been projected to reach 592 million people in 2035. Consequently, the necessity of insulin will rise manifold and an efficient production system for insulin production is required to meet the market demands. The human insulin precursors that enzymatically converted to human insulin can be produced using Escherichia coli, Saccharomyces cerevisiae, or Pichia pastoris. In this study, Pichia pastoris is used for production human insulin precursor because the resulting recombinant protein can be folded accordingly and secreted to the external environment of the cell that simplifies the purification process. The study was initiated with the insertion of a synthetic gene of human insulin precursor into the pPICZαA to create recombinant pPICZαA-IP plasmid. The recombinant plasmid was transformed into Escherichia coli Top10 which then isolated and digested by the SacI enzyme. The linearize pPICZαA-IP plasmid was transfected into Pichia pastoris X-33 by electroporator. The result of transformation process, a total of 20 colonies of P. pastoris X-33 were selected and inoculated in YPD agar medium containing Zeocin. The two colonies of P. pastoris were characterized by PCR and sequencing showed that the recombinant pPICZαA-IP plasmid was successfully integrated into selected colonies of P. pastoris.