Preparation of the Akt Kinase Fusion Protein
碩士 === 國立中正大學 === 生命科學系暨分子生物研究所暨生物醫學研究所 === 98 === Phostensin is a protein phosphatase 1 F-actin cytoskeleton targeting subunit. Phostensin is a F-actin-binding protein that binds to the pointed ends of actin filament and modulates actin dynamics. In our preliminary results, we found that Akt can ph...
Main Authors: | , |
---|---|
Other Authors: | |
Format: | Others |
Language: | zh-TW |
Published: |
2010
|
Online Access: | http://ndltd.ncl.edu.tw/handle/62769377903313199696 |
id |
ndltd-TW-098CCU05105001 |
---|---|
record_format |
oai_dc |
spelling |
ndltd-TW-098CCU051050012015-10-13T18:25:30Z http://ndltd.ncl.edu.tw/handle/62769377903313199696 Preparation of the Akt Kinase Fusion Protein 製備一個Akt激酶融合蛋白 Yu-Chih Liu 劉昱志 碩士 國立中正大學 生命科學系暨分子生物研究所暨生物醫學研究所 98 Phostensin is a protein phosphatase 1 F-actin cytoskeleton targeting subunit. Phostensin is a F-actin-binding protein that binds to the pointed ends of actin filament and modulates actin dynamics. In our preliminary results, we found that Akt can phosphorylate phostensin. In order to study the effect on biochemical activity of phostensin by Akt phosphorylation, we have to prepare the active Akt kinase. Activation of Akt is occurred at the plasma membrane where Akt is phosphorylated at Thr-308 and Ser473. However, E. coli lacks the kinases that phosphorylate Akt at Thr-308 and Ser473. Therefore, in the studies, we have over-expressed (His)6-Ub-Akt1 (T308D, S473D) fusion protein. This recombinant (His)6-Ub-Akt1 (T308D, S473D) was purified by a series of methods, including Ni2+-Sepharose, Mono-Q ionic exchange and gel filtration chromatography. The homogeneity of (His)6-Ub-Akt1 (T308D, S473D) is more than 85 % after three steps of purification. However, The kinase activity assay indicated that the recombinant (His)6-Ub-Akt1 (T308D, S473D) fusion protein cannot phosphorylate phostensin. Hsien-Bin Huang 黃憲斌 2010 學位論文 ; thesis 49 zh-TW |
collection |
NDLTD |
language |
zh-TW |
format |
Others
|
sources |
NDLTD |
description |
碩士 === 國立中正大學 === 生命科學系暨分子生物研究所暨生物醫學研究所 === 98 === Phostensin is a protein phosphatase 1 F-actin cytoskeleton targeting subunit. Phostensin is a F-actin-binding protein that binds to the pointed ends of actin filament and modulates actin dynamics. In our preliminary results, we found that Akt can phosphorylate phostensin. In order to study the effect on biochemical activity of phostensin by Akt phosphorylation, we have to prepare the active Akt kinase. Activation of Akt is occurred at the plasma membrane where Akt is phosphorylated at Thr-308 and Ser473. However, E. coli lacks the kinases that phosphorylate Akt at Thr-308 and Ser473. Therefore, in the studies, we have over-expressed (His)6-Ub-Akt1 (T308D, S473D) fusion protein. This recombinant (His)6-Ub-Akt1 (T308D, S473D) was purified by a series of methods, including Ni2+-Sepharose, Mono-Q ionic exchange and gel filtration chromatography. The homogeneity of (His)6-Ub-Akt1 (T308D, S473D) is more than 85 % after three steps of purification. However, The kinase activity assay indicated that the recombinant (His)6-Ub-Akt1 (T308D, S473D) fusion protein cannot phosphorylate phostensin.
|
author2 |
Hsien-Bin Huang |
author_facet |
Hsien-Bin Huang Yu-Chih Liu 劉昱志 |
author |
Yu-Chih Liu 劉昱志 |
spellingShingle |
Yu-Chih Liu 劉昱志 Preparation of the Akt Kinase Fusion Protein |
author_sort |
Yu-Chih Liu |
title |
Preparation of the Akt Kinase Fusion Protein |
title_short |
Preparation of the Akt Kinase Fusion Protein |
title_full |
Preparation of the Akt Kinase Fusion Protein |
title_fullStr |
Preparation of the Akt Kinase Fusion Protein |
title_full_unstemmed |
Preparation of the Akt Kinase Fusion Protein |
title_sort |
preparation of the akt kinase fusion protein |
publishDate |
2010 |
url |
http://ndltd.ncl.edu.tw/handle/62769377903313199696 |
work_keys_str_mv |
AT yuchihliu preparationoftheaktkinasefusionprotein AT liúyùzhì preparationoftheaktkinasefusionprotein AT yuchihliu zhìbèiyīgèaktjīméirónghédànbái AT liúyùzhì zhìbèiyīgèaktjīméirónghédànbái |
_version_ |
1718031737948209152 |