Membrane anchor for vacuolar targeting: expression of a human lysosomal enzyme iduronidase (hIDUA) in transgenic tobacco plants.

Seto Tai Chi.Thesis (M.Phil.)--Chinese University of Hong Kong, 2005.Includes bibliographical references (leaves 122-138).Abstracts in English and Chinese.Thesis Committee --- p.iiStatement --- p.iiiAcknowledgements --- p.ivAbstract (in English) --- p.vAbstract (in Chinese) --- p.viiTable of Contents --- p.ixList of Tables --- p.xviList of Figures --- p.xvChapter Chapter 1 --- General Introduction and Literature Review --- p.1Chapter 1.1 --- Introduction --- p.2Chapter 1.2 --- Tobacco seed as bioreactor --- p.4Chapter 1.2.1 --- Advantages of using tobacco seed to produce bioactive human lysosomal enzyme --- p.4Chapter 1.2.2 --- Disadvantages and potential problems of using tobacco seed to produce bioactive human lysosomal enzyme --- p.5Chapter 1.2.2.1 --- Difference of asparagine-linked N-glycosylation between plant and human protein --- p.8Chapter 1.2.2.2 --- Immunogenicity of recombinant protein with plant-derived N-glycan to human --- p.10Chapter 1.2.2.3 --- "Strategy to ""humanize"" plant-derived recombinant human lysosomal enzyme" --- p.10Chapter 1.2.2.4 --- Lack of specific glycan structure一mannose-6-phosphate (M6P) tag addition --- p.11Chapter 1.2.2.5 --- Strategy for M6P tag addition on plant-derived human lysosomal enzyme --- p.12Chapter 1.3 --- The plant secretory pathway --- p.13Chapter 1.3.1 --- Plant vacuole in tobacco seed --- p.16Chapter 1.3.2 --- Soluble protein trafficking in plant cell --- p.17Chapter 1.3.3 --- Integral membrane protein trafficking in plant cell --- p.17Chapter 1.3.4 --- Components involved in integral membrane protein trafficking to PSV crystalloid --- p.19Chapter 1.3.4.1 --- BP-80 (80-kDa binding protein) --- p.19Chapter 1.3.4.2 --- α-TIP (α-tonoplast intrinsic protein) --- p.20Chapter 1.3.5 --- Using specific integral membrane protein trafficking system to target recombinant human lysosomal enzyme to tobacco seed PSV --- p.21Chapter 1.4 --- Homo sapiens α-L-iduronidase (hIDUA) --- p.21Chapter 1.4.1 --- Global situation of lysosomal storage disease一hIDUA deficiency --- p.21Chapter 1.4.2 --- Physiological role --- p.22Chapter 1.4.3 --- Molecular property --- p.24Chapter 1.4.3.1 --- Mutation and polymorphism --- p.24Chapter 1.4.4 --- Lysosomal secretory pathway --- p.24Chapter 1.4.5 --- Biochemical property --- p.25Chapter 1.4.6 --- Clinical application --- p.27Chapter 1.4.6.1 --- Enzyme replacement therapy (ERT) --- p.27Chapter 1.4.6.2 --- Clinical trial --- p.28Chapter 1.4.6.3 --- Economic value --- p.29Chapter 1.4.7 --- Expression system --- p.29Chapter 1.4.7.1 --- Production (overexpression) of rhIDUA in CHO cell system --- p.30Chapter 1.4.7.2 --- Production of rhIDUA in tobacco plant leaf --- p.30Chapter 1.5 --- Project objective and long-term significance --- p.30Chapter 1.5.1 --- Project objective --- p.30Chapter 1.5.2 --- Long-term significance --- p.31Chapter Chapter 2 --- Generation and Characterization of Anti-IDUA Antibodies --- p.32Chapter 2.1 --- Introduction --- p.33Chapter 2.2 --- Materials --- p.33Chapter 2.2.1 --- Chemical --- p.33Chapter 2.3 --- Methods --- p.35Chapter 2.3.1 --- Generation of polyclonal anti-IDUA antibody --- p.35Chapter 2.3.1.1 --- Design of synthetic peptide --- p.35Chapter 2.3.1.2 --- Conjugation of synthetic peptide to carrier protein --- p.39Chapter 2.3.1.3 --- Immunization of rabbit --- p.39Chapter 2.3.2 --- Characterization of polyclonal anti-IDUA antibody in rabbit serum --- p.40Chapter 2.3.2.1 --- Dot-blot analysis --- p.40Chapter 2.3.3 --- Purification of polyclonal anti-IDUA antibody --- p.42Chapter 2.3.3.1 --- Construction of anti-IDUA antibody affinity column --- p.42Chapter 2.3.3.2 --- Affinity-purification of anti-IDUA antibody --- p.42Chapter 2.3.4 --- Western blot detection of denatured rhIDUA --- p.42Chapter 2.4 --- Results --- p.43Chapter 2.4.1 --- Characterization of polyclonal anti-IDUA antibody --- p.43Chapter 2.5 --- Discussion --- p.51Chapter 2.6 --- Conclusion --- p.51Chapter Chapter 3 --- Generation and Characterization of Transgenic Tobacco Plants Expressing rhIDUA Fusions --- p.52Chapter 3.1 --- Introduction --- p.53Chapter 3.1.1 --- Signal peptide of hIDUA (hIDUA SP) --- p.54Chapter 3.1.2 --- Signal peptide of proaleurain (Pro. SP) --- p.54Chapter 3.1.3 --- Hypothesis to be tested in this study --- p.54Chapter 3.2 --- Materials --- p.55Chapter 3.2.1 --- Chemical --- p.55Chapter 3.2.2 --- Primers --- p.55Chapter 3.2.3 --- Bacterial strain --- p.58Chapter 3.2.4 --- The insert-Homo sapiens α-L-iduronidase (hIDUA) cDNA used in this study --- p.58Chapter 3.2.5 --- The vector-pLJ526 used in this study --- p.59Chapter 3.3 --- Methods --- p.61Chapter 3.3.1 --- Construction of chimeric gene construct --- p.61Chapter 3.3.1.1 --- Restriction endonuclease´ؤPfIMIl --- p.61Chapter 3.3.1.2 --- Recombinant DNA and molecular cloning techniques used in this study --- p.61Chapter 3.3.1.3 --- Cloning of pSPIDUA-FLAG --- p.62Chapter 3.3.1.4 --- Cloning of pSPIDUA-control --- p.62Chapter 3.3.1.5 --- Cloning of a universal construct (pUniversal) --- p.62Chapter 3.3.1.6 --- Cloning of pSP-IDUA-T7 --- p.66Chapter 3.3.1.7 --- Cloning of pSP-IDUA-control --- p.66Chapter 3.3.1.8 --- Cloning of chimeric gene construct into Agrobacterium binary vector --- p.66Chapter 3.3.2 --- Expression of chimeric gene construct in tobacco plant --- p.73Chapter 3.3.2.1 --- Tobacco plant --- p.73Chapter 3.3.2.2 --- Electroporation of Agrobacterium --- p.73Chapter 3.3.2.3 --- Agrobacterium-mediated transformation of tobacco plant --- p.74Chapter 3.3.2.4 --- Selection and regeneration of tobacco transformant --- p.75Chapter 3.3.3 --- Characterization of transgenic tobacco plant expressing rhIDUA fusion --- p.75Chapter 3.3.3.1 --- Genomic DNA polymerase chain reaction (PCR) --- p.75Chapter 3.3.3.2 --- Southern blot analysis --- p.76Chapter 3.3.3.3 --- Total RNA reverse transcription-PCR (RT-PCR) --- p.77Chapter 3.3.3.4 --- Northern blot analysis of tobacco leaf --- p.78Chapter 3.3.3.5 --- Western blot analysis --- p.79Chapter 3.3.4 --- Purification of plant-derived rhIDUA fusion --- p.81Chapter 3.3.4.1 --- Construction of affinity column with anti-IDUA antibody --- p.81Chapter 3.3.4.2 --- Affinity-purification of rhIDUA fusion from tobacco mature seed --- p.81Chapter 3.3.5 --- Confocal immunoflorescence study --- p.82Chapter 3.3.5.1 --- Preparation of paraffin section --- p.82Chapter 3.3.5.2 --- Single immunocytochemical labeling --- p.82Chapter 3.3.5.3 --- Double labeling with one monoclonal and one polyclonal antibodies --- p.83Chapter 3.3.5.4 --- Double labeling with two polyclonal antibodies --- p.83Chapter 3.3.5.5 --- Image collection --- p.84Chapter 3.4 --- Results --- p.85Chapter 3.4.1 --- Chimeric gene construction and confirmation --- p.85Chapter 3.4.2 --- Selection and regeneration of tobacco transformant with kanamycin- resistance --- p.86Chapter 3.4.3 --- Genomic DNA PCR screening of tobacco transformant --- p.88Chapter 3.4.4 --- Southern blot analysis of tobacco transformant --- p.91Chapter 3.4.5 --- Total RNA RT-PCR screening of tobacco transformant --- p.93Chapter 3.4.6 --- Northern blot analysis of tobacco transformant --- p.93Chapter 3.4.7 --- Western blot analysis --- p.96Chapter 3.4.7.1 --- Western blot analysis of pSP-IDUA-T7-121 transformant leaf --- p.96Chapter 3.4.7.2 --- Western blot analysis of pSP-IDUA-T7-121 transformant mature seed --- p.98Chapter 3.4.8 --- Affinity-purification of rhIDUA fusion --- p.98Chapter 3.4.9 --- Expression level of rhIDUA fusion --- p.102Chapter 3.4.10 --- Subcellular localization of rhIDUA fusion --- p.102Chapter 3.5 --- Discussion --- p.111Chapter Chapter 4 --- Summary and Future Perspectives --- p.117References --- p.122Appendix 1 --- p.139Appendix II (List of Abbreviations) --- p.14