Biomaterials Design: Creation of a novel orthogonal translationalsystem in E. coli for the site-specific incorporation ofproline analogues in biosynthetic protein materials Open Access
Carpenter, Holly Elizabeth (2008)
Abstract
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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