Crystal structure of the recombinant gooseδ-crystallin and use the site-direct mutagenesis method to recovery the enzyme activity
碩士 === 國防醫學院 === 生物化學研究所 === 95 === δ-crystallin is a taxon –specific crystallin that expressed in lenses of birds and terrestrial reptiles. It plays an important role by conferring special refractive properties in eye lenses. Evolutionally, gene of δ-crystallin that recruited from argininosuccina...
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ndltd-TW-095NDMC01070072015-10-13T16:41:20Z http://ndltd.ncl.edu.tw/handle/19776701512299609414 Crystal structure of the recombinant gooseδ-crystallin and use the site-direct mutagenesis method to recovery the enzyme activity 重組鵝眼δ-晶體蛋白結構解析及利用定位突變改善酶活性研究 Chu-Hung Chung 鍾瞿鴻 碩士 國防醫學院 生物化學研究所 95 δ-crystallin is a taxon –specific crystallin that expressed in lenses of birds and terrestrial reptiles. It plays an important role by conferring special refractive properties in eye lenses. Evolutionally, gene of δ-crystallin that recruited from argininosuccinate lyase (ASL) to lens was duplicated and expressed as δ1 and δ2-crystallin, within them only δ2-crystallin still retain the enzyme activity. To identify the reason of the difference in enzyme activity between these two highly homologous proteins (sequence identity > 93 %), the recombinant goose δ-crystallin with only 10 %activity was used as target for protein engineering, several sites (M9W, V14S, T30Y…etc.) were selected for single, double, multiple mutation, but all resulted in marginal recovery in activity. However, when replacement of 53 residue from HASL the enzyme activity was totally recovered. The crystal structures of recombinant goose δ-crystallin with or without C-terminal His-tag were resolved with resolution of 1.65 and 2.07 Ǻ, respectively. Compared to native goose δ-crystallin (1XWO), the topology of these protein were similar. The major difference were localed in 20’s loop (22-31) and 280’s loop (270-290), which has been reported to be important for enzyme activity. Wild type, multiple mutations and N terminal 53 residue replaced chimera protein, have similar secondary structure. However multiple mutation and N terminal 53 residues replaced chimera protein have 7 and 13 oC decrease in Tm, respectively. Hwei-Jen Lee 李惠珍 2007 學位論文 ; thesis 80 zh-TW |
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碩士 === 國防醫學院 === 生物化學研究所 === 95 === δ-crystallin is a taxon –specific crystallin that expressed in lenses of birds and terrestrial reptiles. It plays an important role by conferring special refractive properties in eye lenses. Evolutionally, gene of δ-crystallin that recruited from argininosuccinate lyase (ASL) to lens was duplicated and expressed as δ1 and δ2-crystallin, within them only δ2-crystallin still retain the enzyme activity. To identify the reason of the difference in enzyme activity between these two highly homologous proteins (sequence identity > 93 %), the recombinant goose δ-crystallin with only 10 %activity was used as target for protein engineering, several sites (M9W, V14S, T30Y…etc.) were selected for single, double, multiple mutation, but all resulted in marginal recovery in activity. However, when replacement of 53 residue from HASL the enzyme activity was totally recovered. The crystal structures of recombinant goose δ-crystallin with or without C-terminal His-tag were resolved with resolution of 1.65 and 2.07 Ǻ, respectively. Compared to native goose δ-crystallin (1XWO), the topology of these protein were similar. The major difference were localed in 20’s loop (22-31) and 280’s loop (270-290), which has been reported to be important for enzyme activity. Wild type, multiple mutations and N terminal 53 residue replaced chimera protein, have similar secondary structure. However multiple mutation and N terminal 53 residues replaced chimera protein have 7 and 13 oC decrease in Tm, respectively.
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author2 |
Hwei-Jen Lee |
author_facet |
Hwei-Jen Lee Chu-Hung Chung 鍾瞿鴻 |
author |
Chu-Hung Chung 鍾瞿鴻 |
spellingShingle |
Chu-Hung Chung 鍾瞿鴻 Crystal structure of the recombinant gooseδ-crystallin and use the site-direct mutagenesis method to recovery the enzyme activity |
author_sort |
Chu-Hung Chung |
title |
Crystal structure of the recombinant gooseδ-crystallin and use the site-direct mutagenesis method to recovery the enzyme activity |
title_short |
Crystal structure of the recombinant gooseδ-crystallin and use the site-direct mutagenesis method to recovery the enzyme activity |
title_full |
Crystal structure of the recombinant gooseδ-crystallin and use the site-direct mutagenesis method to recovery the enzyme activity |
title_fullStr |
Crystal structure of the recombinant gooseδ-crystallin and use the site-direct mutagenesis method to recovery the enzyme activity |
title_full_unstemmed |
Crystal structure of the recombinant gooseδ-crystallin and use the site-direct mutagenesis method to recovery the enzyme activity |
title_sort |
crystal structure of the recombinant gooseδ-crystallin and use the site-direct mutagenesis method to recovery the enzyme activity |
publishDate |
2007 |
url |
http://ndltd.ncl.edu.tw/handle/19776701512299609414 |
work_keys_str_mv |
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