Expression and evaluation of a 297-amino acid fragment of the blood stage protein PfC0760c from Plasmodium falciparum by Zainab Baig BSc (Hons) Biochemistry Submitted in fulfilment of the academic requirements of Master of Science in Biochemistry School of Life Sciences College of Agriculture, Engineering and Science University of KwaZulu-Natal Pietermaritzburg South Africa April 2019 Preface The research contained in this dissertation was completed by the candidate while based in the Discipline of Biochemistry, School of Life Sciences of the College of Agriculture, Engineering and Science, University of KwaZulu-Natal, Pietermaritzburg, South Africa. The research was financially supported by The National Research Foundation. The contents of this work have not been submitted in any form to another university and, except where the work of others is acknowledged in the text, the results reported are due to investigations by the candidate. ii Declaration: Plagiarism I, Zainab Baig, declare that: (i) the research reported in this dissertation, except where otherwise indicated or acknowledged, is my original work; (ii) this dissertation has not been submitted in full or in part for any degree or examination to any other university; (iii) this dissertation does not contain other persons’ data, pictures, graphs or other information, unless specifically acknowledged as being sourced from other persons; (iv) this dissertation does not contain other persons’ writing, unless specifically acknowledged as being sourced from other researchers. Where other written sources have been quoted, then: a) their words have been re-written, but the general information attributed to them has been referenced; b) where their exact words have been used, their writing has been placed inside quotation marks, and referenced; (v) where I have used material for which publications followed, I have indicated in detail my role in the work; (vi) this dissertation is primarily a collection of material, prepared by myself, published as journal articles or presented as a poster and oral presentations at conferences. In some cases, additional material has been included; (vii) this dissertation does not contain text, graphics or tables copied and pasted from the Internet, unless specifically acknowledged, and the source being detailed in the dissertation and in the References sections. _______________________ Signed: Zainab Baig Date: 3 September 2019 iii Abstract Malaria is a mosquito-borne disease caused by parasites of the Plasmodium genus. There is no malaria vaccine available. The most lethal form of malaria in humans is caused by infection with Plasmodium falciparum parasites. The protein PfC0760c from P. falciparum was identified as a potential malaria vaccine candidate based on the presence of alpha-helical coiled-coil motifs. This protein is conserved, and homologous proteins have been identified in Plasmodial species infecting humans, monkeys, mice and birds. PfC0760c has not been characterised and has no known function. Conditions for the recombinant expression of a 297 amino acid fragment (N2360 – N2656) from PfC0760c was optimized. The recombinant PfC0760c protein (denoted further as rPfC0760c297) was expressed as an insoluble 56 kDa his6-tagged fusion protein. Different conditions for the expression of soluble rPfC0760c297 were evaluated, including expression in the presence of ethanol and co-expression with Escherichia coli chaperones. Under different expression conditions, rPfC0760c297 remained insoluble, and was thus solubilized before nickel chelate affinity purification. 1 g cell biomass yielded ~22 mg purified rPfC0760c297 protein. Bioinformatics analyses of PfC0760c revealed three conserved peptides. B-cell epitopes, and several T-cell epitopes were also predicted. PfC0760c is expressed during the blood stages of the P. falciparum lifecycle and is found in the parasite nucleus. The absence of transmembrane domains and functional PEXEL motifs suggests that the protein is unlikely to be secreted. Chicken anti-rPfC0760c297 IgY antibodies were able to detect the homologous protein in P. berghei-infected mouse red blood cells. Far western blot analysis revealed protein-protein interactions between human U937 and mouse J774A.1 monocyte proteins with the rPfC0760c297. Mice immunized with rPfC0760c297 were able to raise antibodies against the protein, however succumbed to a P. berghei mouse malaria challenge infection. Various factors may have contributed to this outcome and further research is required in this endeavour. Further research is required for the development of a suitable malaria vaccine candidate. iv Graphical Abstract N YERY EIYK NILK C PfC0760c NILK expressed region predicted rPfC0760c297 structure A kDaA 1 1 2 2 3 3 4 4 5 5 6 67 7 BB 1.2 300 kDa ) 116 116 1 250 mM 6666 56 kDa rPf0.8C0760c297 200 4545 Challenge 35 0.6 150 SDS A280 35 infection 25 0.4 100 solubilization 25 0.2 50 18 18 14 0 0 14 0 2 4 6 8 10 12 14 16 18 20 Imidazole concentration ( Volume (ml) P. berghei Protein-protein interactions infected RBC Mouse blood A kDa 1 2 3 4 5 6 7 8 9 10 B kDa 1 2 3 4 5 6 7 8 9 10 Chicken anti- kDa 116 116 anti-mouse-IgG HRPO PfC0760c 66 66 rPfC0760cA kDa 297 IgY B kDa 45 45 116 116116 homolog 35 35 anti-Histag-IgG 66 66.266 25 25 45 45 45 35 35 18 18 His6tagged rPfC0760c297 35 14 14 25 2525 Far western blot 18 18.418 BSA Protein 14 v Acknowledgments First and foremost, I would like to thank the Almighty, without whom nothing would be possible. I would like to thank the following people: My parents, M.M. Idrees and Hajra for their continued love and support, for believing in me, for praying for me, and for every possible little thing that made a difference in my life. Every teacher and lecturer who inspired my love for science, and helped lay my academic foundation. My supervisor, Professor J.P. Dean Goldring for his supervision, encouragement and guidance throughout my studies as an undergraduate all the way through to postgraduate. Thank you for instilling in us good lab practice and a healthy amount of OCD. Dr Robert G.E. Krause, Dr Kajal Fowdar and Dr Lauren Eyssen for all their helpful discussions as the project progressed. All fellow students of the Malaria Lab: Mark H, Mlondi, Abdulmalik, Eugene, Mark C, Sinothile and Sheldon, for the support and interesting, healthy debates. Kelvin Addicott, who had previously worked with the protein, and who also did the initial cloning of the recombinant protein and raised the chicken antibodies. Thank you for always being available for a chat regarding the protein. Professor Carola Niesler, Dr Raymond Hewer, Dr M.M. Elias Baig, Dr Farzana Baig, Quraisha Baig, Dr M.I. Adam and Rizwana Mia who always had an ear for me and provided invaluable advice. Ebrahim Ally, who sacrificed time and mice for this project. Your continued friendship based on mutual love for tea and animals is appreciated. Our admin ladies Charmaine, Tanya and Natalie, thank you for taking care of all the admin on our behalf. Jessica Moodley and Yegan Pillay who were always available for technical support. Ma Agnes for ensuring we always had dH2O available. All the cleaning staff for making our lives easier. To my brothers Ibrahim and Muhammad, my sister-in-law Tasfiyah and my dear friends Faiaz, Alex, Tash, Tracy, Mariam, Billie, Selisha, Ryan, Jezelle, Ameer, and Tasmiyah for always being around, for a chat, love and support. To the National Research Foundation and the University of KwaZulu-Natal, without which, this study would not have been made possible. vi Table of Contents Page Preface ................................................................................................................................... ii Declaration: Plagiarism.......................................................................................................... iii Abstract ................................................................................................................................. iv Graphical Abstract .................................................................................................................. v Acknowledgments ................................................................................................................. vi Table of Contents ................................................................................................................. vii List of Tables ....................................................................................................................... xiv List of Figures ....................................................................................................................... xv Abbreviations ..................................................................................................................... xvii Chapter 1: Literature review .................................................................................................. 1 1.1 Introduction ................................................................................................................... 1 1.2 Malaria transmission and control .................................................................................. 2 1.3 The Plasmodium falciparum parasite ............................................................................ 2 1.4 The lifecycle of Plasmodium parasites .......................................................................... 3 1.4.1 The sporozoite stage of the Plasmodium lifecycle ..................................................... 3 1.4.2