Incorporation of unnatural amino acids into a four-base codon of AGGA
Incorporation of unnatural amino acids into a four-base codon of AGGA
- 주제(키워드) Unnatural amino acid , Four-base codon
- 발행기관 아주대학교
- 지도교수 유태현
- 발행년도 2017
- 학위수여년월 2017. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000024299
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Incorporation of unnatural amino acids (UAAs) into site-specific position in protein expands the range of protein engineering. Several strategies have been developed to incorporate UAA using unique codon such as stop codon and quadruplet codon. Generally, various UAA have been introduced in response to amber codon (UAG), by using tyrosyl-tRNA and tyrosyl-tRNA synthetase pair derived from Methanococcus jannaschii in Escherichia coli. However, amber suppression has disadvantage that could not allow multiple incorporation of unnatural amino acids into single protein. Recently, a lot of four-base codons were studied for expanding a new codon. One of the four-base codons, AGGA codon has been exploited, but further research is needed to optimize this system. In this study, we engineered MjtRNA to improve the incorporation efficiency of UAAs in response to AGGA codon. Because the Mj tyrosyl-tRNA synthetase had been reported to have promiscuity toward anticodon, anticodon of Mj tRNACUA was changed to UCCU for decoding AGGA codon. In addition, A38 nucleotide of MjtRNAUCCU was changed to C (MjtRNAUCCU-A38C) in order to reduce misincorporation of arginine in protein. In this study, we demonstrated that this system was useful for incorporation of UAAs quantitatively in response to the AGGA codon. And we applied incorporating different kinds of unnatural amino acid to AGGA codon using MjtRNAUCCU-A38C in Escherichia coli. In addition, UAA incorporation efficiency was improved by using BS02 which the endogenous tRNA_CCU^Arg gene of E. coli strain was knocked out. The success of this approach can expand the scope of protein engineering using UAAs, particularly, in combination with other UAA incorporation methods.
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ABSTRACT
Ⅰ. Introduction
1.1. Unnatural amino acids (UAAs)
1.2. Method for incorporation of UAAs in protein
1.3. Orthogonal tRNA synthetase and tRNA pair engineering
Ⅱ. Materials and Methods
2.1. Plasmid construction
2.2. Expression of proteins with UAAs
2.3. Strain-promoted copper-free click chemistry and western blot analysis
2.4. Protein purification
2.5. Mass spectrometry
2.6. Fluorescence-activated cell sorting (FACS) and Flow cytometry analysis
Ⅲ.Results and discussions
3.1. Incorporation of UAA in response to AGGA codon in protein by using MjTyrRS-MjtRNAUCCU pair
3.2. Misincorporation of arginine into AGGA codon
3.3. Increased effieciency of AGGA suppression by changing A38 nucleotide of MjtRNAUCCU to C.
3.4. Incorporation of various UAAs in response to the AGGA codon in protein
3.5. Efficient incorporation of UAA into multiple AGGA positions by using engineered E.coli strain, BS02
3.6. Selection of MjtRNAUCCU for efficient suppression of AGGA codon
Ⅳ. Conclusions
Ⅴ. References
Ⅵ. Abstract in Korean
Ⅶ. Acknowledgement
