CELLULAR AND PROCESS ENGINEERING TO IMPROVE MAMMALIAN MEMBRANE PROTEIN EXPRESSION By Su Xiao A dissertation is submitted to Johns Hopkins University in conformity with the requirements for degree of Doctor of Philosophy Baltimore, Maryland May 2015 © 2015 Su Xiao All Rights Reserved Abstract Improving the expression level of recombinant mammalian proteins has been pursued for production of commercial biotherapeutics in industry, as well as for biomedical studies in academia, as an adequate supply of correctly folded proteins is a prerequisite for all structure and function studies. Presented in this dissertation are different strategies to improve protein functional expression level, especially for membrane proteins. The model protein is neurotensin receptor 1 (NTSR1), a hard-to- express G protein-coupled receptor (GPCR). GPCRs are integral membrane proteins playing a central role in cell signaling and are targets for most of the medicines sold worldwide. Obtaining adequate functional GPCRs has been a bottleneck in their structure studies because the expression of these proteins from mammalian cells is very low. The first strategy is the adoption of mammalian inducible expression system. A stable and inducible T-REx-293 cell line overexpressing an engineered rat NTSR1 was constructed. 2.5 million Functional copies of NTSR1 per cell were detected on plasma membrane, which is 167 fold improvement comparing to NTSR1 constitutive expression. The second strategy is production process development including suspension culture adaptation and induction parameter optimization. A further 3.5 fold improvement was achieved and approximately 1 milligram of purified functional NTSR1 per liter suspension culture was obtained. This was comparable yield to the transient baculovirus- insect cell system. The third strategy is high throughput miRNA screening. MiRNAs are a novel class of small, non-coding RNAs that can simultaneously silence multiple genes. The ii NTSR1-expressing cell line was subjected to human miRNA mimic library screening and nine miRNA mimics were identified to improve functional expression of NTSR1 by as much as 48%. Interestingly, five out of nine identified miRNA mimics were effective in improving the functional expression of other proteins, including luciferase (cytosolic protein), serotonin transporter (membrane protein) and glypican-3 hFc protein (secreted protein). These indicated that the identified miRNAs could have a wide role in enhancing production of proteins with biomedical interest. As genome-wide siRNA screens has emerged to be a powerful methodology for deducing gene functions in various diseases, we applied this technology on HEK293 cells constitutively expressing luciferase reporter to generate a genome-wide profile for recombinant protein expression process. Up to 362 genes associated with significantly enhanced luciferase expression were discovered and 28 of them were found to be enriched in splicesome pathway. Moreover, top 10 genes leading to greatest improvement of luciferase production were validated and tested with secreted and membrane proteins. Investigation of these genes/pathways may provide profound information to understanding protein biosynthesis process in mammalian cells. Oaz1 gene for example, was chosen for further investigation. Oaz1 encodes ornithine decarboxylase (ODC) antizyme 1, a major negative regulator of ODC and cellular polyamines. In our study, it is found that when antizyme was depleted, ODC enzyme and cellular polyamines levels were up-regulated, leading to enhanced luciferase translation. Advisors: Michael J. Betenbaugh and Joseph Shiloach iii Preface It has long been pursued by pharmaceutical and biotechnology industry to enhance the quality and quantity of recombinant proteins produced from host cells by traditional (e.g. media and bioprocess conditions optimization) and cell engineering approaches. In academia, the expression of recombinant proteins in general and mammalian proteins in particular, is also at the heart of current medical and structural studies. This dissertation consists of six chapters and is mainly focused on the improvement of functional expression of a hard-to-express membrane protein- neurotensin receptor (NTSR1). Chapter 1 provides a review on the ongoing effort to target bottlenecks along transcription, translation, protein processing and secretion pathways, as well as cell growth and survival to improve expression of low yielding proteins in a variety of hosts including bacterial, fungal, insect, and mammalian cells. The contents of Chapter 1 have been published in the Current Opinion in Structural Biology journal. Permission for its use was granted by the publisher, Elsvier (license number 3606050366032). Chapter 2 describes the construction of a stable HEK293 cell line with high-level functional NTSR1 expression and a quantitative comparison with NTSR1 produced from insect cells. The text and illustrations used in Chapter 2 have been published in the PLOS ONE journal and Methods in Molecular Biology journal. Permission was granted by Springer (license number 3606050761810). iv Chapter 3 features the identification of five microRNAs (hsa-miR-22-3p, hsa- miR-22-5p, hsa-miR-18a-5p, hsa-miR-429 and hsa-miR-2110) that can further improve NTSR1 functional expression in HEK293 cells. Their wider application was demonstrated by expression enhancement of other cytosolic, secreted and membrane proteins. The contents of Chapter 3 have been published in the Biotechnology and Bioengineering journal. Permission for its reuse was granted by John Wiley and Sons (license number 3606050872125). Chapter 4 extends the scope to system biology of HEK293 cells and identified 10 significant influential genes for heterologous protein production by genome-wide loss-of- function studies. Chapter 5 focuses on one of the identified top 10 genes (oaz1) and investigated the mechanism of improved protein expression upon oaz1 gene knockdown. A manuscript is being prepared based on contents of chapter 4 and 5. Finally, Chapter 6 concludes this dissertation and suggests the future work to extend the efforts. v Acknowledgements First of all I would like to thank my family, for their encouragement and support in my journey to pursue a doctoral degree. I would like to thank my father, who ignited my dream of becoming an engineer and encouraged me to apply for doctoral education. He has always been an inspiring example of pursuing his dream through thick and thin, and of building his success from ground up. He has always been a strong source of mental support and motivation which carried me through the moments of discouragement and irresolution. I would love to thank my mother, who instilled in me the mind of independence and self-acceptance. She has always been very understanding, supportive and caring along this long journey. Words alone cannot express my deep love and appreciation for her. I would like to express my sincere appreciation to my advisors, Dr. Michael J. Betenbaugh and Dr. Joseph Shiloach for their wisdom, mentorship and support. Their vision and commitment to research has inspired and will keep motivating me to excel in my scientific research. Their guidance in the past five years helped me to mature professionally and personally. It was an great honor and privilege to get to know them and work with them. I would also like to thank all my colleagues both at Johns Hopkins University and NIDDK Biotechnology Core Laboratory for their guidance and advices. Especially, I would love to thank Dr. Loc Trinh, Dr. Alejandro Negrete, Dr. Alex Druz, Dr. Bojiao Yin, Dr. Melissa St Amand, Joseph Priola, Amit Kumar and Sarah Inwood for their help vi and advices in designing and carrying out experiments, data analysis and trouble- shooting. I would also like to thank our collaborators at the Membrane Protein Structure and Function Unit at NIH, especially Dr. Reinhard Grisshammer and Jim White, for their training and guidance towards neurotensin receptor field. Sincere appreciation is also given to Dr. Christopher Tate and Dr. Juni Andréll at MRC Laboratory of Molecular Biology in UK, for sharing with me their insightful understanding and passion for membrane protein studies. I would also like to thank all staffs from NIH Chemical Genomics Center, especially Dr. Scott E. Martin and Yu-Chi Chen, as well as Dr. Myung Hee Park and Dr. Swati Mandal at National Institute of Dental and Craniofacial Research for their commitment as well as intelligent and technical input into our study. It has been truly a rewarding experience working with all of them. Finally, I would like to acknowledge that all funding for the work presented here was provided by the Intramural Research program at the National Institute of Diabetes and Digestive Kidney Diseases at the National Institutes of Health and the Department of Chemical and Biomolecular Engineering at Johns Hopkins University. We are grateful to Dr. Philip J. Reeves (University of Essex, Colchester, UK) for the kind gift of pACMV- tetO plasmid and to Dr. Mitchell Ho (National Cancer Institute, Bethesda, US) for HEK- GPC3-hFc cell line. vii Table of Contents Title page i Abstract ii Preface iv Acknowledgements vi List of Tables ix List of Figures x Chapter 1: Engineering cells to improve recombinant protein expression 1 Chapter 2: Stable expression of the neurotensin receptor NTSR1 with T-REx- 19 293 cells and comparison with baculovirus- insect cell system Chapter 3: Large-scale miRNA mimic screen for improved functional 46 expression of neurotensin