Li Yifeng, Sousa Rui
Protein Production Core Facility, Department of Biochemistry, University of Texas Health Science Center at San Antonio, San Antonio, TX 78229, USA.
Protein Expr Purif. 2012 Mar;82(1):162-7. doi: 10.1016/j.pep.2011.12.008. Epub 2012 Jan 2.
The extremely tight binding between biotin and avidin or streptavidin makes labeling proteins with biotin a useful tool for many applications. BirA is the Escherichia coli biotin ligase that site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (also known as Avi-tag). As a complementary approach to in vivo biotinylation of Avi-tag-bearing proteins, we developed a protocol for producing recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as both thioredoxin and MBP fusions, and was released from the corresponding fusion by TEV protease. The liberated ligase was separated from its carrier using HisTrap HP column. We obtained 24.7 and 27.6 mg BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be highly active in catalyzing in vitro biotinylation. The described protocol provides an effective means for making BirA ligase that can be used for biotinylation of different Avi-tag-bearing substrates.
生物素与抗生物素蛋白或链霉抗生物素蛋白之间的紧密结合，使得用生物素标记蛋白质成为许多应用中的一种有用工具。BirA是大肠杆菌生物素连接酶，它能在一个15个氨基酸的受体肽（也称为Avi标签）内对赖氨酸侧链进行位点特异性生物素化。作为对带有Avi标签的蛋白质进行体内生物素化的一种补充方法，我们开发了一种生产用于体外生物素化的重组BirA连接酶的方案。目标蛋白以硫氧还蛋白和MBP融合蛋白的形式表达，并通过TEV蛋白酶从相应的融合蛋白中释放出来。使用HisTrap HP柱将释放的连接酶与其载体分离。从硫氧还蛋白和MBP融合构建体中，我们每升培养物分别获得了24.7毫克和27.6毫克的BirA连接酶。该重组酶在催化体外生物素化方面表现出高活性。所描述的方案为制备可用于不同带有Avi标签的底物进行生物素化的BirA连接酶提供了一种有效方法。
