Improvement of recombinant protein production in pichia pastoris by promoter engineering

• Main Authors: Chia-Hung Chiu, 邱家鴻
• Other Authors: Guan-Chiun Lee
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/61052985743815323614

碩士 === 國立臺灣師範大學 === 生命科學研究所 === 101 === The methylotrophic yeast Pichia pastoris is a well-established protein production host. It has the advantages of eukaryotes, such as eukaryotic post-translational modifications, efficient protein secretion, fast growth on economical media, and little risk of contamination with endotoxin or virus. The glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter (PGAP) has been used for constitutive expression of heterologous proteins. The constitutive PGAP system permits steady-state gene expression and prevent the transcriptional heterogeneity in inducible expression systems. Engineered promoters with various strengths are useful genetic tools that enable the precise control of gene expression to obtain optimal yield. To improve the production of recombinant protein in P. pastoris, we created a PGAP library through random mutagenesis. Antibiotics Zeocin resistant gene (Streptoalloteichus hindustanus bleomycin gene, Sh ble gene) was used as a reporter. We performed transformation through gene replacement to rule out the multi-copy insertion events which may interfere with the promoter selection. 3 survival mutants were selected from 708 transformants under higher Zeocin concentrations. We identified the important mutation sites in the GAP promoter regions that may cause the variant strengths. These strong mutant promoters could be used to improve the expression of other recombinant proteins.