Specific protein labeling in cells with engineered BirA

描述（由申请人提供）：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）。该方法将采用生物素连接酶（BirA）的再工程化突变体，BirA是一种细菌酶，其位点特异性地生物素化13个氨基酸共有序列内的赖氨酸侧链（“受体肽”;下面以蓝色显示的修饰的赖氨酸残基）。通过改变BirA的生物素结合位点以容纳一系列生物素类似物和其他小分子，我们期望产生酶（“探针连接酶”），其将各种探针位点特异性地缀合至携带受体肽的重组蛋白。带有酮、叠氮化物或炔基的蛋白质缀合的生物素类似物可以分别通过腙形成、施陶丁格连接和[3+2]夏普勒斯环加成与适当官能化的荧光团或其他生物物理探针生物正交连接。 具体目标：1.开发一种在体外工作的探针连接酶。2.在活细胞中工作的探针连接酶的开发。3.应用方法学鉴定谷氨酸受体蛋白质相互作用伴侣。

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.