Functional expression of transglutaminase from Streptomyces mobaraensis in Escherichia coli
碩士 === 嘉南藥理科技大學 === 生物科技系暨研究所 === 96 === Transglutaminases (TGA) are enzymes that catalyze the acyl-transfer reaction between the γ-carboxyamide group of peptide or protein-bound glutaminyl residues, and primary amines. Recently, the protein was wildly used in food, biomedicine and pharmaceuticals....
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ndltd-TW-096CNUP51110142015-10-13T15:42:16Z http://ndltd.ncl.edu.tw/handle/56861052013138807167 Functional expression of transglutaminase from Streptomyces mobaraensis in Escherichia coli 於Escherichia coli.表達具有功能性Streptomyces mobaraensis之轉醯基酵素 Yi-Hsin Lo 羅翊心 碩士 嘉南藥理科技大學 生物科技系暨研究所 96 Transglutaminases (TGA) are enzymes that catalyze the acyl-transfer reaction between the γ-carboxyamide group of peptide or protein-bound glutaminyl residues, and primary amines. Recently, the protein was wildly used in food, biomedicine and pharmaceuticals. In this study, we expressed the TGase from Streptomyces mobaraensis in soluble form in Escherichia coli. Two vectors were used, pET21b for intracellular expression and pETYebF for extracellular expression. In order to evaluate the effects of pro-region and T7-tag, three insert genes, PTGA, T7PTGA, and T7PTGA, will amplified by PCR and constructed into relative vectors. The PTGA was expressed in soluble form intracellularly in E. coli. However, it precipitated soon after Ni2+ column purification. T7PTGA was expressed more efficiently than that of PTGA, and it did not precipitate after Ni2+ purification. It can be stored 4℃ for at least 6 month. YebFPTGA was expressed not only in periplasm but also in inclusion bodied. The extracellular broth did not clearly detect the TGA activity. When culture in LB, 25℃, induced by 0.1mM IPTG, 62U/ml TGA activity can be achieved after dispase activation. When culture in ZYP and autoinduction condition were applied, T7PTGA expressed more efficiently and 140U/ml TGA activity can be achieved. This procedure provide an easy way to acquire a large quantity of TGA and it enable the high-throughput screening of mutant libraries without the restraint of refolding. Chien-Min Chiang 江建民 2008 學位論文 ; thesis 94 zh-TW |
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碩士 === 嘉南藥理科技大學 === 生物科技系暨研究所 === 96 === Transglutaminases (TGA) are enzymes that catalyze the acyl-transfer reaction between the γ-carboxyamide group of peptide or protein-bound glutaminyl residues, and primary amines. Recently, the protein was wildly used in food, biomedicine and pharmaceuticals. In this study, we expressed the TGase from Streptomyces mobaraensis in soluble form in Escherichia coli. Two vectors were used, pET21b for intracellular expression and pETYebF for extracellular expression. In order to evaluate the effects of pro-region and T7-tag, three insert genes, PTGA, T7PTGA, and T7PTGA, will amplified by PCR and constructed into relative vectors. The PTGA was expressed in soluble form intracellularly in E. coli. However, it precipitated soon after Ni2+ column purification. T7PTGA was expressed more efficiently than that of PTGA, and it did not precipitate after Ni2+ purification. It can be stored 4℃ for at least 6 month. YebFPTGA was expressed not only in periplasm but also in inclusion bodied. The extracellular broth did not clearly detect the TGA activity. When culture in LB, 25℃, induced by 0.1mM IPTG, 62U/ml TGA activity can be achieved after dispase activation. When culture in ZYP and autoinduction condition were applied, T7PTGA expressed more efficiently and 140U/ml TGA activity can be achieved. This procedure provide an easy way to acquire a large quantity of TGA and it enable the high-throughput screening of mutant libraries without the restraint of refolding.
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author2 |
Chien-Min Chiang |
author_facet |
Chien-Min Chiang Yi-Hsin Lo 羅翊心 |
author |
Yi-Hsin Lo 羅翊心 |
spellingShingle |
Yi-Hsin Lo 羅翊心 Functional expression of transglutaminase from Streptomyces mobaraensis in Escherichia coli |
author_sort |
Yi-Hsin Lo |
title |
Functional expression of transglutaminase from Streptomyces mobaraensis in Escherichia coli |
title_short |
Functional expression of transglutaminase from Streptomyces mobaraensis in Escherichia coli |
title_full |
Functional expression of transglutaminase from Streptomyces mobaraensis in Escherichia coli |
title_fullStr |
Functional expression of transglutaminase from Streptomyces mobaraensis in Escherichia coli |
title_full_unstemmed |
Functional expression of transglutaminase from Streptomyces mobaraensis in Escherichia coli |
title_sort |
functional expression of transglutaminase from streptomyces mobaraensis in escherichia coli |
publishDate |
2008 |
url |
http://ndltd.ncl.edu.tw/handle/56861052013138807167 |
work_keys_str_mv |
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