Engineering Advanced Self-Assembling Protein Biomaterials through the Extrapolation and Functionalization of Peptide-Based Systems 公开

Bartlett, Rebecca (Fall 2018)

Permanent URL: https://etd.library.emory.edu/concern/etds/v118rf424?locale=zh
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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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