文章基本信息

标题：A general approach for chemical labeling and rapid, spatially controlled protein inactivation
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作者：Kevin M. Marks ; Patrick D. Braun ; Garry P. Nolan 等
期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：27
页码：9982-9987
DOI：10.1073/pnas.0401609101
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Chemical labeling of proteins inside of living cells can enable studies of the location, movement, and function of proteins in vivo. Here we demonstrate an approach for chemical labeling of proteins that uses the high-affinity interaction between an FKBP12 mutant (F36V) and a synthetic, engineered ligand (SLF'). A fluorescein conjugate to the engineered ligand (FL-SLF') retained binding to FKBP12(F36V) and possessed similar fluorescence properties as parental fluorescein. FL-SLF' labeled FKBP12(F36V) fusion proteins in live mammalian cells, and was used to monitor the subcellular localization of a membrane targeted FKBP12(F36V) construct. Chemical labeling of FKBP12(F36V) fusion proteins with FL-SLF' was readily detectable at low expression levels of the FKBP12(F36V) fusion, and the level of fluorescent staining with FL-SLF' was proportional to the FKBP12(F36V) expression level. This FL-SLF'-FKBP12(F36V) labeling technique was tested in fluorophore assisted laser inactivation (FALI), a light-mediated technique to rapidly inactivate fluorophore-labeled target proteins. FL-SLF' mediated FALI of a {beta}-galactosidase-FKBP12(F36V) fusion protein, causing rapid inactivation of >90% of enzyme activity upon irradiation in vitro. FL-SLF' also mediated FALI of a {beta}-galactosidase fusion expressed in living NIH 3T3 cells, where {beta}-galactosidase activity was reduced in 15 s. Thus, FL-SLF' can be used to monitor proteins in vivo and to target rapid, spatially and temporally defined inactivation of target proteins in living cells in a process that we call FK-FALI.
关键词：fluorescent ; FKBP12 ; fluorophore-assisted laser inactivation