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

DOI

10.2991/978-94-6463-112-8_74How to use a DOI?

Keywords

Human precursor insulin; diabetes mellitus; Pichia pastoris; expression

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 methanol-inducible 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.

Copyright

© 2023 The Author(s)

Open Access

Open Access This chapter is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses/by-nc/4.0/), which permits any noncommercial use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license and indicate if changes were made.

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TY  - CONF
AU  - Dudi Hardianto
AU  - Efrida Martius
AU  - Fahroziah Assyifa
AU  - Dihan Laziba
AU  - Uli J. Nasution
AU  - Fransiska C. Sekaringtyas
AU  - Juwartina I. Royani
PY  - 2023
DA  - 2023/03/01
TI  - Production and Characterization of Human Insulin Precursor in Pichia Pastoris X-33
BT  - Proceedings of the 1st International Conference for Health Research – BRIN (ICHR 2022)
PB  - Atlantis Press
SP  - 814
EP  - 823
SN  - 2468-5739
UR  - https://doi.org/10.2991/978-94-6463-112-8_74
DO  - 10.2991/978-94-6463-112-8_74
ID  - Hardianto2023
ER  -