Purification and Biochemical Properties of an N-acetylgalactosamine Specific Lectin from Papaya (Carica papaya) Seeds
碩士 === 臺灣大學 === 農業化學研究所 === 98 === Based on our previous study, a novel N-acetylgalactosamine binding protein in papaya seed (Carica papaya lecin, CPL) was discovered by GalNAc-polyacrylamide based enzyme-linked adsorbent assay (Food Chem. 2009, 113: 1218-1225). Papaya seeds with different maturity...
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ndltd-TW-098NTU054060052015-10-13T18:49:38Z http://ndltd.ncl.edu.tw/handle/01953600833350933720 Purification and Biochemical Properties of an N-acetylgalactosamine Specific Lectin from Papaya (Carica papaya) Seeds 番木瓜種子凝集素之純化與生化特性探討 Yen-Lun Kung 龔彥綸 碩士 臺灣大學 農業化學研究所 98 Based on our previous study, a novel N-acetylgalactosamine binding protein in papaya seed (Carica papaya lecin, CPL) was discovered by GalNAc-polyacrylamide based enzyme-linked adsorbent assay (Food Chem. 2009, 113: 1218-1225). Papaya seeds with different maturity were collected, and the extracts of seeds in green papaya fruits shows relatively high activity. Further purification of CPL was conducted as following: freeze-dried papaya seeds were ground into powder, extracted with 20 folds (w/v) of 50 mM phosphate buffered saline (pH 7.4) at 4°C overnight, centrifuged and collected the supernatant, added ammonium sulfate to 70% saturation. Precipitations were resuspended and dialyzed against PBS, then fractionated by a 50 kDa MWCO ultrafiltration. The retentate was further purified by HiTrap CM FF ion exchange chromatography and Superdex 200 GL gel filtration chromatography. SDS-PAGE and HPLC gel filtration indicated that CPL is a polymer with a molecular mass of 804 ± 30 kDa and composed of two different subunits of 38 and 40 kDa associated by non-covalent bonds. It was heat stable until up to 70oC for 30 min and showed optimum sugar-binding activity from pH 6.0 to 8.0. Also, CPL did not require Ca2+, Mg2+, Mn2+, Zn2+for its activity. Of various sugars tested, the lectin was best inhibited by GalNAc. CPL agglutinated all trypsinized human RBC types, with a slight preference for the A blood group which immunodeterminant is GalNAc. Based on the specificity, we performed an alternative ripid, simple purification method via GalNAc-Sepharose 6B affinity chromatography column, and gave the lectin with a 6000-fold purification as compared to the crude extract. 蘇南維 2010 學位論文 ; thesis 92 zh-TW |
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碩士 === 臺灣大學 === 農業化學研究所 === 98 === Based on our previous study, a novel N-acetylgalactosamine binding protein in papaya seed (Carica papaya lecin, CPL) was discovered by GalNAc-polyacrylamide based enzyme-linked adsorbent assay (Food Chem. 2009, 113: 1218-1225). Papaya seeds with different maturity were collected, and the extracts of seeds in green papaya fruits shows relatively high activity. Further purification of CPL was conducted as following: freeze-dried papaya seeds were ground into powder, extracted with 20 folds (w/v) of 50 mM phosphate buffered saline (pH 7.4) at 4°C overnight, centrifuged and collected the supernatant, added ammonium sulfate to 70% saturation. Precipitations were resuspended and dialyzed against PBS, then fractionated by a 50 kDa MWCO ultrafiltration. The retentate was further purified by HiTrap CM FF ion exchange chromatography and Superdex 200 GL gel filtration chromatography. SDS-PAGE and HPLC gel filtration indicated that CPL is a polymer with a molecular mass of 804 ± 30 kDa and composed of two different subunits of 38 and 40 kDa associated by non-covalent bonds. It was heat stable until up to 70oC for 30 min and showed optimum sugar-binding activity from pH 6.0 to 8.0. Also, CPL did not require Ca2+, Mg2+, Mn2+, Zn2+for its activity. Of various sugars tested, the lectin was best inhibited by GalNAc. CPL agglutinated all trypsinized human RBC types, with a slight preference for the A blood group which immunodeterminant is GalNAc. Based on the specificity, we performed an alternative ripid, simple purification method via GalNAc-Sepharose 6B affinity chromatography column, and gave the lectin with a 6000-fold purification as compared to the crude extract.
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author2 |
蘇南維 |
author_facet |
蘇南維 Yen-Lun Kung 龔彥綸 |
author |
Yen-Lun Kung 龔彥綸 |
spellingShingle |
Yen-Lun Kung 龔彥綸 Purification and Biochemical Properties of an N-acetylgalactosamine Specific Lectin from Papaya (Carica papaya) Seeds |
author_sort |
Yen-Lun Kung |
title |
Purification and Biochemical Properties of an N-acetylgalactosamine Specific Lectin from Papaya (Carica papaya) Seeds |
title_short |
Purification and Biochemical Properties of an N-acetylgalactosamine Specific Lectin from Papaya (Carica papaya) Seeds |
title_full |
Purification and Biochemical Properties of an N-acetylgalactosamine Specific Lectin from Papaya (Carica papaya) Seeds |
title_fullStr |
Purification and Biochemical Properties of an N-acetylgalactosamine Specific Lectin from Papaya (Carica papaya) Seeds |
title_full_unstemmed |
Purification and Biochemical Properties of an N-acetylgalactosamine Specific Lectin from Papaya (Carica papaya) Seeds |
title_sort |
purification and biochemical properties of an n-acetylgalactosamine specific lectin from papaya (carica papaya) seeds |
publishDate |
2010 |
url |
http://ndltd.ncl.edu.tw/handle/01953600833350933720 |
work_keys_str_mv |
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