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Study of structural relationship between human ribosomal proteins P1 and P2.

Chiu, Yu Hin Teddy. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 118-129). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / Table of Content --- p.vi / Abbreviations --- p.x / Naming system for mutant proteins --- p.xi / Abbreviation for amino acid --- p.xii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- What are acidic ribosomal proteins? --- p.1 / Chapter 1.2 --- Why P-proteins are so important? --- p.13 / Chapter 1.3 --- Research objectives --- p.15 / Chapter Chapter 2 --- Materials and Methods --- p.17 / Chapter 2.1 --- List of buffers and media --- p.17 / Chapter 2.1.1 --- Preparation of buffers and media --- p.17 / Chapter 2.1.2 --- Buffers for preparing competent cells --- p.17 / Chapter 2.1.3 --- Media for bacterial culture --- p.17 / Chapter 2.1.4 --- Buffers for nucleic acid electrophoresis --- p.19 / Chapter 2.1.5 --- Buffers for protein electrophoresis --- p.19 / Chapter 2.1.6 --- Buffers for interaction studies using BIAcore 3000 --- p.21 / Chapter 2.2 --- General methods --- p.23 / Chapter 2.2.1 --- Preparation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.2 --- Transformation of Escherichia coli (E.coli.) competent cells --- p.23 / Chapter 2.2.3 --- DNA cloning --- p.24 / Chapter 2.2.3.1 --- DNA cloning by polymerase chain reaction (PCR) --- p.24 / Chapter 2.2.3.2 --- Agarose gel electrophoresis of DNA --- p.25 / Chapter 2.2.3.3 --- Extraction and purification of DNA from agarose gels --- p.25 / Chapter 2.2.3.4 --- Restriction digestion of DNA --- p.25 / Chapter 2.2.3.5 --- Ligation of digested insert and expression vector --- p.27 / Chapter 2.2.3.6 --- Verification of insert by PCR --- p.27 / Chapter 2.2.3.7 --- Mini-preparation of plasmid DNA --- p.28 / Chapter 2.2.4 --- Polyacrylamide gel electrophoresis (PAGE) of protein --- p.29 / Chapter 2.2.4.1 --- SDS-polyacrylamide gel electrophoresis (SDS-PAGE) --- p.29 / Chapter 2.2.4.2 --- Tricine SDS-polyacrylamide gel electrophoresis --- p.30 / Chapter 2.2.4.3 --- Native polyacrylamide gel electrophoresis --- p.31 / Chapter 2.2.4.4 --- Commassie brilliant blue staining of proteinin polyacrylamide gel --- p.32 / Chapter 2.2.4.5 --- Zinc Imidazole staining of protein in polyacrylamide gel --- p.33 / Chapter 2.2.5 --- Protein concentration determination --- p.33 / Chapter 2.2.6 --- Expression of recombinant proteins --- p.33 / Chapter 2.2.6.1 --- Expression of recombinant proteins using LB --- p.33 / Chapter 2.2.6.2 --- Expression of recombinant proteins using minimal medium --- p.34 / Chapter 2.2.6.3 --- Harvest and lysis of bacterial cell culture --- p.34 / Chapter 2.3 --- Protein purification --- p.36 / Chapter 2.3.1 --- Purification of ribosomal protein P1 and its deletion mutants --- p.36 / Chapter 2.3.1.1 --- Purification of P1 --- p.36 / Chapter 2.3.1.2 --- Purification of P1ΔC25 --- p.36 / Chapter 2.3.1.3 --- Purification of HisMBP-P1ΔC40 and HisMBP-P1ΔC47 --- p.37 / Chapter 2.3.2 --- Purification of ribosomal protein P2 and its deletion mutants --- p.38 / Chapter 2.3.2.1 --- Purification of P2 --- p.38 / Chapter 2.3.2.2 --- Purification of P2ΔC46 and P2ΔC55 --- p.39 / Chapter 2.4 --- "Preparation and purification of protein complexes formed by P1, P2 and their truncation mutants" --- p.40 / Chapter 2.4.1 --- Preparation of complexes by Co-refolding in urea buffer --- p.40 / Chapter 2.4.1.1 --- Preparation of P1 or P1ΔC25 involved complexes --- p.40 / Chapter 2.4.1.2 --- Preparation of P1ΔC40/ P2ΔC46 and P1ΔC47/ P2ΔC46 --- p.41 / Chapter 2.4.2 --- Preparation of complexes by direct mixing --- p.42 / Chapter 2.5 --- Laser light scattering for the determination of molecular weight of protein and their complexes --- p.43 / Chapter 2.5.1 --- Chromatography mode light scattering experiment (SEC/LS) --- p.43 / Chapter 2.6 --- Interaction study of P1 and P2 using BIAcore 3000 surface plasmon resonance (SPR) biosensor --- p.45 / Chapter 2.6.1 --- Immobilization of P2 onto CM5 sensor chips --- p.45 / Chapter 2.6.2 --- Kinetic measurements of P1 and P2 interaction --- p.46 / Chapter Chapter 3 --- Determination of domain boundaries for dimerization of P1/P2 --- p.46 / Chapter 3.1 --- Introduction --- p.48 / Chapter 3.2 --- Preparation of P1,P2 and their truncation mutants --- p.50 / Chapter 3.2.1 --- Construction of P1 and P2 N-terminal domains (NTDs) --- p.50 / Chapter 3.2.2 --- P1 and its truncation mutants were purified in denaturing condition --- p.53 / Chapter 3.2.3 --- "P2, P2AC46 and P2AC55 were purified" --- p.56 / Chapter 3.3 --- Formation of complexes from P1,P2 and their truncation mutants --- p.59 / Chapter 3.3.1 --- "P1, P2 and their truncation mutants interact to yield protein complexes" --- p.49 / Chapter 3.3.2 --- P1AC47/P2AC46 is the smallest N-terminal domain complex --- p.63 / Chapter 3.4 --- Perturbation of P2 NTD upon binding with P1 --- p.65 / Chapter 3.4.1 --- "1H, 15N 一 HSQC spectrum of P2AC46 changed significantly upon binding with P1" --- p.65 / Chapter 3.4.2 --- P1/P2AC46 prepared by co-refolding and direct mixing give the same HSQC spectra --- p.66 / Chapter 3.5 --- Discussion --- p.69 / Chapter Chapter 4 --- Stochiometry of P1/P2 Complex is revealed by Light scattering --- p.72 / Chapter 4.1 --- Introduction --- p.72 / Chapter 4.2 --- P1 and P2 interact in 1:1 molar ratio --- p.77 / Chapter 4.2.1 --- Purified P2 exists as homo-dimer in solution --- p.77 / Chapter 4.2.2 --- The stochiometry of P1/P2 complex is 1:1 --- p.78 / Chapter 4.3 --- Stochiometries of P1 and P2 truncation mutant complexes varied from the full-length counterparts --- p.81 / Chapter 4.3.1 --- P2AC46 and P2AC55 exist as homo-dimer in solution --- p.81 / Chapter 4.3.2 --- "P1/P2AC46, P1AC25/P2 and P1AC40/P2AC46 retain the hetero-dimeric stochiometry of 1:1" --- p.82 / Chapter 4.3.3 --- P2AC55 involved complexes show a different stochiometry --- p.83 / Chapter 4.4 --- Discussion --- p.87 / Chapter Chapter 5 --- Binding kinetics of P1/P2 complex studied by surface plasmon resonance --- p.92 / Chapter 5.1 --- Introduction --- p.92 / Chapter 5.2 --- Kinetic parameters of P1 and P2 interaction is revealed by surface plasmon resonance --- p.95 / Chapter 5.2.1 --- P2 was coupled to CM5 sensor chip surface for kinetic studies --- p.95 / Chapter 5.2.2 --- Reduction of basal response after the 1st binding of P1 --- p.96 / Chapter 5.2.3 --- P1 induced a great change in response unit than P2 upon binding with immobilized P2 --- p.99 / Chapter 5.2.4 --- Kinetic parameters of P1 and P2 interaction was studied by introducing P1 to the sensor chip surface --- p.101 / Chapter 5.2.5 --- Dissociation constant derived from 1:1 Langmuir binding isotherm --- p.102 / Chapter 5.2.6 --- Dissociation constant derived from responses at equilibrium (Req) --- p.103 / Chapter 5.3 --- Discussion --- p.106 / Chapter Chapter 6 --- Conclusion and discussion of the study --- p.112 / References --- p.118 / Appendix --- p.130

Identiferoai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_326279
Date January 2008
ContributorsChiu, Yu Hin Teddy., Chinese University of Hong Kong Graduate School. Division of Biochemistry.
Source SetsThe Chinese University of Hong Kong
LanguageEnglish, Chinese
Detected LanguageEnglish
TypeText, bibliography
Formatprint, xii, 132 leaves : ill. (some col.) ; 30 cm.
RightsUse 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/)

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