Improvement of recombinant protein production in pichia pastoris by promoter engineering
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 c...
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ndltd-TW-101NTNU51120482016-03-18T04:42:08Z http://ndltd.ncl.edu.tw/handle/61052985743815323614 Improvement of recombinant protein production in pichia pastoris by promoter engineering 藉由啟動子工程改進pichia pastoris蛋白質表達系統之產量 Chia-Hung Chiu 邱家鴻 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. Guan-Chiun Lee 李冠群 2013 學位論文 ; thesis 60 zh-TW |
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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.
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author2 |
Guan-Chiun Lee |
author_facet |
Guan-Chiun Lee Chia-Hung Chiu 邱家鴻 |
author |
Chia-Hung Chiu 邱家鴻 |
spellingShingle |
Chia-Hung Chiu 邱家鴻 Improvement of recombinant protein production in pichia pastoris by promoter engineering |
author_sort |
Chia-Hung Chiu |
title |
Improvement of recombinant protein production in pichia pastoris by promoter engineering |
title_short |
Improvement of recombinant protein production in pichia pastoris by promoter engineering |
title_full |
Improvement of recombinant protein production in pichia pastoris by promoter engineering |
title_fullStr |
Improvement of recombinant protein production in pichia pastoris by promoter engineering |
title_full_unstemmed |
Improvement of recombinant protein production in pichia pastoris by promoter engineering |
title_sort |
improvement of recombinant protein production in pichia pastoris by promoter engineering |
publishDate |
2013 |
url |
http://ndltd.ncl.edu.tw/handle/61052985743815323614 |
work_keys_str_mv |
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