Promiscuous protein biotinylation by Escherichia coli biotin protein ligase
Promiscuous protein biotinylation by Escherichia coli biotin protein ligase
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DOI：
10.1110/ps.04911804
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发表时间：
2004-11-01
期刊：
影响因子：
8
通讯作者：
Cronan, JE
中科院分区：
文献类型：
--
作者：
Choi-Rhee, E;Schulman, H;Cronan, JE
生物素蛋白质连接酶（BPL）是具有非凡特异性的酶。BirA，大肠杆菌的BPL生物素化仅单个细胞蛋白。我们报告了一个突变体BirA，附着生物素的大量细胞蛋白在体内和牛血清白蛋白，氯霉素乙酰转移酶，免疫球蛋白的重链和轻链，RNA酶A在体外。突变体BirA也在体内和体外自我生物素化。野生型BirA蛋白在这些反应中的活性要低得多。的生物素化反应是proximitydependent的，在更大程度的生物素化被视为当突变连接酶偶联到受体蛋白比当受体在溶液中是免费的。这种方法可以通过现有的亲和素/链霉亲和素技术容易地检测和回收相互作用的蛋白质。
Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximitydependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.