Synthesis of protein-based polymers with the potential to form physically and covalently cross-linked networks Pubblico
Patterson, Melissa Ann (2011)
Published
Abstract
Materials science is targeting the construction of 'smart' biomaterials where stimulus by physiologically relevant cues induces an event at the molecular level, which acts cooperatively to bring about a macromolecular response. The first part of the work described herein has focused on development of a cloning strategy for the biosynthesis of high molecular weight, multi-domain polymers, derived from the pentapeptide (VPGVG) repeat motif characterizing native elastin. An attractive strategy for protein-based hydrogel development involves genetically fusing dissimilar polypeptide blocks with unique temperature-dependent hydrophobic assembly behavior and mechanical properties. Development of a biosynthetic strategy that is modular and seamless is reported as well as the temperature-dependent phase behavior of amphiphilic block copolymers as determined by DSC.
The remainder of the dissertation describes work toward developing a series of E. coli expression strains that are competent for multi-site incorporation of unnatural amino acids. UAAs were selected because their incorporation would provide novel strategies for further derivatization or cross-linking of the elastin-mimetic polypeptides under physiologically relevant conditions. In Chapters 3 and 4, residue-specific incorporation strategies are described for incorporation of Hag and DOPA under conditions that are compatible with high levels of analogue replacement and relatively high yield. The fidelity of analogue substitution was examined by amino acid analysis, mass spectrometry, and NMR studies for qualitative and quantitative measurements of replacement of the natural amino acid. The temperature-dependent assembly behavior of the analogue-containing and wild type elastin-mimetic polymers was investigated by DSC and temperature-dependent turbidity measurements. In addition, the metal-ligand binding and cross-linking behavior of the elastin-mimetic polymer containing DOPA was studied. Finally, Chapter 5 details the use of a site-specific incorporation method for multi-site incorporation of the benzophenone analogue of tyrosine, Bpa. Using an orthogonal suppressor tRNA/synthetase pair, the unnatural amino acid is incorporated into the polypeptide chain in response to the amber codon UAG. A mutant host strain is employed, which is characterized by a ribosomal mutation that alters release factor activity. The efficacy of the incorporation strategy is judged by protein yield, FACS analysis, and mass spectrometry.
Table of Contents
Table of Contents
Synthesis of protein-based polymers with the potential to form
physically and covalently
cross-linked networks
Chapter 1. Introduction...1
1. Protein-based materials...2
2. Amphiphilic block copolymers...6
3. Elastin...8
4. Elastin-mimetic polymers and block copolymers...10
5. Cross-linkable elastin-mimetic polymers...15
6. Introduction of non-canonical amino acids...17
7. References...22
Chapter 2. Generation of amphiphilic block copolymers derived from
elastin-mimetic sequences...29
1. Introduction...30
2. Experimental Methods...39
a. Materials...39
b. General methods...40
c. DNA cassette concatemerization...41
d. Assembly of DNA concatemer cassettes into triblock gene
fusions...45
e. Construction of the expression vector...48
f. Expression and purification of elastin-mimetic block
copolymers...52
g. Differential scanning calorimetry...54
3. Results and Discussion...56
4. Conclusion...79
5. References...80
Chapter 3. Introduction of alkene functionality into
elastin-mimetic polypeptides via an E.coli expression
system...83
1. Introduction...84
2. Experimental Methods...157
a. Materials...157
b. General methods...158
c. Construction of the elastin-Y gene...159
d. Cloning of the elastin-Y gene into the expression
plasmid...161
e. Synthetase plasmid construction...163
f. Bacterial growth and expression:...164
(1) Small scale expression in JW2581-1 strain...164
(2) Large scale (1 L) expression...166
g. Purification of the elastin-Y polymers...168
h. Physical and analytical measurements:...169
(1) Mass spectrometry of thermolysin digested
polypeptides...169
(2) Nuclear magnetic resonance (NMR) spectroscopy...170
(3) Temperature-dependent turbidity...171
(4) Metal-ligand binding of elastin-DOPA polymer...171
(5) Formation of an elastin-DOPA hydrogel...172
3. Results and Discussion...173
4. Conclusions...195
5. References...196
Chapter 5. Site-specific, multi-site incorporation of a
photo-cross-linkable amino acid analogue via a novel amber
suppression strategy...201
1. Introduction...202
2. Experimental Methods...210
a. Materials...210
b. General methods...211
c. Construction of plasmids:...212
(1) Elastin-UAG expression plasmids...212
(2) sfGFP expression plasmid...215
d. Bacterial growth and expression...215
e. TALON metal-affinity column purification...217
f. FACS analysis...218
g. Thermolysin digestion...219
h. Mass spectrometry...220
3. Results and Discussion...221
4. Conclusion...234
5. References...235
Chapter 6. Conclusions...238
Appendix 1. Sequences of primers utilized in plasmid
sequencing...243
Appendix 2. Sequences of interest...245
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