Crystal Structure of Engineered Endolysin EC340 derived from Bacteriophage targeting Gram-Negative Bacteria
- 주제(키워드) Gram-negative bacteria , endolysin , cecropin A , antibacterial agent , crystal structure
- 주제(DDC) 547
- 발행기관 아주대학교
- 지도교수 서민덕
- 발행년도 2023
- 학위수여년월 2023. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000032770
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
The development of antibiotics has made it possible to cure many infections. Unfortunately, overuse of antibiotics has led to a rapid increase in antibiotic-resistant strains. Bacteriophage-encoded endolysins have emerged as a new class of antibacterial agents to combat surging antibiotic resistance. We were determined the structure of mtEC340M, which is an engineered endolysin EC340 of the PBEC131 phage that infects E. coli. The mtEC340M has 2.4 Å resolution diffraction data and consisting of 8 α-helices and 2 loops. The three active residues of mtEC340M were predicted by structural comparison with peptidoglycan-degrading lysozyme. LNT113 added cecropin A to the N-terminus to enhance antibacterial activity in the engineered mtEC340M. In this paper, the crystal structure of mtEC340M was identified and structural differences were confirmed by modeling LNT113. We would like to contribute to the development of Gram-negative bacteria-derived endolysin-based antibacterial agents based on structural differences between mtEC340M and LNT113.
more목차
1. Introduction 1
2. Materials and Methods 7
2.1 Materials 7
2.1.1 Materials 7
2.1.2 Agents and apparatus 8
2.2 Method 9
2.2.1 Overexpression and solubility test 9
2.2.2 Purification 10
2.2.3 Circular dichroism (CD) spectroscopy 11
2.2.4 Size exclusion chromatography (SEC) and Multi Angle Light Scattering (MALS) 12
2.2.5 Crystallization 13
2.2.6 X-ray diffraction and data collection 14
2.2.7 Structural determination. 14
2.2.8 Homologous structural comparison 14
3. Results and discussion 15
3.1 Overexpression and solubility of endolysin proteins 15
3.2 Purification of endolysin proteins 16
3.2.1 Purification of mtEC340M 16
3.2.2 Purification of LNT113 16
3.3 Oligomeric states of mtEC340M 19
3.4 Secondary structures using CD 21
3.5 Crystallization of endolysin proteins 24
3.5.1 Crystallization and structural determination of mtEC340M 24
3.5.2 Crystallization and structural prediction of LNT113 25
3.5.3 Structures of mtEC340M and LNT113 25
3.5.4 Active site of mtEC340M endolysin 33
4. Conclusion 37
5. Reference 38
