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Split Inteins As Versatile Tools in Applications of Downstream Purification And Split Inteins As Versatile Tools in Applications of Downstream Purification and Bioconjugation Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Yamin Fan Graduate Program in Chemical Engineering The Ohio State University 2019 Dissertation Committee David W. Wood, Advisor Shang-Tian Yang Andre Palmer Copyrighted by Yamin Fan 2019 Abstract Over the past two decades, inteins have been extensively used in a wide variety of applications in biotechnology. Split inteins are a subset of inteins, which are identified more recently and expressed in two separate segments naturally. They catalyze the splicing reaction in trans upon association of the two halves. Due to their unique features, split inteins offer improved controllability and flexibility in trans-splicing and trans- cleaving over the previous tools based on contiguous inteins. The engineered split inteins would allow the development of efficient self-cleaving affinity tags for purification applications and new methods for protein conjugation. In this work, an engineered split intein derived from Nostoc punciforme (Npu) was applied in a column-free purification strategy in combination with the aggregating tag, elastin-like polypeptide (ELP), as an initial capture step for recombinant proteins expressed in E. coli. Meanwhile, on-column purification strategy using the same engineered split intein was employed for the production of value-added biosimilar target, Granulocyte-colony stimulating factor (G-CSF). To adapt the split intein-based purification platform for the production of protein therapeutics expressed in mammalian cells, multiple leader sequences were designed and screened for optimal expression and secretion of intein-tagged precursor proteins. Moreover, the extein dependency of this ii engineered Npu split intein was thoroughly characterized by using in solution cleaving kinetics study and Förster Resonance Energy Transfer (FRET) based high-throughput method. The information gathered guides for fast and consistent cleavage reactions among various target proteins and provides insight to the cleavage mechanism. In this work, the trans-splicing properties of split inteins were also exploited for developing novel bioconjugation methods onto fluorescent nanodiamonds. Two split inteins, Gos-TerL and GP41.1, were used for the development of N-terminal and C- terminal oriented bioconjugation schemes, respectively. The new methods would allow rapid and spontaneous immobilization of proteins onto fluorescent nanodiamond surfaces for applications such as biomedical imaging or drug delivery. iii Dedication This document is dedicated to my beloved family. iv Acknowledgments This work would not have been possible without the support of many people. I would like to thank my family, especially my parents, whose love and support are with me in whatever I decide to do. I am grateful that they have taken good care of themselves while I am away from home to pursue my degree and dream. I would also like to extend my deepest gratitude to my beloved husband, Weijie Mai. He is my role model and the person who keeps me motivated and curious in research and life. His enormous love and support all the time enables me to survive through all difficulties over the past five years of my Ph.D.. I would like to express my deepest appreciation to my advisor, Professor David W. Wood, for his continuous support and guidance. He has always been trusting me and encouraging me for my research. He also provided me with valuable opportunities to attend conferences and connected me with people from industry and academia. Without his support, I would not be able to attend internships in summers and eventually pursue a career in pharmaceutical industry that I have been looking forward to for many years. Besides my advisor, I would like to thank the rest of my thesis committee members, Professor Shang-Tian Yang, Professor Andre Palmer, who also served on the committees for my qualification and candidacy exams. I would also like to thank Professor Jeffrey v Chalmers for serving on my candidacy committee. Their insightful comments and questions have incented me to improve my research from various perspectives. I thank our collaborators in Columbus Nanoworks Inc, Dr. Arfaan Rampersaud, Issac Rampersaud and David Albertson, for working together for the project in Chapter 5. My sincere thanks also go to my previous and current labmates. In particular, I would like to thank Jackelyn Miozzi, Joseph Taris, Brian Marshall for stimulating discussions throughout the projects and giving me help constantly. The experience would not have been as fun without the accompany of them in the lab and weekly lunches to Chick-Fil-A. I am also extremely grateful to Dr. Tzu-Chiang Han, Dr. Ashwin Lahiry, Dr. Merideth Cooper, Dr. Samuel Stimple for mentoring me and training me all the skills and techniques during my Ph.D.. They are not only great mentors, but also important friends of my life. I would also thank the rest of the current lab members, Kevin McGarry, Hongyu Yuan, Tarek Mazeed, Maria DeBastiani, Natalya Lavrenchuk, Maria Zindarsic, Issac Delev, Christy Caporale, Farah Deeba, Lex Tallan and Joel Silleck, for all of their help and accompany. Finally, I would like to appreciate the fun and support from my friends in Columbus. They are more than friends to me and keep me accompanied like families. I would also thank my friends in other places of the world, especially Shannon Zhang. You guys keep my life meaningful and colorful. vi Vita June 2014 ………………………………………..……………….… B.S. Bioengineering, Zhejiang University, China September 2014 to present …………………………………Graduate Research Associate, Department of Chemical and Biomolecular Engineering, The Ohio State University, U.S. Publications 1. Fan Y., Miozzi M. M., Stimple S. D., Han T.C. and Wood D.W. (2018), Column- free purification methods for recombinant proteins using self-cleaving aggregating tags. Journal of Polymers. DOI: 10.3390/polym10050468 2. Lahiry A., Fan Y., Stimple S. D., Raith M. and Wood D. W. (2017), Inteins as tools for tagless and traceless protein purification. J. Chem. Technol. Biotechnol. DOI:10.1002/jctb.5415 3. Yang D T., Lu X., Fan Y., et al. Evaluation of nanoparticle tracking for characterization of fibrillar protein aggregates[J]. AIChE Journal, 2014. DOI: 10.1002/aic.1434 Fields of Study Major Field: Chemical Engineering vii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments............................................................................................................... v Vita .................................................................................................................................... vii List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xiv Chapter 1 Introduction ........................................................................................................ 1 1.1 Inteins ........................................................................................................................ 1 1.2 Inteins as self-cleaving tags for downstream purification of recombinant proteins . 5 1.3 Inteins as self-splicing tools for protein labeling onto nanoparticles ..................... 10 1.4 Dissertation ............................................................................................................. 12 Chapter 2 Development of purification strategies using self-cleaving tag ....................... 15 2.1 Introduction ............................................................................................................. 15 2.2 Materials and Methods ............................................................................................ 26 2.2.1 Chemicals and Reagents .................................................................................. 26 viii 2.2.2 Plasmids construction ...................................................................................... 26 2.2.3 Protein expression ............................................................................................ 27 2.2.4 Lysis and recovery ........................................................................................... 27 2.2.5 ELP-mediated protein purification .................................................................. 28 2.2.6 NpuN* resin production .................................................................................... 29 2.2.7 On-column purification using intein resin on AKTA Pure 25 ......................... 30 2.2.8 Intein cleavage analysis ................................................................................... 30 2.2.9 Protein quantification ....................................................................................... 31 2.2.10 Activity assays ............................................................................................... 31 2.3 Results ..................................................................................................................... 33 2.3.1 Column-free
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