Production and Characterization of Human Insulin Precursor in Pichia Pastoris X-33

Abstract

The prevalence of Insulin Dependent Diabetes Mellitus (IDDM) has increased significantly in the last decades, resulting in an increased demand for insulin. In addition, new routes of oral and inhaled insulin require higher doses thus increasing insulin requirements. Insulin is the primary drug in patients with type 1 diabetes mellitus (T1DM). In some cases, type 2 diabetes mellitus (T2DM) requires insulin for treatment. The researchers have widely developed another expression system to meet the ever-increasing demand for insulin with higher production capacities. Human insulin precursor (HIP) production can use several microorganisms such as Escherichia coli, Saccharomyces cerevisiae, and Pichia pastoris. In this study, P. pastoris X-33 was used to produce human insulin precursor. Pichia pastoris becomes the promising yeast host for recombinant protein expression because of its ability to reach high cell densities by its robust methanolinducible alcohol oxidase 1 (AOX1) promoter and simple development process, which provide to high quality and a high percentage of recombinant proteins, both intracellular and secretory. In this study, several zeocin-resistant clones were characterized by PCR and sequencing using a specific human insulin precursor gene to detect plasmid integration into the P. pastoris genome. In addition, a test of the effect of zeocin concentration on the growth of the transformation was carried out. The expression of HIP protein in P. pastoris X-33 was characterized by SDS-PAGE and Elisa. The result of PCR and sequencing showed that the HIP gene was successfully integrated into selected colonies of P. pastoris X-33. All of 20 transformant colonies were able to grow at 100 to 2000 μg per mL and selected colonies of P. pastoris X-33 can produce HIP protein.