Engineering the bacterial translation for incorporation of unnatural amino acids at the protein N-terminus
Engineering the bacterial translation for incorporation of unnatural amino acids at the protein N-terminus
- 주제(키워드) Unnatural amino acid , tRNA engineering , Biotechnology
- 발행기관 아주대학교
- 지도교수 유태현
- 발행년도 2019
- 학위수여년월 2019. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000028623
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
There has been an expansion of repertoire of unnatural amino acids that can be incorporated into recombinant proteins in vivo, but all of them are limited to the elongation process of protein synthesis. However, even though the N-terminal position of proteins can be utilized for diverse purposes, in particular chemical modifications, no method has been reported for positioning unnatural amino acids at the translation initiation site. In this study, I have designed a protein initiation system active for unnatural amino acids by engineering an orthogonal tyrosyl-tRNA from Methanococcus jannaschii, which had been developed with aminoacyl-tRNA synthetases for incorporating unnatural amino acids for the translation elongation step. To make the system orthogonal to the natural one, one of stop codons, amber codon, was chosen for the new translation initiation system. A couple of tRNA variants were generated by modifying the acceptor stem, the anticodon loop, and the T loop, aiming making interactions with formyltransferase and initiation factor 2 without hurting the aminoacylation of unnatural amino acids. One engineered tRNA supported the initiation of protein synthesis with O-propagyl tyrosine (OpgY) with overexpression of the formyltransferase. The incorporation of OpgY was confirmed by mass spectrometry and N-terminal sequencing analyses. However, one of natural amino acids, gluamine, was also incorporated into the amber codon, even though its efficiency was lower than that of OpgY. To minimize the misincorporation, attempts have been made to engineer the tRNA further.
more목차
1. Introduction 1
1.1 Protein synthesis initiation and initiator tRNA 1
1.2 Engineering the protein translation initiation system 5
1.3 Unnatural amino acids 6
1.4 Purpose of this study 8
2. Materials and Methods 9
2.1 Plasmids used in this study 9
2.2 Incorporation of unnatural amino acid and protein expression 12
2.3 Western blot analysis and click chemistry 12
2.4 Protein purification 14
2.5 Mass spectrometry 14
3. Results and Discussion 15
3.1 tRNA engineering for translation initiation 15
3.2 Incorporation of UAAs into start codon of GFP model protein 18
3.3. Optimization for overexpression of formyltransferase and initiation factor 2 21
3.4. Investigation that Mj-tRNA2 can be used as the elongator tRNA 23
3.5. Mass spectrometry of Z-domain protein 27
3.6. Validation of the first amino acid of protein 29
3.7. Engineering Mj-tRNA2 to decrease the interaction with E.coli glutamyl-tRNA synthetase 31
3.8 Combinational Mj-tRNA library design 34
4. Conclusion 36
5. References 37
6. Abstract in Korean 43
