Biomaterials Design: Creation of a novel orthogonal translational system in E. coli for the site-specific incorporation of proline analogues in biosynthetic protein materials Open Access

Carpenter, Holly Elizabeth (2008)

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New techniques have recently been presented that enable co-translational, in vivo incorporation of amino acid analogues in proteins through an expansion of the genetic code. In general terms, methods to expand the genetic code have included the selection and directed evolution of orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that function in a heterologous host, such that a non-canonical amino acid is inserted in response to a termination codon using non-sense suppression. While successful incorporation of non-canonical amino acids has been achieved, non-sense suppression has limited efficiency, particularly for multi-site incorporation of amino acid analogues. Therefore, a different approach was chosen that involved capture of codons that are rarely utilized in the native host organism. Given this framework, the CCC codon of the proline family box was chosen as a target for incorporation of non-canonical proline analogues in an E. coli expression system where proline or proline analogues were directly incorporated at the site of multiple CCC codons to produce an elastin-mimetic protein material. Production of an elastin-mimetic material serves as a model for synthetic collagen biosynthesis where proline and hydroxyproline are directly incorporated at multiple sites into the same polypeptide.

Library construction of MjProRS variants and high-throughput screening of the libraries using a fluorescence-activated cell sorting (FACS)-based method were optimized for use in isolation of a synthetase mutant that would be specific for (2 S,4 R)-4-hydroxyproline. Design of a synthetase enzyme that exhibited a preference for the imino acid analogue over the natural proline substrate would be a significant advance toward the design of synthetic collagen production where proline and hydroxyproline incorporation would occur simultaneously in a bacterial host using separate aminoacyl-tRNA synthetase/tRNA pairs.

Chapter 5 outlines the design and expression of recombinant human tropoelastin in E. coli. Human tropoelastin is an attractive biomaterial target as it is the natural soluble precursor to elastin in higher mammals. Recombinant tropoelastin production in bacteria offers a cost-effective and high volume production method for use of tropoelastin or tropoelastin variants (with amino acid analogue incorporation) in biomaterial applications.

Table of Contents

Chapter 1. Introduction 1

Biomaterials: Elastin and Collagen 2

Genetic Engineering of Biomaterials 9

Directed Evolution: Design and genetic engineering of novel, orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs for multi-site incorporation of proline analogues in E. coli 11

Screening methodologies for engineering site-specificity in orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs for multi-site incorporation of proline analogues in E. coli 15

References 21

Chapter 2. Generation of a Synthetic Gene Encoding the Elastin-Mimetic Polypeptide, Elastin-CCC 28

Introduction 29

Materials 33

Methods: Construction of the elastin-CCC gene 34

Expression plasmid construction 37

Cloning elastin-CCC into the expression plasmid 38

Results and Discussion 41

Conclusions 53

References 54

Chapter 3. Design and Genetic Engineering of a Novel Orthogonal Translational

System in E. coli 56

Introduction 57

Materials 63

Methods: Preparation of the bacterial host strain 65

Plasmid and gene construction 66

Confirming orthogonality: Construction of "mix-and-match" plasmids 73

Functional assay 74

Protein expression and purification 74

MALDI-TOF-MS analysis 80

List of primers 80

Results and Discussion: Preparation of the bacterial host strain 83

Protein expression 84

"Mix-and-match" experiment 96

Protein purification 100

Incorporation of proline analogues 103

Functional assay for orthogonality 108

Bacterial host strain, UQ27 ProRSts, ProA 110

Conclusions 110

References 112

Chapter 4. Development of a Fluorescence-Activated Cell Sorting (FACS) Based Screening System for use in Altering Substrate Specificity in the M. jannaschii Prolyl-tRNA Synthetase Enzyme 115

Introduction 116

Materials 129

Methods: Construction of the elastin-CCC-GFPuv fusion reporter 132

Construction of the elastin-CCC-superfolder GFP fusion reporter 134

Cloning and mutagenesis of DsRed 135

Cloning and mutagenesis of GFPuv 137

Rational design: site-directed mutagenesis of MjProRS 139

Library construction: Random mutagenesis of MjProRS 141

Library construction: Site-saturation metagenesis of MjProRS 143

List of primers 145

Fluorescence-activated cell sorting (FACS) and analysis 147

Protein expression 153

Protein purification 159

MALDI-TOF-MS analysis 160

Results and discussion: Site-directed mutagenesis of MjProRS 161

Elastin-CCC-GFPuv reporter systems 162

Random mutagenesis and screening of MjProRS variants 166

Site-saturation mutagenesis and screening of MjProRS 169

GFPuv and DsRed as potential reporter proteins 181

Elastin-CCC-superfolder GFP fusion reporter 189

Conclusions 205

References 207

Chapter 5. Biomaterials Design: Human tropoelastin 210

Introduction 211

Materials 218

Methods: Plasmid construction and cloning 220

Protein expression 222

Purification of tropoelastin 225

Results and Discussion 230

Conclusions 237

References 238

Appendix 1. Sequences of Interest 241

Appendix 2. Recipes for Making Studier Minimal Media 247

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