Chong Lin-Tat., Thesis (M.Phil.)--Chinese University of Hong Kong, 2006., Includes bibliographical references (leaves 76-78)., s in English and Chinese., Chapter Chapter 1 --- introduction, Chapter 1.1 --- Severe acute respiratory syndrome Coronavirus (SARS CoV) --- p.13, Figure 1.1 Genome organization and putative functional ORFs of SARS CoV --- p.14, Chapter 1.2 --- SARS main protease, Chapter 1.2.1 --- Three dimensional structure --- p.15, Figure 1.2 Ribbon illustration of the SARS-coronavirus main protease --- p.17, Figure 1.3 Surface representations of P1 and P2 substrate-binding pocket of main protease --- p.18, Chapter 1.2.2 --- Substrate specificities --- p.19, Table 1.1. Eleven predicted cleavage sites of SARS CoV main protease --- p.21, Chapter 1.3 --- Protein-based FRET assay system --- p.22, Figure 1.4. The principle of fluorescent resonance energy transfer (FRET) --- p.24, Chapter 1.4 --- Objectives --- p.25, Chapter Chapter 2 --- Materials and Methods, Chapter 2.1 --- General Techniques, Chapter 2.1.1 --- Preparation and transformation of competent E. coli DH5a and23 BL21 (DE3)pLysS --- p.26, Chapter 2.1.2 --- Minipreparation of plasmid DNA (Invitrogen) --- p.27, Chapter 2.1.3 --- Spectrophotometric quantitation DNA --- p.28, Chapter 2.1.4 --- Agarose gel electrophoresis, Chapter 2.1.5 --- Purification of DNA from agarose gel (Invitrogen), Chapter 2.1.6 --- Restriction digestion of DNA fragments --- p.29, Chapter 2.1.7 --- Ligation of DNA fragments into vector, Table 2.1. Standard recipe of ligation reaction --- p.30, Chapter 2.1.8 --- SDS-PAGE electrophoresis --- p.31, Table 2.2. Standard recipe of separating gel for SDS-PAGE --- p.32, Table 2.3. Standard recipe of stacking gel for SDS-PAGE --- p.33, Chapter 2.2 --- Sub-cloning and site-directed mutagenesis, Chapter 2.2.1 --- Sub-cloning of SARS Co V main protease --- p.34, Chapter 2.2.2 --- Sub-cloning of Substrate, Chapter 2.2.3 --- Site-directed mutagenesis of substrate variant --- p.35, Table 2.4 Primer sequence for generating substrate variants --- p.36, Table 2.5. Standard recipe of Polymerase Chain Reaction (PCR) --- p.40, Table 2.6. Polymerase Chain Reaction (PCR) profile --- p.41, Chapter 2.3 --- Sample preparation, Chapter 2.3.1 --- Expression of recombinant proteins --- p.42, SARS CoV main protease, Substrate and substrate variants, Chapter 2.3.2 --- Purification of recombinant proteins, Chapter 2.4 --- Protein-based FRET kinetic analysis --- p.45, Chapter 2.5 --- A model for substrate-enzyme binding by docking simulation --- p.46, Chapter Chapter 3 --- Results, Chapter 3.1 --- Preparation of SARS CoV main protease and substrate, Chapter 3.1.1 --- Expression and purification of SARS main protease --- p.48, Figure 3.1. Purification profile of SARS CoV main protease --- p.49, Chapter 3.1.2 --- Expression and purification of substrate and substrate variants --- p.50, Figure 3.2. Purification profile of substrate and substrate variants --- p.51, Chapter 3.2 --- A novel protein-based FRET assay system was established, Chapter 3.2.1 --- "With the cleavage of active main protease, absorbance at 528nm dropped while signal at 485nm were slightly increased" --- p.52, Figure 3.3. Absorbance at 528nm dropped and 485nm increased with the substrate hydrolysis --- p.53, Chapter 3.2.2 --- FRET efficiency ratio (528/485) decreased over time --- p.54, Figure 3.4. FRET efficiency ratio (528/485) decreased over time --- p.55, Chapter 3.2.3 --- Comparable kcat/Km value of SARS CoV main protease was obtained --- p.56, Figure 3.5. Catalytic parameter (kcat/ Km) was determined from the slope of straight Line --- p.57, Chapter 3.3 --- Main protease activity towards substrate variants at different substrate-binding sites (S2'-S2) --- p.58, Table 3.1. Kinetic parameterrs of 76 substrate variants in descending order --- p.59, Chapter 3.3.1 --- S2'substrate-binding site --- p.60, Chapter 3.3.2 --- S1' substrate-b inding site, Chapter 3.3.3 --- S1 substrate-binding site, Chapter 3.3.4 --- S2 substrate-binding site, Figure 3.6. Kinetic analysis of some typical substrate variants against main protease --- p.62, Figure 3.7. SDS-PAGE analysis of some typical substrate variants against main protease --- p.63, Chapter Chapter 4 --- Discussion, Chapter 4.1 --- Quantitative and high-throughput analysis by protein-based FRET assay system --- p.64, Chapter 4.2 --- Substrate specificities of SARS CoV main protease at S2'-S2 subsites, Chapter 4.2.1 --- β-strand conformation was preferred at S2，subsite, Chapter 4.2.2 --- Residues with small aliphatic side chain were preferred at S1 ´ة subsite --- p.65, Chapter 4.2.3 --- "Glutamine at S1 subsite was absolutely conserved, but alternatives were disclosed" --- p.66, Figure 4.1. Glutamine was not absolutely conserved in S1 subsite --- p.67, Chapter 4.2.4 --- Hydrophilic residues were tolerated at S2 subsite --- p.68, Figure 4.2. Hydrophilic residues were tolerated at S2 subsite --- p.70, Table 4.1. Summary of types of residues preferred at individual subsites --- p.71, Chapter 4.3 --- Predicted conformation of substrate towards SARS CoV main protease at S2' and S1' subsites --- p.72, Figure 4.3. Small residues were preferred at S1´ة subsite and Val at S2' subsite was more favoured than the native one --- p.73, Chapter Chapter 5 --- Summary --- p.74, Chapter Chapter 6 --- Future work --- p.75, References --- p.76, http://library.cuhk.edu.hk/record=b5896512, Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/)