Engineering Advanced Self-Assembling Protein Biomaterials through the Extrapolation and Functionalization of Peptide-Based Systems 公开
Bartlett, Rebecca (Fall 2018)
Abstract
Chemically synthesized peptides represent useful building blocks for engineering self-assembling biomaterials because their rapid production permits high-throughput investigation of numerous peptide sequences in a brief time period. However, chemical synthesis limits the length and complexity of the generated peptides, which subsequently limits the potential applications. Despite these restrictions, useful information from successful self-assembling peptides can be extended to the development of more intricate systems through the use of genetic and protein engineering using E. coli as a host organism.
A previously published α-helical peptide was designed to assemble intermolecularly into a two-dimensional nanoporous framework where the thickness of the nanosheets was determined by the length of the peptide. Utilizing molecular cloning techniques, a system was designed for concatemerization of the α-helical peptide to generate nanosheets with greater thickness and improved thermodynamic and mechanical stability. This would support the application of these materials in therapeutic drug delivery or through incorporation into nanodevices.
In an unrelated investigation, HEAT-based concatemers were produced via genetic and protein engineering techniques for the development of thermodynamically stable, high aspect ratio nanotubes with two functionally distinct surfaces. Hypervariable residues on the concave surface are amenable to modification for application purposes. Meanwhile, the exterior surfaces of HEAT-based protein nanotubes were rapidly functionalized with the mCherry fluorescent protein through the formation of a covalent isopeptide bond by the SpyTag/SpyCatcher ligation system. Functionalization of the optimized HEAT-based protein nanotubes displaying the SpyTag peptide can promptly result from the introduction of the SpyCatcher protein fused to any arbitrary functional protein. The asymmetrical and interchangeable functionalization of these nanotubes results in a highly controllable system for various applications.
Table of Contents
Chapter 1. Self-Assembly of α‐Helical Proteins into Thermodynamically Stable Two-Dimensional Nanosheets ...... 1
1.1 Introduction ...... 2
1.2 Two-Dimensional Peptide Assemblies ...... 2
1.3 3FD-IL and 3FD-LL α-Helical Peptide Nanosheets ...... 5
1.4 Genetic Engineering of Biomaterials ...... 12
1.5 Conclusions ...... 15
1.6 References ...... 16
Chapter 2. Development of a System for the Generation of 3FD-LL Concatemers ...... 27
2.1 Introduction ...... 28
2.2 Results and Discussion ...... 31
2.2.1 Genetic Engineering of 3FD-LL Concatemer Sequences ...... 31
Design of Adapter and Insertion Genes ...... 31
Construction of the Expression Vector ...... 36
Generation of the 3FD-LL Monomer Gene Sequence ...... 40
Concatemerization of 3FD-LL Sequence ...... 42
2.2.2 Generation of the 3FD-LL Concatemer Proteins ...... 45
Expression of 3FD-LL Concatemers ...... 45
Cell Lysis ...... 48
Protein Solubilization ...... 48
Inclusion Body Purification ...... 52
Purification of the 3FD-LL Concatemers ...... 54
2.3 Conclusions ...... 56
2.4 Experimental ...... 57
2.4.1 Materials ...... 57
2.4.2 General Methods ...... 58
2.4.3 Genetic Engineering of 3FD-LL Concatemer Sequences ...... 60
2.4.4 Production of the 3FD-LL Concatemer Proteins ...... 70
2.4.5 Tables ...... 79
2.5 References ...... 82
Chapter 3. Production of 3FD-LL Concatemer Nanosheet Assemblies ...... 83
3.1 Introduction ...... 84
3.2 Results and Discussion ...... 86
3.2.1 Initial Assembly and Optimization of 3FD-LL Concatemers ...... 86
Assembly of HisPurTM 3FD-LL Concatemers ...... 86
Assembly of HisPurTM 3FD-LL Concatemers with TFE ...... 102
3.2.2 Characterization of HPLC Purified 3FD-LL Trimer Nanosheets ...... 121
Circular Dichroism Spectropolarimetry ...... 123
Transmission Electron Microscopy ...... 127
Atomic Force Microscopy ...... 129
3.3 Conclusions ...... 132
3.4 Experimental ...... 134
3.4.1 Materials ...... 134
3.4.2 General Methods ...... 135
3.4.3 Initial Assembly and Optimization of 3FD-LL Concatemers ...... 136
3.4.4 Assembly and Characterization of HPLC Purified 3FD-LL Trimer ...... 138
3.4.5 Tables ...... 142
3.5 References ...... 143
Chapter 4. Structurally Defined Helical Nanotubes for the Construction of Functionally Asymmetric Hybrid Nanomaterials ...... 145
4.1 Introduction ...... 146
4.2 Tandem Repeat Proteins (TRPs) ...... 148
4.3 HEAT_R1 Peptide Assemblies ...... 153
4.4 HEAT_6R Protein Assemblies ...... 158
4.5 SpyTag/SpyCatcher Ligation System ...... 162
4.6 Conclusions ...... 168
4.7 References ...... 169
Chapter 5. Design and Production of SpyTag_HEAT and mCherry_SpyCatcher ...... 174
5.1 Introduction ...... 175
5.2 Results and Discussion ...... 179
5.2.1 Generation of ST_HEAT ...... 179
Sequence Design ...... 179
Protein Expression and Purification ...... 181
5.2.2 Generation of mCh_SC ...... 185
Sequence Design ...... 185
Protein Expression and Purification ...... 189
5.2.3 Initial Verification of Linking Between ST_HEAT and mCh_SC ...... 197
Linking of ST_HEAT Lysate with mCh_SC Lysate ...... 197
Linking of HisPurTM Eluents of ST_HEAT and mCh_SC ...... 199
5.3 Conclusions ...... 201
5.4 Experimental ...... 202
5.4.1 Materials ...... 202
5.4.2 General Methods ...... 203
5.4.3 SpyTag_HEAT Production ...... 204
5.4.4 mCherry_SpyCatcher Production ...... 209
5.4.5 Verification of Linking Between ST_HEAT and mCh_SC ...... 216
5.4.6 Tables ...... 218
5.5 References ...... 220
Chapter 6. Characterization of ST_HEAT Nanotubes and Functionalization with mCherry_SpyCatcher ...... 223
6.1 Introduction ...... 224
6.2 Results and Discussion ...... 226
6.2.1 Assembly and Characterization of SpyTag_HEAT Nanotubes ...... 226
Low Concentration HisPurTM ST_HEAT Assemblies ...... 226
High Concentration HisPurTM ST_HEAT Assemblies ...... 232
High Concentration Assemblies of HPLC Purified ST_HEAT ...... 235
Low Concentration Assemblies of HPLC Purified ST_HEAT ...... 239
6.2.2 Functionalization of ST_HEAT Nanotubes with mCh_SC Protein ...... 244
Functionalization of HisPurTM ST_HEAT with mCh_SC ...... 245
Functionalization of HPLC Purified ST_HEAT with mCh_SC ...... 248
Optimizing Amount of mCh_SC for Maximum Coverage ...... 254
Ultracentrifugation for Removal of Excess mCh_SC ...... 256
Centrifugal Filtration for Removal of Excess mCh_SC ...... 263
Fluorimetry Analysis ...... 268
Correlative Light Electron Microscopy (CLEM) ...... 272
6.3 Conclusions ...... 274
6.4 Experimental ...... 277
6.4.1 Materials ...... 277
6.4.2 General Methods ...... 278
6.4.3 Assembly and Characterization of SpyTag_HEAT Nanotubes ...... 279
6.4.4 Functionalization of ST_HEAT Nanotubes with mCh_SC ...... 284
6.4.5 Tables ...... 295
6.5 References ...... 296
Appendix A. Sequences ...... 302
Appendix B. Mass Spectra ...... 313
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