Specific protein labeling in cells with engineered BirA

描述(申请人提供)：提出了用小分子探针(例如，荧光团、交联剂、光开关和自旋标记)对重组蛋白质进行体内标记的新方法。这一方法将使用生物素连接酶(BIRA)的重新工程突变体，BIRA是一种细菌酶，可定点生物素化13个氨基酸共同序列(“受体肽”；修饰的赖氨酸残基，如下图蓝色所示)中的赖氨酸侧链。通过改变BIRA的生物素结合位点以适应一系列生物素类似物和其他小分子，我们希望产生酶(“探针连接酶”)，将各种探针与含有受体多肽的重组蛋白定点结合。蛋白质偶联生物素类似物可以分别通过缩合、Staudinger连接和[3+2]锐环加成等方式与适当官能化的荧光团或其他生物物理探针进行生物正交连接。 具体目标：1.开发一种体外工作的探针连接酶。2.开发一种在活细胞中工作的探针连接酶。3.应用该方法寻找谷氨酸受体的蛋白质相互作用伙伴。

DESCRIPTION (provided by applicant): New methodology is proposed for the in vivo labeling of recombinant proteins with small-molecule probes (e.g., fluorophores, crosslinkers, photo switches, and spin labels). This methodology will employ re-engineered mutants of biotin ligase (BirA), a bacterial enzyme that site-specifically biotinylates a lysine side-chain within a 13-amino acid consensus sequence (the "acceptor peptide"; modified lysine residue shown in blue, below). By altering the biotin binding site of BirA to accommodate a range of biotin analogues and other small molecules, we expect to generate enzymes ("probe ligases") that site-specifically conjugate various probes to recombinant proteins bearing the acceptor peptide. Protein-conjugated biotin analogues beating ketone, azide, or alkyne groups can be bio-orthogonally ligated to appropriately functionalized fluorophores or other biophysical probes via hydrazone formation, Staudinger ligation, and [3+2] Sharpless cycloaddition, respectively. Specific aims: 1. Development of a probe ligase that works in vitro. 2. Development of a probe ligase that works in living cells. 3. Application of the methodology to identification of protein interaction partners for glutamate receptor.