Production and Characterization of Recombinant Insulin in Pichia pastoris GS115

• Main Author: Farabi, Irfan
• Format: Final Project
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/37921
• Institution: Institut Teknologi Bandung

Insulin is a polypeptide hormone that has a major function to regulate blood sugar level. Defects in insulin secretion system, insulin activities and insulin receptor activities can lead to diabetes mellitus. In Indonesia, there were approximately 8.5 million people suffered from diabetes mellitus in 2013. The recombinant insulin used for diabetes mellitus therapy is obtained from foreign pharmaceutical companies. Therefore, as an effort to initiate the availability of national human recombinant insulin, this research was directed to produce and characterize recombinant human insulin precursor in yeast Pichia pastoris GS115. A recombinant plasmid pICZ?A-HIns containing synthetic human insulin precursor gene under the regulation of by AOX1 promoter and fused with oligonucleotide encoding MF-? signal sequence was transformed into P. pastoris GS115. The P. pastoris GS115 transformants were first screened on medium containing various zeocin concentration (100 to 2000 µg/mL) and then followed by colony PCR to identify the presence of human insulin precursor expression cassette. The resulted P. pastoris GS115 transformants carrying the human insulin precursor with muts (methanol utilization slow) phenotype and capable of growing up to 2000 µg/mL zeocin were then subjected to expression study. SDS-PAGE analysis of P. pastoris GS115-HIns culture supernatant showed that the recombinant human insulin precursor was secreted with molecular weight of ?8 kDa. Furthermore, the highest human insulin precursor production was observed after four days by the addition of 3% methanol.