Engineering Proteins with GFP: Study of Protein-Protein Interactions in Vivo, Protein Expression and Solubility
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Engineering Proteins with GFP: Study of Protein-Protein Interactions In vivo, Protein Expression and Solubility Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Mohosin M. Sarkar, M. Sc. Graduate Program in Chemistry The Ohio State University 2009 Dissertation Committee: Thomas J. Magliery, Advisor Dennis Bong Ross E. Dalbey Christopher M. Hadad Copyright by Mohosin M. Sarkar 2009 Abstract Protein–protein interactions (PPIs) play a key role in most biological processes. Many of these interactions are necessary for cell survival. To understand the molecular mechanisms of biological processes, it is essential to study and characterize protein-protein interactions, identify interacting partners and protein interaction networks. There are a number of methods that have been developed to study protein-protein interactions in vitro and in vivo, such as yeast-2-hybrid, fluorescence resonance energy transfer, co-immunoprecipitation, etc. Split protein reassembly is an in vivo probe of protein interactions that circumvents some of the problems with yeast 2- hybrid (indirect interactions, false positives) and co-immunoprecipitation (loss of weak and transient interactions, decompartmentalization). Split GFP reassembly is especially attractive because the GFP chromophore forms spontaneously on protein folding in almost every cell type. However, existing split systems have limitations of evolving cellular fluorescence slowly (3-4 days), failure to evolve at all for some interactions, and also failure to work at a physiological temperature. Among different variants of GFP tested, we found that split folding-reporter GFP (frGFP, a hybrid of EGFP and GFPuv) evolves fluorescence much faster (24 - 30 h) with associating peptides and also evolves fluorescence for the RING domain BRCA1/BARD1 wild type pair. Thirty six known cancer-associated BRCA1 RING domain mutants were tested with split-frGFP system for their role in BRCA1/BARD1 interactions. Some of these mutations resulted in significant reduction of complex reassembly and cellular fluorescence. Split frGFP fragments were further improved by directed evolution (error-prone PCR and ii DNA shuffling) to obtain fragments for fast and efficient fluorescence reassembly. The evolved fragments were able to generate fluorescence in as little as 12-16 h at 30 °C and in 10-14 h at 37 °C. This system was successfully tested for the detection of interactions of several therapeutically important protein pairs (such as Bcl-xL/Bim, Bcl-2/Bim, p53/hDM2, XIAP/Smac), which have key roles in apoptosis and cancer. Response to known inhibitors of these interactions was also tested using this system. These results suggest that the efficient split GFP (esGFP) fragments we developed will be very useful for in vivo screening of small molecule or cyclic peptide libraries to develop effective modulators of protein-protein interactions in their native cellular context from direct fluorescence reassembly. Human paraoxonase-1 (huPON1) has been known for some time for its broad hydrolytic specificity against organophosphorus (OP) pesticides and nerve agents, such as, sarin, soman and tabun, etc. The large-scale expression of the soluble protein and the improvement of the stability and catalytic activity are the most critical challenges for huPON1 to be used as a drug for detoxification of OP pesticides and nerve agents. As a human protein, it is considered to be a potent candidate for the development of a catalytic bioscavenger for effective pre- and post- exposure treatment of OP intoxication. HuPON1 is very unstable and prone to aggregation when expressed in E. coli. PON1’s hydrophobic leader sequence, hydrophobic surfaces on the HDL binding sites and the lack of post-translational modifications in bacteria are considered to be some of the reasons for its lower stability in E. coli. We applied rational and semirational approaches to re-engineer huPON1 for higher expression and solubility in E. coli. At the same time, applying approaches of chaperone co-expression and MBP (maltose binding protein) fusion and optimizing purification conditions, we were able to express active, wild-type human PON1 and the engineered variants in large-scale with a high degree of purity and solubility. iii Dedication To my parents And To Ruhnaz and Anousha for their love and support iv Acknowledgements During the course of my graduate school, I have been encouraged, inspired and supported by numerous outstanding individuals whose contribution made it possible to complete my thesis. This work would never have been possible without many of their support and help. I would like to take an opportunity here to acknowledge each and everyone’s support and contribution and express my sincere gratitude towards them. I would like to sincerely thank my advisor Professor Thomas J. Magliery for his excellent guidance and support and for giving me the opportunity to be a part of his lab. His depth of knowledge and his thoughtful insights about science and ways of addressing scientific problems always intrigued me. The opportunity to work with him has been a valuable experience through which, I must admit, I have learned a great deal from him. I would like to express my sincere gratitude to him for all the assistance, support and encouragements. I am deeply indebted to him. During my research work I have been privileged to have worked with a number of talented people from whom I have received invaluable advice and inspiration. I would like express my sincere gratitude to Dr. Sean V. Taylor for giving me the opportunity to learn molecular biology in his lab. It has been a great experience to work with him and learn general molecular biology experiments and enzyme kinetics. I would like to thank and extend my appreciation to Professor Christopher M. Hadad, Professor Terry L. Gustafson, Professor George P. Wang, Professor Richard Sayre (Danforth Plant Science Center, Missouri), Dr. David E. Lenz (USAMRICD) for their helpful suggestions and advice on the paraoxonase-1 work. I would also like to thank Professor Christopher P. Jaroniec for valuable advice and suggestions on the v biosynthesis of the Transthyretin peptide. I would like to thank Professor Dustin Maly for providing us with pro- and anti-apoptotic proteins and inhibitor and for valuable suggestions to work with them. I would like to express my sincere gratitude towards all Magliery Lab members for being very supportive of me and for all the help during the course of my research. I would like to thank George Matic and Deepti Mathur (Cornell University) for their help with the huPON1 and the BRCA1/ BARD1 works. In particular, I would like to thank and express my sincere appreciation to Christina Harsch and David Mata for their support and help, for the helpful discussions and feedback and for all the PON1 works that we have done together. I would also, like to thank, Dr. Vivekanand Shete, Dr. Lihua Nie, Brandon Sullivan, Chau Nguyen, Shila Sen, Sarah Johnston and all other lab members for being supportive and helpful. Especially, I would like to thank Dr. Jason Lavinder, whom I have spent most of my time in the lab with in my graduate school career. I have learned a great deal of valuable technicalities about biochemical bench work from him. Also, I would like to thank him for his valuable suggestions and helpful discussions when, seemingly, things were not going in the right direction and, of course, for blaming it always on the luck when nothing was going in his way on the pool table. I would like to sincerely thank my committee members, Professor Ross E. Dalbey, Professor Christopher M. Hadad, and Professor Dennis Bong for their time and valuable advice. I would like to express my gratitude to The Ohio State University, The National Institute of Health (NIH) Center of Excellence for Catalytic Bioscavenger Medical Defense Research for their support and generous funding. Finally, I would like to thank my family members and friends for their unwavering support and encouragement. I am thankful to my parents Abdus Shahid and Anwara Shahid for their invaluable love and support. Words are not just enough to express my gratitudes towards them. Especially, I would like to thank and express my sincere gratitude to my brother Shahadat vi Hossain for all the support and guidance he extended since my childhood to these days. I would like to thank my wife, Ruhnaz, for her invaluable support and sacrifice she made and for being there for me. My three-year-old daughter, Anousha, has always been a constant source of encouragement, inspiration and strength to get back to work whenever I needed. Without them I could never have made it this far. vii Vita 1989 ..................................................................... Ashek Mahmud College, Jamalpur, Bangladesh 1996 ..................................................................... B. Sc, Applied Chemistry, Univeristy of Dhaka, Dhaka, Bangladesh 2001-2003 ............................................................ M. Sc., Chemistry, Kent State University, Ohio 2004-2009 ............................................................ Graduate Associate, Department of Chemistry, The Ohio State University, Ohio Publications Philippe S. Nadaud, Mohosin Sarkar, Bo Wu, Cait E. Macphee, Thomas J. Magliery, Christopher P. Jaroniec. Expression and purification of a recombinant amyloidogenic peptide from transthyretin for solid-state NMR spectroscopy. Protein