Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2
碩士 === 國立清華大學 === 分子與細胞生物研究所 === 98 === Tachypleus plasma lectin two (TPL-2) derived from the hemolymph of Taiwanese horseshoe crab, has been found to be able to recognize Gram-positive and negative bacteria. Previously, recombinant TPL-2 was expressed by yeast P. pastoris, but the expression level...
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ndltd-TW-098NTHU50610622015-11-04T04:01:50Z http://ndltd.ncl.edu.tw/handle/13605039970133012943 Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2 重組鱟血漿凝集素2之細菌及配體結合活性探討 Low, Ee Ling 劉俞伶 碩士 國立清華大學 分子與細胞生物研究所 98 Tachypleus plasma lectin two (TPL-2) derived from the hemolymph of Taiwanese horseshoe crab, has been found to be able to recognize Gram-positive and negative bacteria. Previously, recombinant TPL-2 was expressed by yeast P. pastoris, but the expression level and purification yield were low. Therefore, the aim of this study is to increase TPL-2 expression by fusing an affinity tag for investigation of biological functions. Recombinant TPL-2 was designed and engineered to be consisted of an N-terminal Rhizopus oryzae starch binding domain (RoSBD), an R. oryzae glucoamylase linker (RoLK), and a C-terminal TPL-2 to give ALT2. ALT2 was further modified on two N-glycosylation sites at Asn 140 and 150 to make ALT2-dm. ALT2-dm was overexpressed in P. pastoris from laboratory scale up to 100 L, and starch matrix adsorption was proven to be feasible for the purification of and a purification yield of 79.8 mg/L was achieved. Functional analyses revealed that ALT2-dm recognized specific bacteria and the pathogen associated molecular patterns (PAMPs) such as lipoteichoic acid (LTA) in Gram-positive bacteria and lipopolysaccharide (LPS) in Gram-negative bacteria. In addition, ALT2-dm was capable of recognizing a conserved moiety in the prokaryotic cell wall which in turn led to the development of high sensitivity detection methods. The major contribution of this study include achievement of high level expression of TPL-2, modulation of protein glycosylation, characterization of protein-glycan interaction, and discovery of a novel function of recombinant TPL-2. Chang, Margaret Dah-Tsyr 張大慈 2010 學位論文 ; thesis 79 en_US |
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碩士 === 國立清華大學 === 分子與細胞生物研究所 === 98 === Tachypleus plasma lectin two (TPL-2) derived from the hemolymph of Taiwanese horseshoe crab, has been found to be able to recognize Gram-positive and negative bacteria. Previously, recombinant TPL-2 was expressed by yeast P. pastoris, but the expression level and purification yield were low. Therefore, the aim of this study is to increase TPL-2 expression by fusing an affinity tag for investigation of biological functions. Recombinant TPL-2 was designed and engineered to be consisted of an N-terminal Rhizopus oryzae starch binding domain (RoSBD), an R. oryzae glucoamylase linker (RoLK), and a C-terminal TPL-2 to give ALT2. ALT2 was further modified on two N-glycosylation sites at Asn 140 and 150 to make ALT2-dm. ALT2-dm was overexpressed in P. pastoris from laboratory scale up to 100 L, and starch matrix adsorption was proven to be feasible for the purification of and a purification yield of 79.8 mg/L was achieved. Functional analyses revealed that ALT2-dm recognized specific bacteria and the pathogen associated molecular patterns (PAMPs) such as lipoteichoic acid (LTA) in Gram-positive bacteria and lipopolysaccharide (LPS) in Gram-negative bacteria. In addition, ALT2-dm was capable of recognizing a conserved moiety in the prokaryotic cell wall which in turn led to the development of high sensitivity detection methods. The major contribution of this study include achievement of high level expression of TPL-2, modulation of protein glycosylation, characterization of protein-glycan interaction, and discovery of a novel function of recombinant TPL-2.
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author2 |
Chang, Margaret Dah-Tsyr |
author_facet |
Chang, Margaret Dah-Tsyr Low, Ee Ling 劉俞伶 |
author |
Low, Ee Ling 劉俞伶 |
spellingShingle |
Low, Ee Ling 劉俞伶 Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2 |
author_sort |
Low, Ee Ling |
title |
Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2 |
title_short |
Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2 |
title_full |
Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2 |
title_fullStr |
Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2 |
title_full_unstemmed |
Characterization of bacteria and ligand binding activities of recombinant Tachypleus plasma lectin 2 |
title_sort |
characterization of bacteria and ligand binding activities of recombinant tachypleus plasma lectin 2 |
publishDate |
2010 |
url |
http://ndltd.ncl.edu.tw/handle/13605039970133012943 |
work_keys_str_mv |
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