Muti-site specific incorporation of non-canonical amino acids for biomaterials design Público

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 Methods

38

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 Discussion

68

4. Conclusions

90

5. References

92

Chapter 3. System optimization for the multi-site specific incorporation of photo-crosslinkable amino acid analogues

96

1. Introduction

97

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 discussion

116

4. Conclusions

138

5. References

139

Chapter 4. System application: Synthesis of photo-crosslinkable elastin diblock copolymers

143

1. Introduction

144

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 discussion

164

4. Conclusions

178

5. References

180

Chapter 5. Conclusion

184

1. Conclusion

185

2. References

191

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

About this Dissertation

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School	Laney Graduate School
Department	Chemistry
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry
Palavra-chave	unnatural amino acid biomaterial elastin
Committee Chair / Thesis Advisor	Conticello, Vincent, Emory University
Committee Members	Lutz, Stefan, Emory University Salaita, Khalid, Emory University

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