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C M Gabe Thesis Student200902650 2 Pdfx Production and purification of recombinant amelogenins for investigating proteopathic mechanisms associated with amelogenesis imperfecta Claire Madialin Gabe Submitted in accordance with the requirements for the degree of Doctorate of Philosophy The University of Leeds Faculty of Medicine and Health School of Dentistry November 2018 - ii - Intellectual Property and Publication Statements The candidate confirms that the work submitted is his/her own, except where work which has formed part of jointly-authored publications has been included. The contribution of the candidate and the other authors to this work has been explicitly indicated below. The candidate confirms that appropriate credit has been given within the thesis where reference has been made to the work of others. The published work is integrated in the Materials and Methods, Results and Discussion sections of the thesis. The method Sections 2.1.1.1 (expression of recombinant amelogenin), 2.1.1.4 and 2.1.2.3 (polyacrylamide gel electrophoreses), 2.1.2.1 (nickel column chromatography), and the Figures 41, 42, 43 and 73 are based on work from the jointly authored publication: GABE, C. M., BROOKES, S. J. & KIRKHAM, J. 2017. Preparative SDS PAGE as an Alternative to His-Tag Purification of Recombinant Amelogenin. Frontiers in Physiology, 8, 424. All three authors Miss Claire Madialin Gabe (CMG), Dr. Seven Joseph Brookes (SJB) and Prof. Jennifer Kirkham (JK) contributed to the design of the experiments. CMG carried out the practical work (data acquisition). All three authors CMG, SJB and JK contributed to drafting the manuscript and approved the final submitted version. This copy has been supplied on the understanding that it is copyright material and that no quotation from the thesis may be published without proper acknowledgement. The right of Claire Madialin Gabe to be identified as Author of this work has been asserted by her in accordance with the Copyright, Designs and Patents Act 1988. © 2018 The University of Leeds and Claire Madialin Gabe - iii - Acknowledgements It has been a great and enjoyable experience to work under the supervision of Dr. Steven Brookes and Prof. Jennifer Kirkham. I will always be thankful to them for their friendliness, invaluable help and constant support, patience and great scientific advice thoughout my PhD. I have enjoyed very much research as part of the Enamel research team. I would like to thank as well Dr. Sarah Myers for her excellent help and support, Mr. Matty Percial for his great help on day-to-day basis, and other colleagues from Oral Biology department. I would like to thank Dr. Thuy Do for her teaching of bioinformatics software. I am very thankful to Dr. Xuebin Yang, PGRT, whose support has been very important in achieveing my PhD. I am grateful to all my colleagues and friends in Oral Biology, whom it has been a pleasure to meet and work with on a daily basis. I am grateful to my wonderful friends Lucia, Sam, Jorge, Oscar and Lizzie, Rodrigo and many others for their kindness and support, thanks to whom my Leodisian PhD years were very enjoyable. I am grateful to Sasha, Adriana, Ricardo for introducing me to the exciting world of research and bringing to me an inspiring experience, thanks to which I knew I would want to enrol in PhD. I would like to dedicate this thesis to Phyllis and Alan, for their constant love and help, to whom I owe a lot of my English language, I would like to dedicate this thesis to my parents and whole family, to whom I am grateful for their love and constant support, in all circumstances, which have been important to reach this step. Last but not least, I would like to dedicate my thesis as well to my beautiful Goddaughter Emilie, hoping that it can inspire her in the future, whichever will be her choices. - iv - Abstract In a mouse model of amelogenesis imperfecta (Al), an amelogenin p.Y64H mutation was reported to cause the abnormal retention of amelogenin in the ameloblast secretory pathway. This was hypothesised to be due to enhanced pathological aggregation of mutant amelogenin. The aim of this thesis was to develop purification methodologies to deliver large amounts of wild-type (WT) and mutant recombinant amelogenins (r-amelogenins) and develop microplate based binding assays to study protein-protein interactions of these recombinants to elucidate the effect of the mutation on aggregation. His-tagged r-amelogenin was extracted from Escherichia coli (E. Col) with 3% acetic acid. This extract was subject to nickel affinity chromatography (targeting the His-tag); the 'gold standard' technique for purifying recombinant proteins from bacterial contaminants. The His­ tag was then removed but cleavage was only -50% efficient. Cleaved r-amelogenin unexpectedly still bound the nickel column (presumably due to the presence of di and tri histidine motifs in the amelogenin sequence) which precluded its isolation from uncleaved contaminant. Size exclusion chromatography was also trialled and also found to be ineffective. Finally, preparative SOS PAGE was found to produce cleaved r-amelogenin at single band purity on analytical SOS PAGE. After optimising the purification regime, simple and cost-effective microplate binding assays were developed initially using amelogenin rich porcine enamel matrix derivative (EMO) as a surrogate. Initially the aim was to immobilise EMO on the plate and then measure the binding of FITC-labelled EMO by simple end point fluorescence measurements. An alternative method trialled UV spectrophotometry to monitor the loss of EMO from free solution in real time as it bound EMO immobilised to the well surfaces. This latter method provided an adaptable, simple and cost-effective means of monitoring amelogenin binding and aggregation. It provided pilot data suggesting that p.Y64H mutant r-amelogenin was clearly more aggregative than WT r-amelogenin. -v- Table of Contents Chapter 1 Introduction ................................................................................... - 1 - 1.1 Dental enamel ...................................................................................... - 1 - 1.1.1 Structure and function of mature enamel .................................... - 1 - 1.1.2 Development of enamel: cellular and extra-cellular events ......... - 4 - 1.1.2.1 Embryonic origins of enamel tissue .................................... - 4 - 1.1.2.2 Presecretory stage: Inner enamel epithelium cells differentiate into ameloblasts ................................................................. - 7 - 1.1.2.3 Secretory stage: ameloblasts acquire secretory function in contact with pre-existing dentine, then secrete enamel matrix . .................................................................................- 8 - 1.1.2.4 Transition and maturation stages: reduction of secretory activity and secondary mineralisation ............................... - 10 - 1.2 Secretory stage enamel matrix proteins ............................................. - 13 - 1.2.1 Amelogenin, the major enamel matrix protein .......................... - 13 - 1.2.1.1 Sexual dimorphism .......................................................... - 13 - 1.2.1.2 Transcription variants of amelogenin (alternative splicing) - 14 - 1.2.1.3 Properties of amelogenin protein...................................... - 17 - 1.2.1.3.1 Primary structure ..................................................... - 17 - 1.2.1.3.2 Secondary structure ................................................. - 19 - 1.2.1.3.3 Tertiary structure ...................................................... - 20 - 1.2.1.3.4 Quaternary structure: amelogenin self-assembly ..... - 20 - 1.2.2 Amelogenin self-assembly ....................................................... - 20 - 1.2.2.1 Amelogenin propensity to aggregate ................................ - 20 - 1.2.2.2 Amelogenin hierarchical assembly ................................... - 21 - 1.2.2.2.1 Domains involved in amelogenin recognition and self- assembly ................................................................. - 21 - 1.2.2.2.2 Intracellular assembly of amelogenin in vivo ............ - 22 - 1.2.2.2.3 Extracellular assembly of amelogenin observed in vivo ...........................................................................- 23 - 1.2.2.2.4 Characterisation of amelogenin self-assembly in vitro ...........................................................................- 24 - 1.2.3 Matrix biochemistry and processing of amelogenin .................. - 30 - 1.2.3.1 Amelogenin processing throughout secretory stage ......... - 30 - -vi- 1.2.3.1.1 Protelolytic processing during secretion ................... - 30 - 1.2.3.1.2 Effect of amelogenin processing on mineral binding and role of amelogenin in enamel matrix mineralisation .. - 33 - 1.2.3.2 Amelogenin degradation during maturation stage............. - 35 - 1.2.4 Other structural proteins of the developing enamel matrix : ameloblastin and enamelin ....................................................... - 37 - 1.2.4.1 Ameloblastin ..................................................................... - 37 - 1.2.4.2 Enamelin .......................................................................... - 38 - 1.2.5 Amelogenin transit through the ameloblast ER for secretion ..... - 40 - 1.2.5.1 Ameloblasts: cells specialised in protein secretion require an efficient ER trafficking machinery ..................................... - 40 - 1.2.5.2 Protein-protein interactions associated with amelogenin as it transits the ameloblast secretory pathway
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