一种经过工程改造的芳基叠氮化物连接酶，用于通过光交联对蛋白质-蛋白质相互作用进行位点特异性映射。
An engineered aryl azide ligase for site-specific mapping of protein-protein interactions through photo-cross-linking.
作者信息
Baruah Hemanta, Puthenveetil Sujiet, Choi Yoon-Aa, Shah Samit, Ting Alice Y
机构信息
Department of Chemistry, Room 18-496, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
出版信息
Angew Chem Int Ed Engl. 2008;47(37):7018-21. doi: 10.1002/anie.200802088.
Abstract
We re-engineered the small-molecule binding site of E. coli lipoic acid ligase (LplA) to accept a fluorinated aryl azide probe in place of lipoic acid. The mutated ligase can covalently attach the aryl azide to recombinant proteins fused to a 17-amino acid recognition sequence for LplA (called "LAP"). Labeling is highly specific, modifying LAP fusion proteins to the exclusion of all endogenous mammalian proteins. In cell lysate, we labeled FKBP with aryl azide and demonstrated rapamycin-dependent photocrosslinking to FRB.
摘要
我们对大肠杆菌硫辛酸连接酶（LplA）的小分子结合位点进行了重新设计，使其能够接受氟化芳基叠氮化物探针以取代硫辛酸。突变后的连接酶可将芳基叠氮化物共价连接到与LplA的17个氨基酸识别序列（称为“LAP”）融合的重组蛋白上。标记具有高度特异性，可修饰LAP融合蛋白而不影响所有内源性哺乳动物蛋白。在细胞裂解物中，我们用芳基叠氮化物标记了FKBP，并证明了雷帕霉素依赖性的光交联到FRB。
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