Purification and characterization of defense-related proteins from Hokkaido large black soybean and emperor banana.

Ho, Sai Man.Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.Includes bibliographical references (leaves 144-164).Abstracts in English and Chinese.TABLE OF CONTENTS --- p.iiABSTRACT --- p.xii撮要 --- p.xvLIST OF ABBREIVIATIONS --- p.xviLIST OF TABLES --- p.xviiLIST OF FIGURES --- p.xixChapter Chapter 1 --- General IntroductionChapter 1.1 --- Overview of lectins --- p.1Chapter 1.1.1 --- History of lectins --- p.1Chapter 1.1.2 --- Definitions of lectins --- p.2Chapter 1.1.3 --- Classification and nomenclature of lectins based on structure --- p.2Chapter 1.1.4 --- Classification and nomenclature of lectins based on carbohydrate-bindingspecificity --- p.4Chapter 1.1.5 --- Structure of plant lectins --- p.4Chapter 1.1.6 --- Biological function of plant lectins --- p.5Chapter 1.1.6.1 --- Anti-viral activity of plant lectiins --- p.5Chapter 1.1.6.2 --- Lectins as plant defense proteins --- p.6Chapter 1.1.6.3 --- Insecticidal activity of plant lectins --- p.7Chapter 1.1.6.4 --- Anti-fungal activity of plant lectins --- p.7Chapter 1.1.6.5 --- Mitogenic activity of plant lectins --- p.7Chapter 1.1.6.6 --- Anti-tumor and anti-proliferative activity of plant lectins --- p.9Chapter 1.1.7 --- Background of legume lectins --- p.11Chapter 1.1.7.1 --- Structure of legume lectins --- p.11Chapter 1.1.7.2 --- Functions and activities of legume lectins --- p.12Chapter 1.2 --- Overview of serine protease inhibitors in plants --- p.14Chapter 1.2.1 --- Classification of serine protease inhibitor --- p.15Chapter 1.2.2 --- The main functions of plant serine protease inhibitors --- p.17Chapter 1.2.3 --- Commercial application of serine protease inhibirtors --- p.19Chapter 1.2.3.1 --- Medical application --- p.19Chapter 1.2.3.2 --- Transgenic application in agriculture --- p.22Chapter 1.3 --- Overview of Pathogenesis-related proteins in plants --- p.25Chapter 1.3.1 --- Overview of PR-5 family Thaumatin-like proteins (TLPs) --- p.27Chapter 1.3.1.1 --- Structural similarities among TLPs --- p.28Chapter 1.3.1.2 --- Antifungal activity of TLP --- p.31Chapter 1.3.2 --- Overview of Chinase-like proteins (CLPs) --- p.33Chapter 1.3.2.1 --- Classification of chitinase --- p.34Chapter 1.3.2.1.1 --- On the basis of amino acid sequence of glycosyl hydrolase --- p.34Chapter 1.3.2.1.2 --- On the basis of amino acid sequence of plant chitinase --- p.35Chapter 1.3.2.2 --- Antifungal activity of CLP --- p.36Chapter 1.3.3 --- Anti-freeze property of PR proteins --- p.38Chapter 1.3.4 --- Application of PR proteins in agriculture --- p.40Chapter 1.4 --- Rationale of the present study --- p.42Chapter Chapter 2 --- Materials and MethodsChapter 2.1 --- Materials --- p.43Chapter 2.2 --- Preparation of crude extract --- p.44Chapter 2.2.1 --- Hokkaido large black soybean --- p.44Chapter 2.2.2 --- Emperor banana --- p.45Chapter 2.3 --- Purification --- p.45Chapter 2.4 --- Chromatography --- p.46Chapter 2.4.1 --- DEAE-cellulose chromatography --- p.46Chapter 2.4.2 --- Affi-gel Blue gel --- p.47Chapter 2.4.3 --- SP-Sepharse --- p.48Chapter 2.4.4 --- Mono Q HR 5/5 and Mono S HR 5/5 --- p.49Chapter 2.4.5 --- Superdex 75 and superdex 200 --- p.50Chapter 2.5 --- Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) --- p.50Chapter 2.6 --- Protein concentration determination --- p.54Chapter 2.7 --- Preparation of rabbit reticulocyte lysate --- p.54Chapter 2.8 --- Determination of N-terminal amino acid sequence --- p.56Chapter 2.9 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.56Chapter 2.10 --- Thermal stability determination assays --- p.57Chapter 2.10.1 --- Stability at various temperatures --- p.57Chapter 2.10.2 --- Stability at 100°C --- p.57Chapter 2.11 --- Assay of pH dependence of hemagglutinating activity --- p.58Chapter 2.12 --- Assay of ion dependence of hemagglutinating activity --- p.58Chapter 2.13 --- Assay of antifungal activity --- p.58Chapter 2.14 --- Assay of trypsin inhibitory activity --- p.60Chapter 2.15 --- Assay of antibacterial activity --- p.61Chapter 2.16 --- Assay for cytotoxic activity on cancer cell lines --- p.61Chapter 2.17 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.62Chapter 2.18 --- Assay of mitogenic activity --- p.63Chapter Chapter 3 --- Purification and Characterization of Defense-Related Proteins from their Respective SourcesChapter 3.1 --- Purification and Characterization of a Lectin from the Seeds of Hokkaido large black soybeanChapter 3.1.1 --- Introduction --- p.65Chapter 3.1.2 --- Results --- p.66Chapter 3.1.3 --- Purification --- p.68Chapter 3.1.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.69Chapter 3.1.3.2 --- Anion-exchange chromatography on DEAE-cellulose --- p.70Chapter 3.1.3.3 --- Anion-exchange chromatography on Mono Q column --- p.71Chapter 3.1.3.4 --- Gel filtration on Superdex 200 column --- p.72Chapter 3.1.3.5 --- Hemagglutinating activity at each purification step --- p.73Chapter 3.1.4 --- Characterization of Lectin --- p.74Chapter 3.1.4.1 --- Molecular mass determination --- p.74Chapter 3.1.4.2 --- N-terminal amino acid sequencing --- p.76Chapter 3.1.4.3 --- Assay of inhibition of hemagglutinating activity by different carbohydrates --- p.77Chapter 3.1.4.4 --- Thermal stability --- p.78Chapter 3.1.4.5 --- Assay of pH dependence of hemagglutinating activity --- p.80Chapter 3.1.4.6 --- Assay of ion dependence of hemagglutinating activity --- p.81Chapter 3.1.4.7 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.82Chapter 3.1.4.8 --- Assay of mitogenic activity --- p.83Chapter 3.1.4.9 --- Assay of antibacterial activity --- p.84Chapter 3.1.5 --- Discussion --- p.86Chapter 3.2 --- Purification and Characterization of a Trypsin inhibitor from the Seeds of Hokkaido large black soybeanChapter 3.2.1 --- Introduction --- p.93Chapter 3.2.2 --- Results --- p.94Chapter 3.2.3 --- Purification --- p.95Chapter 3.2.3.1 --- Anion-exchange chromatography on Mono Q column --- p.96Chapter 3.2.3.2 --- Gel filtration on Superdex 75 column --- p.98Chapter 3.2.3.3 --- Trypsin inhibitory activity at each purification step --- p.99Chapter 3.2.4 --- Characterization of trypsin inhibitory --- p.100Chapter 3.2.4.1 --- Molecular mass determination --- p.100Chapter 3.2.4.2 --- N-terminal amino acid sequencing --- p.102Chapter 3.2.4.3 --- Assay for HIV-1 reverse transcriptase (RT) inhibitory activity --- p.103Chapter 3.2.4.4 --- Antiproliferative effect on MCF-7 and Hep G2 cells --- p.104Chapter 3.2.4.5 --- pH and thermal stability --- p.105Chapter 3.2.5 --- Discussion --- p.106Chapter 3.3 --- Purification and Characterization of a Thaumatin-like protein and Chitinase-like protein from Emperor BananaChapter 3.3.1 --- Introduction --- p.108Chapter 3.3.2 --- Results --- p.109Chapter 3.3.3 --- Purification --- p.111Chapter 3.3.3.1 --- Affinity chromatography on Affi-gel Blue gel --- p.112Chapter 3.3.3.2 --- Cation exchange chromatography on Mono S column --- p.113Chapter 3.3.3.3 --- Gel filtration on Superdex 75 column --- p.114Chapter 3.3.3.3.1 --- Fraction MS 2 --- p.114Chapter 3.3.3.3.2 --- Fraction MS 4 --- p.115Chapter 3.3.3.3.3 --- Fraction MS 5 --- p.118Chapter 3.3.4 --- Characterization of the thaumatin-like protein --- p.121Chapter 3.3.4.1 --- N-terminal amino acid sequence determination --- p.121Chapter 3.3.4.2 --- Assay for antifungal activity --- p.122Chapter 3.3.4.3 --- Thermal stability --- p.124Chapter 3.3.4.4 --- pH stability --- p.125Chapter 3.3.4.5 --- Resistance to trypsin digestion --- p.125Chapter 3.3.4.6 --- Anti-HIV-1 reverse transcriptase activity --- p.126Chapter 3.3.4.7 --- Discussion --- p.127Chapter 3.3.5 --- Characterization of the two chitinase-like protein --- p.131Chapter 3.3.5.1 --- N-terminal amino acid sequence determination --- p.131Chapter 3.3.5.1.1 --- Emperor banana MS2 CLP --- p.131Chapter 3.3.5.1.2 --- Emperor banana MS4 CLP --- p.132Chapter 3.3.5.2 --- Assay for antifungal activity --- p.133Chapter 3.3.5.3 --- Discussion --- p.136Chapter Chapter 4 --- general discussion --- p.138References --- p.14