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  • 09월 04일 17시 이후 : 초록수정 불가능, 일정확인 및 검색만 가능

제114회 대한화학회 학술발표회, 총회 및 기기전시회 안내 Traceless and site-specific fluorescent labeling of membrane anchored proteins in live cells

2014년 8월 28일 15시 49분 02초
BIO.P-638 이곳을 클릭하시면 발표코드에 대한 설명을 보실 수 있습니다.
10월 15일 (수요일) 16:00~19:00
저자 및
이의연, 권영은*
동국대학교 의생명공학과, Korea
The understanding of protein interaction and cellular dynamics is an important step to get insight of biological processes. Therefore, a recent life science research has increasing demands on studying physiological events on the molecular level in real-time. For this purpose, live cell imaging using fluorescent proteins (FPs) has been widely utilized to visualize target proteins under a microscopy by constructing fusion proteins. However, several limitations of FPs, such as slow maturation kinetics or issues with photo-stability under laser illumination, have led researchers to examine new technologies beyond FP-based imaging. In this study, we have utilized Npu DnaE split-intein mediated protein trans-splicing (PTS) reaction for site-specific fluorescent labeling of cell surface proteins. Trans-splicing reaction is a self-processing reaction and can be used to introduce various synthetic probes to target proteins. We prepared a model cell surface protein fused to N-terminal fragment of Npu DnaE intein and a C-terminal fragment of Npu DnaE intein fused to a fluorescent probe. Here, we adopted an engineered Npu intein. The engineered C-intein is considerably shorter compared to wild-type C-inteins, to make the preparation of C-intein becomes easier and to make this approach more practical. We demonstrated that engineered Npu intein mediated labeling reaction is comparable to wild-type Npu intein. No external energy was required for the labeling reaction. This approach can be used for dual color labeling systems by using orthogonal split-inteins and will lead to new methologies that investigate protein localization, transportation, and cell signaling.