Purification and Characterization of Histidine Tagged Vacuolar H+ -pyrophosphatase Expressed in Yeast
碩士 === 國立清華大學 === 生命科學系 === 90 === H+-PPase (E.C.3.6.1.1) existed primarily in higher plants and several bacteria and requires PPi as energy source, supplying energy to the secondary active transport system. Fusion proteins containing a string of his-tag made it easy to be purified from...
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ndltd-TW-090NTHU01050982015-10-13T10:34:05Z http://ndltd.ncl.edu.tw/handle/90239210369255337647 Purification and Characterization of Histidine Tagged Vacuolar H+ -pyrophosphatase Expressed in Yeast 酵母菌表現組胺酸修飾液泡焦磷酸水解酵素之純化與定性分析 Yi-Lung Hsu 徐逸龍 碩士 國立清華大學 生命科學系 90 H+-PPase (E.C.3.6.1.1) existed primarily in higher plants and several bacteria and requires PPi as energy source, supplying energy to the secondary active transport system. Fusion proteins containing a string of his-tag made it easy to be purified from both eukaryotic and prokaryotic organisms. The high purity and large-scale yield were useful for structural studies and immunogenic peptides preparation. In the present study, we first successfully purified vacuolar H+-pyrophosphatase heterologously expressed in yeast microsome. The technique of purification we used is immobilized-metal affinity chromatography, Ni2+-NTA (Nitrilotriacetic acid). Analysis with the SDS-PAGE (sodium dodecyl sulfate- polyacrylamine gel electrophoresis) and the Western blot of purified his-tagged vacuolar H+-PPase showed that only one single polypeptide with molecular mass of about 73 kDa was obtained. After analyzing enzymatic kinetics, we obtained a Km of 100 uM and Vmax of 111 umol PPi mg-1h-1. The maximum enzymatic activity was measured with the substrate of Mg2+/ PPi ratio of 1:1 at pH 8.0. Taken together, the main properties of vacuolar H+-PPase heterologously expressed were similar to the endogenous one from mung bean seedlings. The results demonstrate the method of heterologous expression of V-PPase is feasible. By the intrinsic spectroscopy, we only knew that K+ and Ca2+ bind to V-PPase resulting in conformational changes. Further work is to investigate the mechanism of binding K+ and Ca2+ to V-PPase by using isotope, CD, and so on. Rong-Long Pan 潘榮隆 2002 學位論文 ; thesis 63 zh-TW |
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碩士 === 國立清華大學 === 生命科學系 === 90 === H+-PPase (E.C.3.6.1.1) existed primarily in higher plants and several bacteria and requires PPi as energy source, supplying energy to the secondary active transport system. Fusion proteins containing a string of his-tag made it easy to be purified from both eukaryotic and prokaryotic organisms. The high purity and large-scale yield were useful for structural studies and immunogenic peptides preparation. In the present study, we first successfully purified vacuolar H+-pyrophosphatase heterologously expressed in yeast microsome. The technique of purification we used is immobilized-metal affinity chromatography, Ni2+-NTA (Nitrilotriacetic acid). Analysis with the SDS-PAGE (sodium dodecyl sulfate- polyacrylamine gel electrophoresis) and the Western blot of purified his-tagged vacuolar H+-PPase showed that only one single polypeptide with molecular mass of about 73 kDa was obtained.
After analyzing enzymatic kinetics, we obtained a Km of 100 uM and Vmax of 111 umol PPi mg-1h-1. The maximum enzymatic activity was measured with the substrate of Mg2+/ PPi ratio of 1:1 at pH 8.0. Taken together, the main properties of vacuolar H+-PPase heterologously expressed were similar to the endogenous one from mung bean seedlings. The results demonstrate the method of heterologous expression of V-PPase is feasible. By the intrinsic spectroscopy, we only knew that K+ and Ca2+ bind to V-PPase resulting in conformational changes. Further work is to investigate the mechanism of binding K+ and Ca2+ to V-PPase by using isotope, CD, and so on.
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author2 |
Rong-Long Pan |
author_facet |
Rong-Long Pan Yi-Lung Hsu 徐逸龍 |
author |
Yi-Lung Hsu 徐逸龍 |
spellingShingle |
Yi-Lung Hsu 徐逸龍 Purification and Characterization of Histidine Tagged Vacuolar H+ -pyrophosphatase Expressed in Yeast |
author_sort |
Yi-Lung Hsu |
title |
Purification and Characterization of Histidine Tagged Vacuolar H+ -pyrophosphatase Expressed in Yeast |
title_short |
Purification and Characterization of Histidine Tagged Vacuolar H+ -pyrophosphatase Expressed in Yeast |
title_full |
Purification and Characterization of Histidine Tagged Vacuolar H+ -pyrophosphatase Expressed in Yeast |
title_fullStr |
Purification and Characterization of Histidine Tagged Vacuolar H+ -pyrophosphatase Expressed in Yeast |
title_full_unstemmed |
Purification and Characterization of Histidine Tagged Vacuolar H+ -pyrophosphatase Expressed in Yeast |
title_sort |
purification and characterization of histidine tagged vacuolar h+ -pyrophosphatase expressed in yeast |
publishDate |
2002 |
url |
http://ndltd.ncl.edu.tw/handle/90239210369255337647 |
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