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UNIVERSITY of CALIFORNIA, IRVINE an Expanded Genetic Code UNIVERSITY OF CALIFORNIA, IRVINE An expanded genetic code for the characterization and directed evolution of tyrosine-sulfated proteins DISSERTATION submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILSOPHY in Biomedical Engineering by Xiang Li Dissertation Committee: Assistant Professor Chang C. Liu, Chair Associate Professor Wendy Liu Associate Professor Jennifer A. Prescher 2018 Portion of Chapter 2 © John Wiley and Sons Portion of Chapter 3 © Springer Portion of Chapter 4 © Royal Society of Chemistry All other materials © 2018 Xiang Li i Dedication To My parents Audrey Bai and Yong Li and My brother Joshua Li ii Table of Content LIST OF FIGURES ..................................................................................................................VI LIST OF TABLES ................................................................................................................. VIII CURRICULUM VITAE ...........................................................................................................IX ACKNOWLEDGEMENTS .................................................................................................... XII ABSTRACT .......................................................................................................................... XIII CHAPTER 1. INTRODUCTION ................................................................................................ 1 1.1. INTRODUCTION .................................................................................................................. 1 1.2. OVERVIEW OF DISSERTATION ............................................................................................. 2 1.2. REFERENCES ..................................................................................................................... 3 CHAPTER 2. BIOLOGICAL APPLICATIONS OF EXPANDED GENETIC CODES ............... 5 2.1. INTRODUCTION .................................................................................................................. 6 2.2. PROTEIN BIOLOGY ............................................................................................................. 7 2.2.1 Therapeutic Proteins through Bioconjugation ............................................................. 8 2.2.2 Addressing Posttranslationally Modified Proteins ....................................................... 8 2.3. CELL BIOLOGY ................................................................................................................ 11 2.3.1. Light-activated Crosslinking and Control ................................................................. 11 2.3.2. Fluorescent Reporters .............................................................................................. 13 2.3. SYNTHETIC BIOLOGY ....................................................................................................... 14 2.4. LABORATORY EVOLUTION ............................................................................................... 16 2.5. OUTLOOK ........................................................................................................................ 18 2.6. ACKNOWLEDGEMENTS .................................................................................................... 18 2.7. REFERENCES ................................................................................................................... 18 CHAPTER 3. SITE-SPECIFIC INCORPORATION OF SULFOTYROSINE USING AN EXPANDED GENETIC CODE ................................................................................................ 30 3.1. INTRODUCTION ................................................................................................................ 31 3.2. MATERIALS ..................................................................................................................... 32 3.2.1. E. coli strains ........................................................................................................... 32 3.2.2. Plasmids .................................................................................................................. 32 3.2.3. Stock solutions ........................................................................................................ 33 3.2.4. Media ...................................................................................................................... 34 3.2.5. Agar Plates .............................................................................................................. 34 3.2.6. Transformation Reagents ......................................................................................... 34 3.3. METHODS........................................................................................................................ 35 3.3.1. Construction of plasmids ......................................................................................... 35 3.3.2. Preparation of electrocompetent bacterial cells. ....................................................... 35 3.3.3. Transformation via electroporation. ......................................................................... 37 3.3.4. Protein expression.................................................................................................... 38 3.4. NOTES............................................................................................................................. 38 iii 3.5. REFERENCES ................................................................................................................... 40 3.6. FIGURE LOG .................................................................................................................... 42 CHAPTER 4. A SECOND-GENERATION EXPRESSION SYSTEM FOR TYROSINE SULFATED PROTEINS AND ITS APPLICATION IN CROP PROTECTION ....................... 46 4.1. INTRODUCTION ................................................................................................................ 47 4.2. RESULTS AND DISCUSSION ............................................................................................... 48 4.3. CONCLUSION ................................................................................................................... 52 4.4. AUTHOR CONTRIBUTION .................................................................................................. 52 4.5. COMPETING FINANCIAL INTERESTS ................................................................................... 52 4.6. ACKNOWLEDGEMENTS .................................................................................................... 53 4.7. REFERENCES ................................................................................................................... 53 4.8. SUPPLEMENTAL INFORMATION ........................................................................................ 58 4.8.1. Experimental Details ............................................................................................... 58 4.8.1.1. Construction of second-generation sY protein expression vectors...................... 58 4.8.1.2. Cloning, expression, and analyses of GFP ......................................................... 58 4.8.1.3. Cloning, Expression and Purification of RaxX60 .............................................. 59 4.8.1.4. Gene expression assay in rice ............................................................................ 60 4.8.1.5. Statistical analysis ............................................................................................. 60 4.8.1.6. Proteomic Analysis ........................................................................................... 61 4.8.1.7. Shotgun proteomics .......................................................................................... 61 4.8.1.8. Targeted proteomics.......................................................................................... 62 4.8.1.9. Calculations of Relative sY status ..................................................................... 62 4.8.1.10. Circular Dichroism.......................................................................................... 64 4.8.1.11. Post-treatment experiment ............................................................................... 64 4.8.1.12. Commercial peptides....................................................................................... 64 4.8.1.13. Sequences of GFP-1UAG ............................................................................... 65 4.8.1.14. Sequences of GFP-3UAG ............................................................................... 65 4.8.2. Supplemental figures ............................................................................................... 66 4.8.3. Supplemental tables ................................................................................................. 69 4.8.4. Supplemental references .......................................................................................... 73 CHAPTER 5. CHARACTERIZATION OF A SULFATED ANTI-HIV ANTIBODY USING AN EXPANDED GENETIC CODE ................................................................................................ 75 5.1. INTRODUCTION ................................................................................................................ 76 5.2. METHODS........................................................................................................................ 78 5.2.1. Construction of expression plasmids for E51 Fabs and small hairpin RNAs. ...........
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