Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase
Targeted and proximity-dependent promiscuous protein biotinylation by a mutant Escherichia coli biotin protein ligase
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DOI：
10.1016/j.jnutbio.2005.03.017
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发表时间：
2005-07-01
影响因子：
5.6
通讯作者：
Cronan, JE
中科院分区：
文献类型：
--
作者：
Cronan, JE
建立了一种酶法生物素化蛋白质的方法。使用大肠杆菌的生物素蛋白质连接酶（BirA）的突变形式（R118 G），并且认为生物素化是通过由突变蛋白质释放的生物素酰基-5 ' AMP化学酰化蛋白质赖氨酸侧链来进行的。牛血清白蛋白、氯霉素乙酰转移酶、免疫球蛋白链和RNA酶A以及大量的E.大肠杆菌蛋白已经被生物素化。生物素化反应是邻近依赖性的，因为当连接酶与受体蛋白偶联时生物素化的程度比当受体在溶液中游离时大得多。这可能是由于酰化剂生物素酰-5 '-AMP的快速水解所致。因此，当突变连接酶连接到参与蛋白质-蛋白质相互作用的一个配偶体时，它可以用于用生物素特异性标记另一个配偶体，从而允许通过现有的抗生物素蛋白/链霉抗生物素蛋白技术容易地检测和回收蛋白质。(c)2005年爱思唯尔公司All rights reserved.
A method for general protein biotinylation by enzymatic means has been developed. A mutant form (R118G) of the biotin protein ligase (BirA) of Escherichia coli is used and biotinylation is thought to proceed by chemical acylation of protein lysine side chains by biotinoyl-5' AMP released from the mutant protein. Bovine serum albumin, chloramphenicol acetyltransferase, immunoglobulin chains and RNAse A as well as a large number of E. coli proteins have been biotinylated. The biotinylation reaction is proximity dependent in that the extent of biotinylation is much greater when the ligase is coupled to the acceptor protein than when the acceptor is free in solution. This is presumably due to rapid hydrolysis of the acylation agent, biotinoyl-5'-AMP. Therefore, when the mutant ligase is attached to one partner involved in a protein-protein interaction, it can be used to specifically tag the other partner with biotin, thereby permitting facile detection and recovery of the proteins by existing avidin/streptavidin technology. (c) 2005 Elsevier Inc. All rights reserved.