McClymont, Karen (2017) Structural studies of the endotoxin sensing protein Factor C. PhD thesis. http://theses.gla.ac.uk/8058/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Structural studies of the endotoxin sensing protein Factor C Karen McClymont Submitted in fulfilment of the requirements for the Degree of Doctor of Philosophy Institute of Molecular, Cell and Systems Biology College of Medical, Veterinary and Life Sciences University of Glasgow March, 2017 2 Abstract Human exposure to Gram-negative bacterial endotoxin can result in life-threatening conditions including sepsis and toxic shock syndrome. Given the importance of avoiding exposure to endotoxin, a sensitive test is required to ensure that medical devices and injectable medicines are not contaminated. Limulus amebocyte lysate (LAL), produced from the blood of horseshoe crabs, contains an endotoxin-sensing protein, Factor C, that initiates a coagulation cascade. This lysate is used to test for contamination of medical products, with formation of a clot indicating the presence of endotoxin in the sample. Alternative methods to the LAL test have been explored to overcome problems with the test. These include the detrimental effects that harvesting has on the wild population of horseshoe crabs. More recently, tests relying on the direct detection of enzymatically active Factor C have been introduced, some of which utilize Factor C produced recombinantly. Despite the importance of Factor C to the health industries, no detailed explanation of its mechanism of endotoxin recognition has been published. The aim of this project was therefore to develop a better understanding of how Factor C is activated by endotoxin binding and to identify conformational changes that take place as a result. Two main strategies were employed for structural characterisation and functional analysis of Factor C. First, fragments of Factor C protein were produced recombinantly in Escherichia coli to determine the structures of the individual domains and identify the endotoxin binding region more precisely. Second, expression of the whole Factor C protein in eukaryotic expression systems was attempted to produce material for experiments that would shed light on any overall conformational change that occurs. Factor C fragments were successfully expressed in E. coli and purified. Circular dichroism spectroscopy identified that the majority of these fragments were folded, and changes in the spectra of some fragments in the presence of lipopolysaccharide identified potential binding sites. A preliminary structure of the first pair of complement control domains has been determined by nuclear magnetic resonance spectroscopy. Comparison of this structure with other LPS binding proteins will pave the way for future work to characterise the endotoxin binding site of Factor C. 3 Contents Abstract ................................................................................................................................... 2 List of tables ............................................................................................................................ 8 List of figures .......................................................................................................................... 9 Acknowledgements ............................................................................................................... 11 Author’s Declaration ............................................................................................................. 12 Definitions/Abbreviations ..................................................................................................... 13 1 Introduction ................................................................................................................... 17 1.1 Bacterial Endotoxin ................................................................................................ 17 1.2 Horseshoe Crabs ..................................................................................................... 19 1.2.1 Amebocytes ..................................................................................................... 22 1.2.2 Coagulation Cascade ....................................................................................... 24 1.2.3 Limulus Amebocyte Lysate Test ..................................................................... 25 1.3 Factor C .................................................................................................................. 27 1.4 Experimental Basis ................................................................................................. 31 1.5 Project Aims ........................................................................................................... 35 1.6 Experimental Approach .......................................................................................... 36 2 Limulus polyphemus Gene ............................................................................................ 38 2.1 Overview ................................................................................................................ 38 2.2 Choice of Factor C Sequence ................................................................................. 38 2.3 Assembly of the Limulus polyphemus Factor C Coding Sequence ........................ 38 2.3.1 Identification of Limulus polyphemus Genomic Sequences ........................... 39 2.3.2 Factor C Sequence Alignment ........................................................................ 44 2.4 Design and Assembly of the Synthetic Gene Construct ......................................... 46 3 Recombinant Expression in E. coli ............................................................................... 49 3.1 Overview ................................................................................................................ 49 3.2 Protein Production .................................................................................................. 50 3.3 Vector Construction ............................................................................................... 50 4 3.3.1 Expression Plasmid ......................................................................................... 50 3.3.2 Ligation Independent Cloning ........................................................................ 51 3.3.3 Thermodynamically Balanced Inside-Out PCR-Based Gene Synthesis ...... 54 3.3.4 lpFC Complement Control Protein Domains .................................................. 59 3.3.5 DNA Sequencing ............................................................................................ 61 3.4 Protein Expression .................................................................................................. 61 3.4.1 Protein Test Expressions ................................................................................. 62 3.4.2 Large Scale Protein Expression ...................................................................... 62 3.5 Protein Purification ................................................................................................. 63 3.5.1 Cell Lysis ........................................................................................................ 63 3.5.2 Centrifugation ................................................................................................. 63 3.5.3 Ni2+ Affinity Chromatography ........................................................................ 64 3.5.4 Inclusion Bodies Protein Purification ............................................................. 64 3.5.5 Buffer Exchange and Sample Concentration .................................................. 65 3.5.6 TEV-Cleavage ................................................................................................. 65 3.5.7 Gel Filtration Chromatography ....................................................................... 65 3.5.8 Reversed Phase High-Performance Liquid Chromatography ......................... 66 3.5.9 Lyophilisation ................................................................................................. 66 3.6 Protein Analysis ..................................................................................................... 67 3.6.1 Circular Dichroism Spectroscopy ................................................................... 67 3.6.2 Nuclear Magnetic Resonance Spectroscopy ................................................... 67 3.6.3 X-Ray Crystallography ................................................................................... 67 3.7 Factor C Protein Constructs ................................................................................... 69 3.7.1 Tachypleus tridentatus Recombinant Factor C LPS Binding Fragments ....... 69 3.7.2 Limulus polyphemus recombinant Factor C LPS binding fragments .............. 74 3.8 Summary ...............................................................................................................