PROTEIN ENGINEERING VIA IN VIVO PHENYLALANINE ANALOGS
TABLE OF CONTENTS CHAPTER 1 UNNATURAL AMINO ACIDS IN BIOMATERIALS AND PROTEIN ENGINEERING INTRODUCTION 2 REFERENCES 16 CHAPTER 2 BIOSYNTHESIS OF PROTEINS INCORPORATING A VERSATILE SET OF PHENYLALANINE ANALOGS INTRODUCTION 24 MATERIALS AND METHODS 26 RESULTS AND DISCUSSION 29 PAGE ANALYSIS OF THE EFFECTS PHERS ON ANALOG INCORPORATION INTO MDHFR 29 QUANTITATIVE ANALYSIS OF ANALOG INCORPORATION BY AMINO ACID ANALYSIS 29 CONFIRMATION OF ANALOG INCORPORATION BY MALDI TOF ANALYSIS 29 LARGE SCALE EXPRESSION OF MDHFR CONTAINING UNNATURAL ANALOGS 32 ANALYSIS OF MDFHR CONTAINING ANALOGS DEMONSTRATES NEW UV SIGNATURES 32 CONCLUSION 36 REFERENCES 37 CHAPTER 3 A DESIGNED PHENYLALANYL TRNA SYNTHETASE VARIANT ALLOWS EFFICIENT IN VIVO INCORPORATION OF ARYL KETONE FUNCTIONALITY INTO PROTEINS INTRODUCTION 42 MATERIALS AND METHODS 45 RESULTS AND DISCUSSION 51 MUTATIONAL PREDICTIONS BASED ON ORBIT CALCULATIONS 51 PAGE ANALYSIS OF MUTANT SYNTHETASE EFFECTS ON INCORPORATION OF 2 INTO MDHFR 51 MALDI TOF ANALYSIS OF INCORPORATION OF 2 55 SELECTIVE MODIFICATION OF DHFR 2 WITH BIOTIN HYDRAZIDE 55 CONCLUSION 59 REFERENCES 60 CHAPTER 4 ENGINEERING RELAXED SUBSTRATE SPECIFICITY INTO E COLI PHENYLALANYL TRNA SYNTHETASE TO INCORPORATE A DIVERSE SET OF NON NATURAL AMINO ACIDS INTRODUCTION 64 MATERIALS AND METHODS 69 RESULTS AND DISCUSSION 74 EXPRESSION AND PURIFICATION OF PHERS VARIANTS 74 ACTIVATION OF ANALOGS BY VARIANT ENZYMES IN VITRO 74 IN VIVO EVALUATION BY DHFR TRYPTIC PEPTIDE ANALYSIS 78 CONCLUSION 85 REFERENCES 86 CHAPTER 5 EFFICIENT PHOTOCROSSLINKING OF AN ARTIFICIAL EXTRACELLULAR MATRIX PROTEIN VIA IN VIVO INCORPORATION OF ARYLAZIDE FUNCTIONALITY INTRODUCTION 93 MATERIALS AND METHODS 97 RESULTS AND DISCUSSION 100 PROTEIN EXPRESSION AND PURIFICATION 100 INFRARED SPECTROSCOPY OF CS5 ELF N3 100 FILM PRODUCTION 105 MECHANICAL TESTING 105 CONCLUSION 109 REFERENCES 76 CHAPTER 6 PATTERNING AND CELL BINDING PROPERTIES OF A PROTEIN PHOTORESIST PRODUCED IN E COLI INTRODUCTION 115