Development of a novel protease assay method by using an enzymatic cascades of engineered zymogens
Development of a novel protease assay method by using an enzymatic cascades of engineered zymogens
- 주제(키워드) Protease assay method , Engineered zymogen , Enzymatic cascade
- 발행기관 아주대학교
- 지도교수 유태현
- 발행년도 2016
- 학위수여년월 2016. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000021658
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Members of the matrix metalloproteinase (MMP) family have been implicated in disease states such as arthritis, periodontal disease, and tumor cell invasion and metastasis. MMPs were identified as therapeutic targets for cancers, and reviews have been published for the roles of MMPs in cancers. We previously reported a protease sensor platform developed by using an engineered ß-lactamase zymogen, and it was used for assaying MMP-2 activity. The assay method has a signal amplification step by ß-lactamase and thus showed a high sensitivity. In order to improve the method, we, in this study, have engineered a caspase-3 zymogen (pro-caspase-3) that can be activated by MMP-2 and then developed a new MMP-2 assay method by using an enzymatic cascade composed of the pro-caspase-3 enzyme and a ß-lactamase zymogen which can be activated by caspase-3. This method includes two signal amplification steps by caspase-3 and ß-lactamase and showed a detection of limit (LOD) of 29 pM, which is, to our knowledge, the lowest LOD reported so far. Two engineered zymogens can be easily engineered for other protease, and thus we believe that the signal cascade platform will be developed for assaying various proteases.
more목차
1. Introduction 1
1.1 Matrix metalloprotease-2 (MMP-2) and caspase-3 1
1.2 Protease assay methods based on fluorescence resonance energy transfer (FRET) 5
1.3 Protease assay methods based on zymogens 7
2. Materials and methods 9
2.1 Plasmid construction 9
2.2 Expression and purification of proteins 10
2.3 Protease assay 10
2.4 Analysis 12
3. Results and Discussion 16
3.1 Pro-caspase-3 that can be activated by MMP-2 18
3.2 Assay of MMP-2 using the engineered pro-caspase-3 23
3.3. ß-lactamase zymogen that can be activated by caspase-3 25
3.4 Assay of MMP-2 using the ß-lactamase zymogens by enzymatic cascade 33
4. Conclusion 35
5. References 37
ABSTRACT IN KOREAN 40
ACKNOWLEDGEMENT 42
