Codon reassignment with reactive amino acid analogues and site-specific conjugation of proteins
Codon reassignment with reactive amino acid analogues and site-specific conjugation of proteins
- 주제(키워드) Unnatural amino acid , site-specific conjugation , protein modification
- 발행기관 아주대학교
- 지도교수 유태현
- 발행년도 2018
- 학위수여년월 2018. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000027908
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The biological protein synthesis system has been engineered to incorporate unnatural amino acid into proteins, and this has opened up new routes for engineering proteins with novel compositions. The novel compositions allow that proteins are modified site-specifically by bio-orthogonal method. While such systems have been successfully applied in research, there remains a need to develop new approaches with respect to the wider application of unnatural amino acids. In this study, we reported a strategy for incorporating unnatural amino acids into proteins by reassigning one of the Arg sense codons, the AGG codon and four-base codon, AGGA codon. Using this method, several unnatural amino acids were quantitatively incorporated into the AGG and AGGA sites. And we applied the method to multiple AGG and AGGA sites, and even to tandem AGG and AGGA sequences. The method developed and described here could be used for engineering proteins with diverse unnatural amino acids, particularly when employed in combination with other methods. Moreover, monoclonal cell-penetrating antibody, cytotransmab was conjugated to biomacromolecule site-specifically using unnatural amino acid and it was demonstrated that the cytotransmab conjugate deliver the biomacromolecule into the cytosol.
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Abstract i
List of figures vi
Chapter I. General introduction 1
1. Amino acid analogue 2
1.1. Protein biosynthesis 2
1.2. Expanding the diversity of protein modification via unnatural amino acids 6
1.3. Chemical synthesis of peptides and proteins with unnatural amino acids 9
1.4. In vivo incorporation of unnatural amino acids into proteins 11
1.5. The use of unnatural amino acids 21
2. Antibody conjugation 24
2.1. Antibody conjugates 24
2.2. Therapeutic antibody conjugates 25
3. Aims of this study 28
Chapter II. Incorporation of unnatural amino acids in response to AGG codon 30
1. Introduction 31
2. Materials and methods 36
2.1. Strain Construction 36
2.2. Plasmid Construction 37
2.3. Incorporation of Unnatural Amino Acids into Protein 38
2.4. Copper-catalyzed Click Reaction and Western Blot Analysis 39
2.5. Protein purification 40
2.6. Synthesis of THPTA 41
2.7. Mass spectrometry analysis 42
3. Results and discussion 44
3.1. tRNACCUArg is not necessary for growth of E. coli 44
3.2. Overexpression of E. coli prolyl-tRNA synthetase for preventing misaminoacylation 46
3.3. Impacts of unnatural amino acids in response to AGG codons of proteome 50
3.4. Knockout of argW gene allows efficient incorporation of unnatural amino acids 52
3.5. Incorporation of unnatural amino acids in response to multiple AGG sites 57
4. Conclusion 64
Chapter III. Incorporation of unnatural amino acids in response to AGGA codon 65
1. Introduction 66
2. Materials and methods 69
2.1. Plasmid construction 69
2.2. Expression of proteins with UAAs 70
2.3. Strain-promoted copper-free click chemistry and western blot analysis 70
2.4. Protein purification 71
2.5. Mass spectrometry 72
3. Results and discussion 74
3.1. The MjTyrRS-MjtRNA_UCCU pair enabled incorporation of UAAs in response to the AGGA codon 74
3.2. Arg was incorporated in response to the AGGA codon in the presence of MjtRNA_UCCU 79
3.3. The A38C change in MjtRNA_UCCU increased the efficiency of AzF incorporation into the AGGA codon site 82
3.4. Various UAAs were incorporated into the protein in response to the AGGA codon 86
3.5. Knocking out the argW gene (tRNA_CCU^Arg) increased the incorporation efficiency of UAA into multiple AGGA positions 89
4. Conclusion 94
Chapter IV. Site-specific conjugation of cell-penetrating antibody and biomolecules 96
1. Introduction 97
2. Materials and methods 100
2.1. siRNAs for the conjugation 100
2.2. Construction of plasmids expressing thio-cytotransmab 100
2.3. Expression and purification of antibody 101
2.4. Cytotransmab-siRNA conjugation by non-specific method 102
2.5. Cytotransmab and siRNA (PE24) conjugation by site-specific method 102
2.6. Expression, purification of PE24 103
2.7. Cytotoxicity 104
3. Results and discussion 106
3.1. Synthesis of cytotransmab-siRNA conjugates 106
3.2. Site-specific conjugation of cytotransmab and siRNA 109
3.3. Site-specific conjugation of herceptin and PE24 115
4. Conclusion 119
Chapter V. Summary 120
References 124
Abstract in Korean 138
