OPTIMIZATION OF RECOMBINANT HUMAN INSULIN PRECURSOR PRODUCTION IN PICHIA PASTORIS KM71

• Main Author: Nelson Anwar, Stefanus
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/33693
• Institution: Institut Teknologi Bandung

The number of people with diabetes in the world has reached almost 400 million, and Indonesia has become the second highest country for prevalence of diabetes in the Southeast Asia. As the number of diabetics always increases, in turn the insulin demand for diabetes therapy will also increase. The objective of this research was to construct a yeast Pichia pastoris KM71 which carries human insulin precursor gene (HIns) and to produce recombinant human insulin precursor in Pichia pastoris KM71. Pichia pastoris KM71 was transformed by pPICZ?-HIns plasmid which contains synthetic human insulin precursor gene encoding a polypeptide of 63 amino acid residues. Pichia pastoris KM71 transformants carrying multiple copies of HIns gene were selected on YPD (Yeast extract-Peptone- Dextrose) plates containing 500, 1,000, and 2,000 µg/mL Zeocin. The presence of HIns gene in P. pastoris KM71 chromosome was then evaluated by PCR (Polymerized Chain Reaction). The resulted 676 base pairs DNA fragment from PCR represented the size of HIns gene. Recombinant human insulin precursor expression was induced by addition of 2% methanol every 24 hours for seven days. Tricine SDS-PAGE analysis showed that the yeast Pichia pastoris KM71-HIns secreted Hins. The secreted Hins seemed to be gylosylated as judged by its molecular weight (?8 kDa) which is higher than the deduced amino acid sequence. Insulin precursor expression profiles of five Pichia pastoris KM71-HIns showed variation of expression levels which were subjected to induction periods.