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Protein Engineering Utilising Single Amino Acid Deletions Within Photinus Pyralis Firefly Luciferase

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Protein Engineering Utilising Single Amino Acid Deletions Within Photinus Pyralis Firefly Luciferase Cardiff University School of Bioscience Protein Engineering Utilising Single Amino Acid Deletions Within Photinus Pyralis Firefly Luciferase Lisa M. Halliwell September 2015 Cardiff School of Biosciences The Sir Martin Evans Building Museum Avenue Cardiff CF10 3AX A thesis submitted for the degree of Doctor of Philosophy for Cardiff University i Abstract The bioluminescence reaction is catalysed by firefly luciferase, converting the substrates D- luciferin, ATP and molecular oxygen with Mg2+ to produce light and this reaction has had wide ranging implications within a number of fields from industry to academia. The discovery of luciferase has been revolutionary in the real time in vivo study of cells given that it requires no energy for excitation, delivering a high signal to background ratio providing a highly sensitive assay. This protein, to date, has been utilised in molecular cell biology, cellular imaging, microbiology and numerous other fields. The extensive application of this protein has paved the way for the generation of toolbox of variants with altered properties. Protein engineering involving substitution mutations made within Photinus pyralis (Ppy) FLuc has led to the discovery of a number of novel variants however there is a bank of growing evidence displaying the power of deletions as an alternative for the development of proteins with altered properties since deletions can sample structural diversity not accessible to substitutions alone. A novel mutagenic strategy was implemented to incorporate single amino acid deletions within thermostable firefly luciferase (x11FLuc) targeting loop structures (M1-G10, L172- T191, T352-F368, D375-R387, D520-L526, K543-L550). Of 43 deletion mutants obtained, 41 retained bioluminescent activity and other characteristics such as resistance to thermal inactivation. Surprisingly, only 2 variants, ΔV365 and ΔV366, exhibited a complete loss of activity showing that the luciferase protein is largely tolerant to single amino acid deletions. In order to identify useful mutants in the extensive library, a 96-well format luminometric cell lysate assay was developed which indicated the effect of deletions was largely region specific, for example, N- terminal deletions did not alter the activity of x11FLuc, whilst deletions within L172- T191, D375-R387, D520-L526 and the C- terminal loop reduced overall activity. On the other hand, deletions within T352-F368 enhanced overall bioluminescent activity and remarkably exhibited other important characteristics such as increases in specific activity and a reduced KM for luciferin. Therefore, a novel motif (omega loop) was identified as important for FLuc activity after full characterization of mutants. ii Characterisation of the deletion mutants originating from the omega loop (T352-F368), ΔP359 and ΔG360 both presented a reduced KM for luciferin, whilst ΔA361, ΔV362, ΔG363 presented an increase in KM towards ATP as compared to x11FLuc. Thus, deletions in the omega loop, in the main, improved activity and altered reaction kinetics, in particular ΔG363 retained 53% of initial activity after 250s. As such, it is considered that the field of protein engineering should not only overlook the utility of single amino acid deletions, since such mutagenic strategies may sample structural space not achieved by substitutions alone and mutations within less popularized secondary structures such as omega loops are can act as a useful tool in the improvement of proteins. iii Acknowledgements I would like to start by saying a thank you to the research group in which I have been a part, in particular, Angela Marchbank, and Joanne Kilby, whom throughout this past year have gone out of their way to support this project be it through answering my endless technical questions to ensuring that the materials needed throughout were always in stock and available. A special thank you must go to my mentor, Amit Jathoul, without whose help this thesis and the data within would not have been possible. I am so grateful for his consistent patience, encouragement and commitment to this project and I have found his enthusiasm and overwhelming knowledge on the field of bioluminescence and luciferase protein engineering to be both inspiring and a constant source of motivation. Another special thank you must go to my supervisor, Prof Jim Murray. Thank you for allowing me to explore a topic in which I have always had a keen interest but more than that, thank you for the continued guidance, support and understanding you have provided me over the last 4 years. Lastly, thank you to my wonderful family and my equally wonderful boyfriend. Thank you for the putting up with the mood swings, for providing never ending cups of coffee and for just being there when I felt that this thesis would not be possible. Thank you for your endless support over the completion of this work and as such, this is for you. iv Table of Contents Chapter 1 ................................................................................................................................. 1 Introduction .............................................................................................................................. 1 1.1. Bioluminescence ........................................................................................................... 1 1.1.1. Bioluminescent Systems ........................................................................................ 1 1.1.2. The Beetle Luciferases ........................................................................................... 4 1.1.3. The Coleopteran Bioluminescent Reaction ........................................................... 6 1.1.4. Structure of Firefly Luciferase ............................................................................... 9 1.1.5. Characteristic Kinetics of Firefly Bioluminescence ............................................ 14 1.1.6. Further Reactions of Firefly Luciferase ............................................................... 16 1.1.7. Bioluminescence and Colour Modulation ........................................................... 17 1.1.8. Current Protein Engineering Strategies Utilized for Improved Characteristics of Firefly Luciferase ........................................................................................................... 19 1.1.9. Applications for Bioluminescence ....................................................................... 25 1.2. Dogma and New Directions in Firefly Luciferase Protein Engineering ..................... 27 1.2.1. The Central Dogma .............................................................................................. 27 1.2.2. Protein engineering .............................................................................................. 27 1.2.3. Strategies for Protein Design and Engineering .................................................... 27 1.2.4. Current Protein Engineering Incorporating Deletion Mutations ......................... 31 1.2.5. Deletions of Amino Acids within Firefly Luciferase .......................................... 32 1.3. Aims and Objectives ................................................................................................... 34 Chapter 2 ............................................................................................................................... 35 Materials and Methods ........................................................................................................... 35 2.1. Materials ..................................................................................................................... 35 2.1.1. Chemicals ............................................................................................................. 35 2.1.2. Bacterial Cell Strains and Plasmids ..................................................................... 38 2.1.3. Bacterial growth media ........................................................................................ 38 2.1.4. Molecular Reagents ............................................................................................. 38 2.1.5 Oligonucleotide primers ........................................................................................ 39 2.2. General Molecular Biology and Recombinant DNA Methods ................................... 42 2.2.1. DNA Sequencing ................................................................................................. 42 2.2.2. Purification of Plasmid DNA ............................................................................... 42 2.2.3. Agarose Gel Electrophoresis ................................................................................ 43 v 2.2.4. PCR with GoTaq polymerase .............................................................................. 43 2.2.5. Site directed mutagenesis Phusion polymerase ................................................... 43 2.2.6. Restriction digestion ............................................................................................ 44 2.2.7. Ligation ................................................................................................................ 44 2.2.8. Preparation of electro-competent cells ................................................................. 45 2.2.9. Transformation by electroporation ...................................................................... 45 2.2.10. Transformation by heat shock ............................................................................ 46 2.2.11.
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