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University of Southampton Research Repository University of Southampton Research Repository Copyright © and Moral Rights for this thesis and, where applicable, any accompanying data are retained by the author and/or other copyright owners. A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. This thesis and the accompanying data cannot be reproduced or quoted extensively from without first obtaining permission in writing from the copyright holder/s. The content of the thesis and accompanying research data (where applicable) must not be changed in any way or sold commercially in any format or medium without the formal permission of the copyright holder/s. When referring to this thesis and any accompanying data, full bibliographic details must be given, e.g. Thesis: Author (Year of Submission) "Full thesis title", University of Southampton, name of the University Faculty or School or Department, PhD Thesis, pagination. Data: Author (Year) Title. URI [dataset] UNIVERSITY OF SOUTHAMPTON FACULTY OF NATURAL & ENVIRONMENTAL SCIENCES Biological Sciences Volume 1 of 1 Using GFP to investigate protein localisation, function and global cellular response by Benjamin Yarnall Thesis for the degree of Doctor of Philosophy September_2017 UNIVERSITY OF SOUTHAMPTON ABSTRACT FACULTY OF NATURAL & ENVIRONMENTAL SCIENCES Biological Sciences Thesis for the degree of Doctor of Philosophy USING GFP TO INVESTIGATE PROTEIN LOCALISATION, FUNCTION AND GLOBAL CELLULAR RESPONSE By Benjamin Paul Yarnall Integral membrane proteins (IMPs) make up 20-30 % of genes in all walks of life. They are major determinants of disease pathology, making them prime therapeutic targets for cancer, bacterial infection and genetic disorders. Despite this, they are underrepresented in the literature; this is often attributed to complications with IMP expression, purification and characterisation. This thesis aimed to tackle the difficulties with IMP characterisation. Exploitation of tagged versions of the IMPs has opened up new research fields, initially based on simple observations. The tag used is the well characterised GFP which has allowed for the simple optimisation of conditions for protein over-expression, determination of intracellular localisation of H. sapiens SWEET sugar transporter, development of a ligand binding method that does not rely on properties of the ligand and demonstrated that protein over-expression using the Escherichia coli pET system only occurs in mutant forms of this organism. The implication being that protein over- expression only occurs via genetically modified organisms. Ubiquitously expressed, the MFS is one of the largest protein superfamilies, with roles including metabolite and xenobiotic transport. They have been implicated in the development of antimicrobial resistance in E. coli, making them a clinically relevant target. An expression library of 63 GFP-tagged proteins was produced, before screening for optimal expression conditions. The larger amounts of transporter obtained via this approach enabled the implementation of a ligand-binding assay using the technique of thermophoresis. This approach produced novel binding substrates as well as identifying a new binding event for the well characterised drug efflux transporter mdfA. Significantly, the approach has shown that cyclic AMP also binds to mdfA and another MFS transporter kgtP, potentially identifying a novel role for these transporters in the catabolite repression process. The work presented in this thesis has shown the versatility of a reporter system like GFP to uncover fundamental properties at the cellular level (protein localisation experiments), the biochemical level (optimisation of protein over-expression and ligand binding studies) but also at the cellular level (E. coli’s use of mutants during protein over-expression). This research is a building block to identifying new drug targets to tackle the global problem of antimicrobial resistance. Table of Contents Table of Contents Table of Contents ..................................................................................................................... i Table of Tables ........................................................................................................................ ix Table of Figures ................................................................................................................... xiii Academic Thesis: Declaration of Authorship ..................................................... xxi Acknowledgements ......................................................................................................... xxiii Definitions and Abbreviations ................................................................................... xxv Chapter 1 Using GFP to investigate protein localisation, function and global cellular response ............................................................................. 1 1.1 Characterised membrane proteins are underrepresented in the literature .......................................................................................1 1.2 Green Fluorescent Protein discovery and physiology .......................4 1.3 Alternative fluorescent proteins .....................................................8 1.4 Using GFP fluorescence as a reporter ........................................... 12 1.5 Complications associated with the use of GFP as a reporter .......... 18 1.6 Project aims and applications ...................................................... 18 Chapter 2 Materials and methods ............................................................................. 22 2.1 General molecular biology ........................................................... 22 2.1.1 Polymerase chain reaction (PCR) amplification for expression vector construction ........................................................... 22 2.1.2 Gel electrophoresis to assess DNA and generate purified vector inserts .............................................................................. 24 2.1.3 DNA purification of expression vector inserts following PCR and gel electrophoresis .................................................... 24 2.1.4 Cloning ................................................................................. 25 2.1.4.1 Ligation-dependent T4 cloning of PQ loop genes ............. 25 2.1.4.2 Ligation-independent cloning (LIC) of HsSWEET and MFS genes ............................................................................. 27 2.1.4.3 Confirmation of gene incorporation into expression vectors ........................................................................... 29 2.2 General bacterial materials and methods ..................................... 30 i Table of Contents 2.2.1 Recipes of different media used for bacterial growth ............. 30 2.2.2 Creation of chemically competent E. coli cells ....................... 30 2.2.3 Transformation of expression vectors into competent E. coli . 31 2.2.4 Extraction of plasmid DNA from E. coli.................................. 31 2.3 General protein expression ......................................................... 32 2.3.1 Expression of cloned genes using T7 polymerase regulated vectors ............................................................................ 32 2.3.2 Affinity chromatography purification of bacterially expressed protein ............................................................................ 32 2.3.3 SDS-PAGE electrophoresis analysis of bacterially expressed protein ............................................................................ 33 2.3.4 Concentration of expressed protein ...................................... 33 2.3.5 Gel filtration analysis of bacterially expressed protein ........... 34 2.3.6 Western blot immunoanalysis of bacterially expressed protein ............................................................................ 34 2.3.7 GFP quantification using protein fluorescence ....................... 35 2.4 Expression, purification and characterisation of HsSWEET ............ 36 2.4.1 Tertiary structural predictions of HsSWEET using amino acid sequence ......................................................................... 36 2.4.2 Cloning of HsSWEET and PQ-loop proteins into vectors for subcellular localisation and protein expression ................ 36 2.4.3 Subcellular localisation of HsSWEET in human HEK and RPE cell lines ................................................................................ 40 2.4.4 Predictions of HsSWEET subcellular localisation using primary sequence ......................................................................... 41 2.4.5 Comparison of GFP-tagged HsSWEET expression at different temperatures ................................................................... 41 2.4.6 Batch purification of HsSWEET with different detergents to identify solubilisation efficiency ....................................... 42 2.4.7 Using GFP-tagged HsSWEET to compare the yield of expression conditions growth comparison ......................................... 43 2.4.8 Gel filtration of HsSWEET ...................................................... 43 2.4.9 Tm thermostability test of HsSWEET titrated with glucose ....... 44 ii Table of Contents 2.5 Expression, purification and characterisation of MFS proteins ....... 45 2.5.1 Cloning of MFS proteins into the E. coli expression vector H6- msfGFP............................................................................. 45 2.5.2 Comparing expression yield of MFS proteins using
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