Muti-site specific incorporation of non-canonical amino acids for biomaterials design Pubblico
Wu, I-Lin (2013)
Abstract
Site-specific incorporation of selective unnatural functional groups into proteins has provided a means to expand the protein diversity, creating proteins with unique chemical properties useful for a broad range of new applications. However, the preparation of protein polymers derived from the sequence-repetitive polypeptides requires new development in which non-canonical amino acids are incorporated at multiple and specific locations in the polypeptides sequences. We describe herein a simple and efficient method to facilitate the multi-site selective insertions of non-canonical amino acids at structurally defined positions within recombinant polypeptides and provide opportunities for engineering proteins modified extensively with selected amino acid analogues. In this approach, Escherichia coli MRA30, a bacterial host strain with an attenuated activity of release factor 1 (RF1), was assessed for its ability to support the incorporation of a diverse range of non-canonical amino acids in response to multiple encoded amber codons (UAG) within genes derived from superfolder GFP and elastin-mimetic protein polymers. Suppression efficiency and protein yield depended on the identity of the orthogonal aminoacyl-tRNA synthetase/ tRNACUA pair and the non-canonical amino acid. Elastin-mimetic protein polymers were prepared where non-canonical amino acids were incorporated up to twenty-two specific sites with high substitution efficiency. The identities and positions of the variant residues were confirmed by mass spectrometric analysis of the full-length polypeptides and the proteolytic cleavage fragments from the thermolysin digestion. Based on the developed system, we further demonstrate the generation of novel amphiphilic elastin diblock copolymers that could undergo temperature-dependent segregation and self-assemble into core-shell-corona nanoparticles with photo-crosslinkable activities. The photo-crosslinking experiments were monitored by dynamic light scattering and microscopes. Our data suggest that this multi-site suppression approach permits the preparation of protein-based materials in which novel chemical functionality can be introduced at precisely pre-defined positions within the polypeptide sequence.
Table of Contents
Multiple site-specific incorporation of non-canonical amino acids for novel biomaterials design
Chapter 1. Introduction
1
1. Incorporation of non-canonical amino acids
2
2. Protein-based biomaterials
6
3. Elastin
11
4. Elastin-mimetic polypeptides
14
5. Amphiphilic block copolymers
16
6. References
22
Chapter 2. System development for the multi-site specific incorporation of non-canonical amino acids
25
1. Introduction 26 2. Experimental Methods38
a. Materials
38
b. General methods
39
c. Construction of the expression vector
40
d. Construction of sfGFP expression plasmids
44
e. Construction of the elastin-mimetic peptides, Elastin-UAG genes
45
f. Construction of the Elastin-UAG expression plasmids
48
g. Construction of the orthogonal aminoacyl-tRNA synthetase/tRNA pairs
49
h. Protein expression and purification
51
i. Flow Cytometry
55
j. Thermolysin digestion
56
k. Mass spectrometry
57
l. Temperature-dependent turbidity
58
3. Results and Discussion68
4. Conclusions90
5. References92
Chapter 3. System optimization for the multi-site specific incorporation of photo-crosslinkable amino acid analogues
96
1. Introduction97
2. Experimental Methods:104
a. Materials
104
b. General methods
105
c. Construction of the M. jannaschii tRNA plasmid
106
d. Construction of the M. jannaschii aminoacyl-tRNA synthetase / tRNA plasmid
106
e. Bacterial growth and expression
107
f. TALON® metal-affinity column purification
108
g. Flow cytometry
110
h. Thermolysin digestion
110
i. Mass spectrometry
111
j. Photo-crosslink experiments
112
3. Results and discussion116
4. Conclusions138
5. References139
Chapter 4. System application: Synthesis of photo-crosslinkable elastin diblock copolymers
143
1. Introduction144
2. Experimental Methods:
152
a. Materials
152
b. General methods
152
a. Construction of the Elastin-diblock plasmids
153
b. Protein expression and purification
155
c. Transmission electron microscopy
156
d. Temperature-dependent turbidity
157
e. Dynamic light scattering
157
f. Atomic force microscopy
158
g. Thermolysin digestion and Mass spectrometry
158
3. Results and discussion164
4. Conclusions178
5. References180
Chapter 5. Conclusion
184
1. Conclusion185
2. References191
Appendix. Sequences of Interest
193
Appendix 1. Sequencing primers utilized in the study
194
Appendix 2. Sequences of the MbPylRS
195
Appendix 3. Sequences of the MjTyrRS
196
Appendix 4. Sequences of the wild-type sfGFP-CCC
197
Appendix 5. Sequences of the wild-type sfGFP-(UAG)4
198
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