Specific protein labeling in cells with engineered BirA

使用工程 BirA 标记细胞中的特定蛋白质

• 批准号: 6855424
• 负责人: ALICE Y TING
• 金额: $ 29.93万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-17 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6855424
• 关键词: biotechnology; biotin; fluorescent dye /probe; glutamate receptor; ionophores; ligase; molecular probes; nanotechnology; protein engineering; protein protein interaction; recombinant proteins; small molecule

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.

描述（由申请人提供）：提出了用小分子探针（如荧光团、交联剂、光开关和自旋标记）在体内标记重组蛋白的新方法。该方法将采用生物素连接酶（BirA）的重新工程突变体，BirA是一种细菌酶，可在13个氨基酸的一致序列（“受体肽”；下图蓝色显示的修饰赖氨酸残基）中特异性地对赖氨酸侧链进行生物素化。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，我们期望产生酶（“探针连接酶”），将各种探针特异性地结合到含有受体肽的重组蛋白上。蛋白质偶联的生物素类似物可以通过形成腙、Staudinger连接和[3+2]Sharpless环加成分别与适当功能化的荧光团或其他生物物理探针进行生物正交连接。