Lee Chi-Fung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 154-167). / Abstracts in English and Chinese. / Thesis Committee --- p.I / Statement --- p.II / Acknowledgements --- p.II / Abstract --- p.IV / 摘要 --- p.VI / Content --- p.VIII / Abbreviations --- p.VIX / List of figures and tables --- p.XVII / Chapter Chapter 1 - --- Introduction --- p.1 / Chapter 1.1 --- How are the thermophilic proteins stabilized? --- p.2 / Chapter 1.1.1 --- Hydrophobic interactions --- p.2 / Chapter 1.1.2 --- Hydrogen bonds --- p.4 / Chapter 1.1.3 --- Electrostatic interactions --- p.6 / Chapter 1.1.4 --- Reduction in ΔCP --- p.9 / Chapter 1.2 --- Models of study: Thermococcus celer and yeast L30e --- p.12 / Chapter 1.2.1 --- Thermococcus celer ribosomal protein L30e --- p.12 / Chapter 1.2.2 --- Yeast ribosomal protein L30e --- p.13 / Chapter 1.2.3 --- Comparison between the two proteins --- p.13 / Chapter 1.3 --- Objective of this study --- p.20 / Chapter Chapter 2 - --- Materials and Methods --- p.21 / Chapter 2.1 --- General techniques --- p.21 / Chapter 2.1.1 --- Preparation and transformation of competent E. coli DH5α and BL21(DE3)pLysS --- p.21 / Chapter 2.1.2 --- Minipreparation of plasmid DNA (Invitrogen) --- p.22 / Chapter 2.1.3 --- Spectrophotometric quantitation of DNA --- p.24 / Chapter 2.1.4 --- Agarose gel electrophoresis --- p.24 / Chapter 2.1.5 --- Purification of DNA from agarose gel (Invitrogen) --- p.25 / Chapter 2.1.6 --- Restriction digestion of DNA fragments --- p.26 / Chapter 2.1.7 --- Ligation of DNA fragments into vector --- p.26 / Chapter 2.1.8 --- SDS-PAGE electrophoresis --- p.28 / Chapter 2.1.9 --- Native-PAGE electrophoresis --- p.32 / Chapter 2.2 --- Protein Engineering of Proteins --- p.35 / Chapter 2.2.1 --- Polymerase chain reaction (PCR) --- p.35 / Chapter 2.2.2 --- Site-directed mutagenesis of T. celer L30e --- p.37 / Chapter 2.2.3 --- Protein engineering of yeast L30e --- p.42 / Chapter 2.3 --- "Sub-cloning of mutation PCR fragment into expression vector, pET8c" --- p.45 / Chapter 2.4 --- Expression of recombinant proteins --- p.45 / Chapter 2.5 --- Purification of T. celer and its mutants --- p.46 / Chapter 2.5.1 --- Extraction of proteins by sonication --- p.46 / Chapter 2.5.2 --- Purification by ion-exchange chromatography --- p.47 / Chapter 2.5.3 --- Purification by affinity chromatography --- p.48 / Chapter 2.5.4 --- Purification by size exclusion chromatography --- p.49 / Chapter 2.6 --- Purification of yeast L30e and its mutants --- p.49 / Chapter 2.6.1 --- Extraction of proteins by sonication --- p.49 / Chapter 2.6.2 --- Purification yeast L30e variants by washing the inclusion bodies --- p.50 / Chapter 2.6.3 --- Purification by column chromatography --- p.51 / Chapter 2.7 --- Thermodynamic studies of proteins --- p.52 / Chapter 2.7.1 --- Guanidine-induced denaturation --- p.52 / Chapter 2.7.2 --- Thermal-induced denaturation --- p.53 / Chapter 2.7.3 --- Determination of protein stability curves by denaturant unfolding --- p.54 / Chapter 2.7.4 --- ΔCP and protein stability curve determination by thermal unfolding --- p.55 / Chapter 2.8 --- Media and buffer recipes --- p.56 / Chapter 2.8.1 --- Medium for bacterial culture --- p.56 / Chapter 2.8.2 --- Reagents for competent cell preparation --- p.58 / Chapter 2.8.3 --- Nucleic acid electrophoresis buffers --- p.58 / Chapter 2.8.4 --- Buffers for T. celer L30e variants purification --- p.59 / Chapter 2.8.5 --- Buffers for yeast L30e variants purification --- p.59 / Chapter 2.8.6 --- Reagents of SDS-PAGE --- p.60 / Chapter Chapter Three - --- Purification of T. celer and Yeast L30e --- p.63 / Chapter 3.1 --- Purification of T. celer L30e and its mutants --- p.63 / Chapter 3.2 --- Purification of yeast L30e and its mutants --- p.72 / Chapter Chapter Four - --- Thermodynamic Studies of T. celer and Yeast L30e --- p.77 / Chapter 4.1 --- Introduction --- p.77 / Chapter 4.2 --- Result --- p.79 / Chapter 4.3 --- Discussion --- p.85 / Chapter Chapter Five- --- Mutagenesis Study of a Charge Cluster in T. celer L30e --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.2 --- Result --- p.92 / Chapter 5.3 --- Structure determination of T. celer L30e mutants --- p.99 / Chapter 5.4 --- Discussion --- p.105 / Chapter Chapter Six - --- Alanine Scanning Mutagenesis of Charge Residues of T. celer L30e --- p.114 / Chapter 6.1 --- Introduction --- p.114 / Chapter 6.2 --- Result --- p.114 / Chapter 6.3 --- Discussion --- p.121 / Chapter Chapter Seven - --- Protein Engineering of T. celer and Yeast L30e --- p.132 / Chapter 7.1 --- Introduction --- p.132 / Chapter 7.2 --- Result --- p.136 / Chapter 7.3 --- Discussion --- p.138 / Chapter Chapter Eight - --- Concluding Remarks --- p.141 / Appendix --- p.143 / Reference --- p.154

Leung Chun-wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 113-122). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Acknowledgment --- p.iii / Abstract --- p.v / Table of contents --- p.ix / List of abbreviations --- p.xiv / List of figures --- p.xvii / List of tables --- p.xix / Chapter 1 --- LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Artificial sweeteners --- p.3 / Chapter 1.2.1 --- SACCHARIN --- p.3 / Chapter 1.2.2 --- cyclamate --- p.4 / Chapter 1.2.3 --- Aspartame --- p.4 / Chapter 1.2.4 --- acesulfame-k --- p.5 / Chapter 1.2.5 --- SUCRALOSE --- p.5 / Chapter 1.3 --- natural sweet plant proteins --- p.7 / Chapter 1.3.1 --- THAUMATIN --- p.7 / Chapter 1.3.2 --- MONELLIN --- p.10 / Chapter 1.3.3 --- CURCULIN --- p.11 / Chapter 1.3.4 --- PENTADIN AND BRAZZEIN --- p.11 / Chapter 1.3.5 --- MIRACULIN --- p.12 / Chapter 1.3.6 --- MABINLIN --- p.12 / Chapter 1.4 --- Genetic Engineering of Sweet Plant Protein --- p.19 / Chapter 1.4.1 --- biotechnological studies on thaumatin --- p.20 / Chapter 1.4.1.1 --- Protein modification and sweetness --- p.20 / Chapter 1.4.1.2 --- Transgenic expression in microbes --- p.21 / Chapter 1.4.1.3 --- Transgenic expression in higher plants --- p.23 / Chapter 1.4.2 --- BIOTECHNOLOGICAL STUDIES ON MONELLIN --- p.24 / Chapter 1.4.2.1 --- Gene modification and transgenic expression in microbes --- p.24 / Chapter 1.4.2.2 --- Transgenic expression in plants --- p.25 / Chapter 1.4.3 --- TRANSGENIC EXPRESSION OF MABINLIN IN PLANTS --- p.26 / Chapter 1.5 --- phaseolin and its regulatory sequences --- p.27 / Chapter 1.6 --- ARABIDOPSIS --- p.29 / Chapter 1.6.1 --- ARABIDOPSIS THALIANA as a model plant --- p.29 / Chapter 1.6.2 --- Transformation methods --- p.29 / Chapter 1.6.2.1 --- Direct DNA uptake --- p.30 / Chapter 1.6.2.2 --- Agrobacterium-mediated transformation --- p.31 / Chapter 1.6.2.3 --- In planta transformation --- p.31 / Chapter 2 --- GENKRAL INTRODUTION AND HYPOTHESIS --- p.22 / Chapter 2.1 --- General Introduction --- p.33 / Chapter 2.2 --- Hypothesis --- p.34 / Chapter 3 --- MOLECULAR STUDIES ON SWEET PROTEIN MARINLIN : THERMAL STABILITY --- p.28 / Chapter 3.1 --- Introduction --- p.38 / Chapter 3.2 --- Materials --- p.40 / Chapter 3.2.1 --- laboratory wares --- p.40 / Chapter 3.2.2 --- Equipments --- p.40 / Chapter 3.2.3 --- Chemicals --- p.40 / Chapter 3.2.4 --- commerical kits --- p.41 / Chapter 3.2.5 --- DNA primers --- p.42 / Chapter 3.2.6 --- DNA plasmids --- p.43 / Chapter 3.2.7 --- bacterial strains --- p.43 / Chapter 3.2.8 --- Plant materials --- p.44 / Chapter 3.2.9 --- Protein and Antibody --- p.44 / Chapter 3.3 --- Methods --- p.45 / Chapter 3.3.1 --- Transformation of Arabidopsis with mbliii and mbli genes --- p.45 / Chapter 3.3.1.1 --- Construction of mutant MBLIII and MBLI genes containing single codon mutation by megaprimer PCR --- p.45 / Chapter 3.3.1.2 --- Cloning of PCR-amplified MBLIII and MBLI cDNAs into vector pD3-8 --- p.48 / Chapter 3.3.1.3 --- In vitro site-directed mutagensis (for the construction of MBLIII and MBLI cDNAs containing single codon mutation) --- p.49 / Chapter 3.3.1.4 --- Cloning of the wild-type and mutated MBLIII and MBLI cDNA into vector pTZ / phas --- p.53 / Chapter 3.3.1.5 --- Confirmation of sequence fidelity and mutated codon in MBLIII and MBLI cDNA by DNA sequencing --- p.53 / Chapter 3.3.1.6 --- Transfer of wild-type MBLIII and MBLI cDNA flanked by phaseolin regulatory sequence into Agrobacterium binary vector --- p.55 / Chapter 3.3.1.7 --- Transformation of Agrobacterium with pBI / phas / MBLIII and pBI / phas / MBLI chimeric gene constructs --- p.57 / Chapter 3.3.1.8 --- Vacuum infiltration transformation of A rabidopsis --- p.58 / Chapter 3.3.1.9 --- Screening of homozygous transgenic Arabidopsis --- p.59 / Chapter 3.3.2 --- Expression analysis of MBLIII transgene --- p.61 / Chapter 3.3.2.1 --- GUS assay of transgenic plants --- p.61 / Chapter 3.3.2.2 --- Genomic DNA isolation from transgenic plants --- p.61 / Chapter 3.3.2.3 --- PCR amplification of transgene --- p.62 / Chapter 3.3.2.4 --- Total RNA isolation from transgenic Arabidopsis --- p.63 / Chapter 3.3.2.5 --- RT-PCR of total RNA from transgenic Arabidopsis --- p.64 / Chapter 3.3.2.6 --- Verification of the presence of mutagenic site and the fidelity of RNA transcript from transgenic Arabidopsis --- p.65 / Chapter 3.3.2.7 --- Protein extraction and tricine SDS-PAGE of putative transgenic protein from Arabidopsis --- p.65 / Chapter 3.3.2.8 --- N-terminal amino acid sequencing --- p.66 / Chapter 3.3.2.9 --- Isoelectric precipitation of MBL --- p.67 / Chapter 3.3.2.10 --- Production of polyclonal antibody against purified MBL --- p.67 / Chapter 3.3.2.11 --- Western-blotting and immunodectection of Arabidopsis protein by anti-MBL polyclonal antibody --- p.69 / Chapter 3.4 --- results & discussion --- p.71 / Chapter 3.4.1 --- Site-specific mutations of Arginine residue in mbliii cdna and glutamine in mbli cdna --- p.71 / Chapter 3.4.1.1 --- Megaprimer PCR --- p.71 / Chapter 3.4.1.2 --- Cloning into the seed-specific expression vector pD38 --- p.74 / Chapter 3.4.1.3 --- In vitro site-directed mutagenesis --- p.76 / Chapter 3.4.2 --- Construction of plant expression vectors containing chimeric MBLIII and MBLI --- p.80 / Chapter 3.4.2.1 --- Cloning of MBLIII and MBLI cDNAs into the seed-specific expression vector pTZ / phas --- p.80 / Chapter 3.4.2.2 --- Cloning into the plant expression vector pBI121 --- p.83 / Chapter 3.4.3 --- Generation of homozygous transgenic Arabidopsis --- p.84 / Chapter 3.4.3.1 --- Screening of transgenic R1 Arabidopsis --- p.84 / Chapter 3.4.3.2 --- Screening of transgenic R2 plants --- p.86 / Chapter 3.4.3.3 --- Screening of homozygous R3 transgenic plants --- p.88 / Chapter 3.4.4 --- Detection of MBLIII transgene in Arabidopsis --- p.89 / Chapter 3.4.4.1 --- Gus Assay --- p.89 / Chapter 3.4.4.2 --- Detection of transgene integration --- p.90 / Chapter 3.4.5 --- DETECTION of MBLIII TRANSCRIPT IN TRANSGENIC arabidopsis --- p.92 / Chapter 3.4.5.1 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.92 / Chapter 3.4.5.2 --- Verification of the presence of the mutant codon and sequence fidelity of the RT-PCR product --- p.94 / Chapter 3.4.6 --- DETECTION OF MBL III PROTEIN IN TRANSGENIC arabidopsis --- p.97 / Chapter 3.4.6.1 --- Expression of MBL protein --- p.97 / Chapter 3.4.6.2 --- Isoelectric precipitation --- p.101 / Chapter 3.4.6.3 --- Assay of titers and quality of primary polyclonal antibody against purified MBL protein --- p.103 / Chapter 3.4.6.4 --- Western blot / Immunodetection --- p.106 / Chapter 4 --- GENERAL DISCUSSION --- p.109 / Conclusion --- p.112 / References --- p.113

Fung Ka Leung. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 83-93). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract --- p.v / 摘要 --- p.vii / Table of Contents --- p.viii / List of Tables --- p.xii / List of Figures --- p.xiii / List of Abbreviations --- p.xv / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- An overview of the secretory pathway in eukaryotic cells --- p.2 / Chapter 1.2 --- The secretory pathway in plants --- p.4 / Chapter 1.2.1 --- Plant cells contain two functionally distinct vacuoles --- p.4 / Chapter 1.2.2 --- Three vesicular pathways to two vacuole --- p.6 / Chapter 1.2.3 --- Transport vesicles in the three vesicular pathways --- p.9 / Chapter 1.2.4 --- Vacuolar sorting determinants (VSDs) --- p.10 / Chapter 1.2.5 --- Vacuolar sorting receptors (VSRs) --- p.12 / Chapter 1.3 --- The PSVs in mature seeds --- p.15 / Chapter 1.3.1 --- Biogenesis of PSV --- p.15 / Chapter 1.3.2 --- The two chimeric integral membrane reporters --- p.16 / Chapter 1.3.3 --- Subcellular localization of the two chimeric integral membrane reporters in PSVs of mature tobacco seeds --- p.17 / Chapter 1.4 --- Project objectives --- p.19 / Chapter Chapter 2 --- Materials and Methods --- p.20 / Chapter 2.1 --- Construction of the YFP-BP-80 and the YFP- a -TIP reporters --- p.21 / Chapter 2.1.1 --- The pYFP-BP-80-K construct --- p.21 / Chapter 2.1.2 --- The pYFP- a -TIP-K construct --- p.22 / Chapter 2.2 --- Construction of GFP-RMR reporter --- p.23 / Chapter 2.2.1 --- Cloning of pGFP-RMR --- p.23 / Chapter 2.2.2 --- Cloning of pGFP-RMR-K --- p.23 / Chapter 2.3 --- Construction of pGONST1-YFP construct --- p.26 / Chapter 2.3.1 --- The pGONSTl-YFP construct --- p.26 / Chapter 2.4 --- Transformation of Agrobacterium by electroporation --- p.27 / Chapter 2.5 --- Tobacco transformation and selection --- p.28 / Chapter 2.5.1 --- Plant materials --- p.28 / Chapter 2.5.2 --- Tobacco transformation --- p.28 / Chapter 2.6 --- Screening of transgenic tobacco plants expressing YFP fusion proteins --- p.30 / Chapter 2.6.1 --- Kanamycin screening --- p.30 / Chapter 2.6.2 --- Extraction of genomic DNA from leaves --- p.30 / Chapter 2.6.3 --- PCR of genomic DNA --- p.31 / Chapter 2.7 --- Southern blot analysis of genomic DNA --- p.32 / Chapter 2.8 --- Western blot analysis of transgenic tobacco plants --- p.33 / Chapter 2.8.1 --- Extraction of total protein from tobacco leaves or seeds --- p.33 / Chapter 2.8.2 --- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis --- p.34 / Chapter 2.9 --- Confocal immunofluorescence studies --- p.35 / Chapter 2.9.1 --- Preparation of sections --- p.35 / Chapter 2.9.2 --- Single labeling --- p.35 / Chapter 2.9.3 --- Double labeling with one polyclonal and one monoclonal antibodies --- p.36 / Chapter 2.9.4 --- Double labeling with two polyclonal antibodies --- p.36 / Chapter 2.9.5 --- Collection of images --- p.37 / Chapter 2.10 --- Chemicals --- p.38 / Chapter 2.11 --- Primers --- p.38 / Chapter 2.12 --- Bacterial strain --- p.38 / Chapter 2.13 --- Antibodies --- p.39 / Chapter 2.14 --- Growing condition of transgenic plants and determining the developmental stage of tobacco flowers --- p.39 / Chapter Chapter 3 --- Results --- p.41 / Chapter 3.1 --- Generation of transgenic tobacco plants --- p.42 / Chapter 3.2 --- PCR screening of transgenic tobacco plants --- p.46 / Chapter 3.3 --- Southern blot analysis --- p.48 / Chapter 3.4 --- Detection of the YFP fusion proteins in transgenic tobacco plants by western blot analysis --- p.50 / Chapter 3.4.1 --- Detection of the YFP fusion proteins in leaves --- p.50 / Chapter 3.4.2 --- Western blot analysis of vegetative tissues --- p.57 / Chapter 3.4.3 --- Western blot analysis of mature seeds --- p.59 / Chapter 3.5 --- Confocal immunofluorescence studies --- p.61 / Chapter 3.5.1 --- Detection of YFP signals in root tip cells --- p.61 / Chapter 3.5.2 --- Detection of YFP signals in developing seeds --- p.65 / Chapter 3.5.3 --- Subcellular localization of the YFP fusion proteins in mature seeds --- p.67 / Chapter Chapter 4 --- Discussion --- p.72 / Chapter Chapter 5 --- Summary and Future Perspectives --- p.77 / Chapter 5.1 --- Summary --- p.78 / Chapter 5.1.1 --- Generation of transgenic tobacco plants expressing the YFP fusion proteins --- p.78 / Chapter 5.1.2 --- Full-length fusion proteins and cleaved soluble YFP were detected in vegetative tissues --- p.79 / Chapter 5.1.3 --- Only cleaved soluble YFP was detected in mature seeds --- p.79 / Chapter 5.1.4 --- The two fusion proteins might localized in different compartments in developing seeds --- p.79 / Chapter 5.1.5 --- Both fusion proteins were localized within the PSVs of mature seeds --- p.80 / Chapter 5.2 --- Future perspectives --- p.81 / References --- p.83

Leung, Hoi Ching. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 114-128). / Abstracts in English and Chinese. / THESIS/ASSESSMENT COMMITTEE --- p.i / STATEMENT FROM AUTHOR --- p.ii / ACKNOWLEDGEMENTS --- p.iii / ABSTRACT --- p.v / TABLE OF CONTENTS --- p.xi / LIST OF FIGURES --- p.xvi / LIST OF TABLES --- p.xviii / LIST OF ABBREVIATIONS --- p.xix / Chapter CHAPTER 1. --- GENERAL INTRODUCTION --- p.1 / Chapter CHAPTER 2. --- LITERATURE REVIEW --- p.4 / Chapter 2.1 --- Nutritional quality of rice --- p.4 / Chapter 2.1.1 --- Classification of seed proteins --- p.4 / Chapter 2.1.2 --- Amino acid composition of rice proteins --- p.5 / Chapter 2.1.3 --- Other nutritional components of rice --- p.6 / Chapter 2.2 --- Rice seed storage proteins --- p.7 / Chapter 2.2.1 --- Properties and classification of seed storage proteins --- p.7 / Chapter 2.2.2 --- Composition and stucture --- p.9 / Chapter 2.2.2.1 --- Glutelin --- p.9 / Chapter 2.2.2.2 --- Prolamin --- p.10 / Chapter 2.2.2.3 --- Albumin and globulin --- p.12 / Chapter 2.2.3 --- "Synthsis, assembly and deposition of rice seed storage proteins" --- p.13 / Chapter 2.2.3.1 --- Storage protein folding and assembly in the ER --- p.14 / Chapter 2.2.3.2 --- Storage protein transport and protein body formation --- p.16 / Chapter 2.2.3.3 --- Protein bodies and their distribution in endosperm --- p.18 / Chapter 2.3 --- Transgenic approaches to improve the nutritional quality of rice seed proteins --- p.19 / Chapter 2.3.1 --- General introduction --- p.19 / Chapter 2.3.2 --- Attempts to improve the nutritional quality of seed proteins --- p.20 / Chapter 2.3.3 --- Rice grain quality improvement by genetic engineering --- p.22 / Chapter 2.3.3.1 --- Increase in the lysine content of rice endosperm --- p.22 / Chapter 2.2.3.2 --- Other examples of rice nutritional quality improvement --- p.25 / Chapter 2.3.4 --- Expression of recombinant protein in transgenic plants --- p.26 / Chapter 2.3.5 --- Effects of recombinant proteins on the high-lysine rice --- p.27 / Chapter 2.4 --- Proteomics --- p.28 / Chapter 2.4.1 --- General overview --- p.28 / Chapter 2.4.1.1 --- Two-dimensional polyacrylamide gel electrophoresis for proteome analysis --- p.29 / Chapter 2.4.1.2 --- Protein visualization --- p.32 / Chapter 2.4.1.3 --- Computer-aided image analysis --- p.34 / Chapter 2.4.1.4 --- Mass spectrometry-based methods for protein identification --- p.35 / Chapter 2.4.1.5 --- Database search --- p.36 / Chapter 2.4.1.6 --- Protein sequence database --- p.37 / Chapter 2.4.2 --- Plant proteomics --- p.40 / Chapter 2.4.2.1 --- Rice proteomics --- p.41 / Chapter 2.4.2.2 --- Comparative proteomics --- p.43 / Chapter 2.5 --- Hypothesis and objectives --- p.45 / Chapter CHAPTER 3. --- MATERIALS AND METHODS --- p.47 / Chapter 3.1 --- Materials --- p.47 / Chapter 3.1.1 --- Chemicals and commercial kits --- p.47 / Chapter 3.1.2 --- Instruments --- p.47 / Chapter 3.1.3 --- Softwares --- p.48 / Chapter 3.1.4 --- Plant materials --- p.48 / Chapter 3.2 --- Methods --- p.49 / Chapter 3.2.1 --- Collection of developing rice seeds --- p.49 / Chapter 3.2.2 --- Extraction of rice seed proteins --- p.51 / Chapter 3.2.2.1 --- Extraction of total protein --- p.51 / Chapter 3.2.3.2 --- Extraction of four fractions of rice seed proteins --- p.51 / Chapter 3.2.3 --- 2D gel electrophoresis --- p.53 / Chapter 3.2.3.1 --- Protein precipitation and quantification --- p.53 / Chapter 3.2.3.2 --- Isoelectric focusing (IEF) --- p.54 / Chapter 3.2.3.3 --- IPG strips equilibration --- p.54 / Chapter 3.2.3.4 --- Second-dimension SDS-PAGE --- p.55 / Chapter 3.2.3.5 --- Silver staining of 2D gel --- p.55 / Chapter 3.2.3.6 --- Image and data analysis --- p.56 / Chapter 3.2.4 --- MALDI-ToF mass spectrometry (Matrix Assisted Laser Desorption Ionization-Time of Flight) --- p.56 / Chapter 3.2.4.1 --- Sample destaining --- p.56 / Chapter 3.2.4.2 --- In-gel digestion with trypsin --- p.57 / Chapter 3.2.4.3 --- Desalination of the digested sample with Zip Tip --- p.58 / Chapter 3.2.4.4 --- Protein identification by mass spectrometry and database searching --- p.58 / Chapter 3.2.5 --- Detection of LRP fusion protein in 2D PAGE --- p.59 / Chapter 3.2.5.1 --- 2D gel electrophoresis --- p.59 / Chapter 3.2.5.2 --- Western blotting using anti-LRP antibody --- p.60 / Chapter 3.2.6 --- Antiserum production --- p.61 / Chapter 3.2.6.1 --- Purification of glutelin and prolamin proteins --- p.61 / Chapter 3.2.6.2 --- Immunization of rabbits and mice --- p.62 / Chapter 3.2.6.3 --- Testing of antibody specificity --- p.62 / Chapter 3.2.7 --- Transmission electron microscopy (TEM) --- p.63 / Chapter 3.2.7.1 --- Sample fixation and section preparation --- p.63 / Chapter 3.2.7.2 --- TEM observation --- p.64 / Chapter 3.2.7.3 --- Immunocytochemical observation --- p.64 / Chapter CHAPTER 4. --- RESULTS --- p.66 / Chapter 4.1 --- Proteomic analysis of high-lysine rice --- p.66 / Chapter 4.1.1 --- Extraction of proteins --- p.66 / Chapter 4.1.2 --- The proteomic profiles of different storage proteins in developing high-lysine rice seeds --- p.67 / Chapter 4.1.3 --- Quantitative analysis of protein spots --- p.76 / Chapter 4.1.4 --- Proteomic analysis of salt-soluble proteins --- p.79 / Chapter 4.1.5 --- Proteomic analysis of alcohol-soluble proteins --- p.81 / Chapter 4.1.6 --- Proteomic analysis of salt-soluble proteins --- p.82 / Chapter 4.1.7 --- Proteomic analysis of water-soluble proteins --- p.89 / Chapter 4.1.8 --- Comparison of changes in expression patterns of specific proteins in the high lysine rice --- p.89 / Chapter 4.2 --- Antibody production --- p.92 / Chapter 4.2.1 --- The production of anti-prolamin and anti-glutelin antibodies --- p.92 / Chapter 4.2.2 --- The specificity of anti-prolamin and anti-glutelin antibodies --- p.93 / Chapter 4.3 --- Transmission electron microscopy observation of rice protein bodies --- p.95 / Chapter 4.3.1 --- Morphology of protein bodies in high-lysine rice --- p.95 / Chapter 4.3.2 --- Subcellular localization of storage proteins and LRP --- p.98 / Chapter CHAPTER 5. --- DISCUSSION --- p.100 / Chapter 5.1 --- Protein profiling of LRP fusion protein and its effects on the expression of other proteins --- p.100 / Chapter 5.2 --- Over-expression of glutelin and its effects on the expression of other proteins --- p.102 / Chapter 5.3 --- Formation of malformed protein bodies and deposition of storage proteins --- p.103 / Chapter 5.4 --- Relationship between changes in protein expression and the Unfolded Protein Response --- p.105 / Chapter 5.5 --- Effects of transgenes on rice grain quality --- p.108 / Chapter 5.6 --- Allergenic effects of transgenic rice --- p.109 / Chapter 5.7 --- Future perspectives --- p.110 / Chapter CHAPTER 6. --- CONCLUSIONS --- p.112 / REFERENCES --- p.114

Cellulose binding domains (CBDs) are found in cellulolytic microorganisms as discrete domains in free cellulases or as cellulosomes, which are extracellular multi-enzyme complexes. CBDs bind to cellulose and help the catalytic domains to access cellulose substrates. CBDs are used as affinity tags for immobilizing cells, proteins or molecules on cellulose matrices. They can also be used in protein engineering to alter protein expression and solubilities. Cohesins and dockerins are domains exclusive to cellulosomes. They interact with high affinity and the interactions are Ca2+-dependent. Chelation of Ca2+ causes irreversible conformational change to dockerins thus disrupting the interactions. The first aim of this project was to validate a putative CBD from endoglucanase EngD of C. cellulovorans, to test its effect on solubility as a fusion in chimeras. The second aim was to use chimeric proteins containing CBD, cohesin, dockerin and LG to establish a system for efficient cell immobilization, expansion and harvest in hollow cellulose fibres. A putative CBD from an enzyme with its natural linker (PTCBDengD), a CBD from a scaffoldin (CBDcbpA), three cohesin domains from different strains (Cip7, Coh6 and CipC1) and an antibody binding protein (LG) were used to construct various chimeric and fusion proteins. The two CBDs were fused to different cohesins and LG respectively and the chimeras? solubility was analyzed. The results showed that fusing with CBDcbpA did not significantly help to increase the solubility of the insoluble domain Coh6 and it even greatly reduced the solubility of the soluble domain CipC1. In contrast, PTCBDengD fusion increased the solubility of Coh6 by three fold and it did not alter the solubility of soluble protein/ domains. These results suggested that PTCBDengD may be a better domain to use as a fusion tag for expression and other biotechnology applications. Cellulose binding specificity of PTCBDengD and its chimeric proteins were tested and SDS-PAGE analysis results clearly demonstrated that PTCBDengD and its chimeras specifically bound to crystalline cellulose Avicel and non-crystalline cellulose Cuprophan. The results confirmed that PTCBDengD is a true CBD. Chimeric protein pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc were used to build the scaffold on Cuprophan hollow cellulose fibre for reversible cell immobilization studies. Cell adhesion assay results showed that the double-chimera systems efficiently immobilize cells onto Cuprophan hollow fibre. Dissociation of LG-Doc from amorphous cellulose Cuprophan-bound CBDcbpA-Cip7 by EDTA treatment resulted in decrease of cell binding by almost 90%; however, re-association of LG-Doc after EDTA dissociation only achieved 50% efficiency of cell immobilization. Dot blot and SDS-PAGE analysis showed that dissociation/ re-association of LG-Doc to Cip7-PTCBDengD could be decreased in was interfered by the presence of cellulose. Preliminary results indicated that crystalline cellulose Avicel may improve dissociation/ re-association efficiency. In conclusion, studies on recombinant proteins validated CBDengD's specific affinity to cellulose and its solubilizing effect on its fusion partner. Chimera pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc are effective in cell immobilization. However, optimization is required to develop recyclable protein scaffolding and complexes on cellulosic matrices.

Transcription factors GATA-1, Fli-1 and FOG-1 are essential proteins for normal megakaryopoiesis, however, the detailed analyses of their functions within developmental stages of megakaryopoiesis are lacking. In my thesis, over expression of gene in target cells was adopted as the main strategy to study the biological functions of these proteins, therefore, an efficient gene delivery method was first developed by using retrovirus.This approach was then utilized to over express GATA-1, Fli-1 and FOG-1 in murine leukemia M1 cells and mouse hematopoietic stem cells (HSCs), and their effects on different developmental stages of megakaryopoiesis were investigated. In the transduced M1 cells, enforced expression of GATA-1 and Fli-1 was found to induce the megakaryocytic development, which was associated with the formation of megakaryocyte (Mk) and the increased expression of Mk specific genes c-Mpl and GPIX. In the transduced mouse HSCs, it was found that the expression of endogenous GATA-1, Fli-1 and FOG-1 was up-regulated throughout Mk differentiation; enforced expression of these transcription factors led to the significantly enhanced Mk development. Megakaryocytes over expressing GATA-1, Fli-1 and FOG-1 were characterized by the increased expression of various Mk-specific genes including GPIX, c-Mpl, platelet factor 4 (PF4), acetylcholinesterase (AChE) and NF-E2, an important transcription factor for terminal megakaryopoiesis; however, GATA-1, Fli-1 and FOG-1 displayed the different abilities in promoting the proliferation of hematopoietic cells and MK differentiation, as well as regulating other transcription factors involved in hematopoiesis. To further elucidate the role of the functional domains of Fli-1, various mutants of Fli-1 were also over expressed in mouse HSCs. The results demonstrated that first, the combination of the activation domain of Fli-1 and its Ets domain is required for early megakaryopoiesis but not sufficient for terminal megakaryopoiesis; second, DNA binding of Fli-1 was not the only requirement for early Mk enhancement, moreover, the interaction between Fli-1 and GATA-1 through the Ets domain and the resultant transcriptional synergy was the essential determinant for Fli?1 ability in Mk development. Taken together, the studies presented in this thesis provided strong in vitro evidence that GATA-1, Fli-1 and FOG-1 indeed play the critical roles in normal megakaryopoiesis.

The aim of this thesis was to produce DNA expression constructs and use them to investigate the feasibility of recombinantly expression proteins for future interaction studies between human RBBP6 and p53 and pRb proteins.

The aim of this thesis was to produce DNA expression constructs and use them to investigate the feasibility of recombinantly expression proteins for future interaction studies between human RBBP6 and p53 and pRb proteins.

Transcription factors GATA-1, Fli-1 and FOG-1 are essential proteins for normal megakaryopoiesis, however, the detailed analyses of their functions within developmental stages of megakaryopoiesis are lacking. In my thesis, over expression of gene in target cells was adopted as the main strategy to study the biological functions of these proteins, therefore, an efficient gene delivery method was first developed by using retrovirus.This approach was then utilized to over express GATA-1, Fli-1 and FOG-1 in murine leukemia M1 cells and mouse hematopoietic stem cells (HSCs), and their effects on different developmental stages of megakaryopoiesis were investigated. In the transduced M1 cells, enforced expression of GATA-1 and Fli-1 was found to induce the megakaryocytic development, which was associated with the formation of megakaryocyte (Mk) and the increased expression of Mk specific genes c-Mpl and GPIX. In the transduced mouse HSCs, it was found that the expression of endogenous GATA-1, Fli-1 and FOG-1 was up-regulated throughout Mk differentiation; enforced expression of these transcription factors led to the significantly enhanced Mk development. Megakaryocytes over expressing GATA-1, Fli-1 and FOG-1 were characterized by the increased expression of various Mk-specific genes including GPIX, c-Mpl, platelet factor 4 (PF4), acetylcholinesterase (AChE) and NF-E2, an important transcription factor for terminal megakaryopoiesis; however, GATA-1, Fli-1 and FOG-1 displayed the different abilities in promoting the proliferation of hematopoietic cells and MK differentiation, as well as regulating other transcription factors involved in hematopoiesis. To further elucidate the role of the functional domains of Fli-1, various mutants of Fli-1 were also over expressed in mouse HSCs. The results demonstrated that first, the combination of the activation domain of Fli-1 and its Ets domain is required for early megakaryopoiesis but not sufficient for terminal megakaryopoiesis; second, DNA binding of Fli-1 was not the only requirement for early Mk enhancement, moreover, the interaction between Fli-1 and GATA-1 through the Ets domain and the resultant transcriptional synergy was the essential determinant for Fli?1 ability in Mk development. Taken together, the studies presented in this thesis provided strong in vitro evidence that GATA-1, Fli-1 and FOG-1 indeed play the critical roles in normal megakaryopoiesis.

Cellulose binding domains (CBDs) are found in cellulolytic microorganisms as discrete domains in free cellulases or as cellulosomes, which are extracellular multi-enzyme complexes. CBDs bind to cellulose and help the catalytic domains to access cellulose substrates. CBDs are used as affinity tags for immobilizing cells, proteins or molecules on cellulose matrices. They can also be used in protein engineering to alter protein expression and solubilities. Cohesins and dockerins are domains exclusive to cellulosomes. They interact with high affinity and the interactions are Ca2+-dependent. Chelation of Ca2+ causes irreversible conformational change to dockerins thus disrupting the interactions. The first aim of this project was to validate a putative CBD from endoglucanase EngD of C. cellulovorans, to test its effect on solubility as a fusion in chimeras. The second aim was to use chimeric proteins containing CBD, cohesin, dockerin and LG to establish a system for efficient cell immobilization, expansion and harvest in hollow cellulose fibres. A putative CBD from an enzyme with its natural linker (PTCBDengD), a CBD from a scaffoldin (CBDcbpA), three cohesin domains from different strains (Cip7, Coh6 and CipC1) and an antibody binding protein (LG) were used to construct various chimeric and fusion proteins. The two CBDs were fused to different cohesins and LG respectively and the chimeras? solubility was analyzed. The results showed that fusing with CBDcbpA did not significantly help to increase the solubility of the insoluble domain Coh6 and it even greatly reduced the solubility of the soluble domain CipC1. In contrast, PTCBDengD fusion increased the solubility of Coh6 by three fold and it did not alter the solubility of soluble protein/ domains. These results suggested that PTCBDengD may be a better domain to use as a fusion tag for expression and other biotechnology applications. Cellulose binding specificity of PTCBDengD and its chimeric proteins were tested and SDS-PAGE analysis results clearly demonstrated that PTCBDengD and its chimeras specifically bound to crystalline cellulose Avicel and non-crystalline cellulose Cuprophan. The results confirmed that PTCBDengD is a true CBD. Chimeric protein pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc were used to build the scaffold on Cuprophan hollow cellulose fibre for reversible cell immobilization studies. Cell adhesion assay results showed that the double-chimera systems efficiently immobilize cells onto Cuprophan hollow fibre. Dissociation of LG-Doc from amorphous cellulose Cuprophan-bound CBDcbpA-Cip7 by EDTA treatment resulted in decrease of cell binding by almost 90%; however, re-association of LG-Doc after EDTA dissociation only achieved 50% efficiency of cell immobilization. Dot blot and SDS-PAGE analysis showed that dissociation/ re-association of LG-Doc to Cip7-PTCBDengD could be decreased in was interfered by the presence of cellulose. Preliminary results indicated that crystalline cellulose Avicel may improve dissociation/ re-association efficiency. In conclusion, studies on recombinant proteins validated CBDengD's specific affinity to cellulose and its solubilizing effect on its fusion partner. Chimera pairs CBDcbpA-Cip7/ LG-Doc and Cip7-PTCBDengD/ LG-Doc are effective in cell immobilization. However, optimization is required to develop recyclable protein scaffolding and complexes on cellulosic matrices.