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: 7545931
• 负责人: ALICE Y TING
• 金额: $ 28.3万
• 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/7545931
• 关键词: AMPA Receptors; Agaricales; Alkynes; Amino Acids; Azides; Benzophenones; Binding Sites; Biotin; Brain; Cell Membrane Permeability; Cells; Cognitive Science; Collaborations; Consensus Sequence; Crosslinker; Dendritic Spines; Development; Engineering; Enzymes; Evolution; Excitatory Synapse; Glutamate Receptor; Growth; Hydrazones; In Vitro; Ketones; Label; Life; Ligase; Ligation; Light; Lysine; Mediating; Methodology; Molecular; N-terminal; Peptides; Phage Display; Proteins; Recombinant Proteins; Screening procedure; Side; Site; Specificity; Spin Labels; Structure; Synapses; Testing; Toxic effect; Work; analog; cross reactivity; crosslink; cycloaddition; density; design; desthiobiotin; extracellular; fluorophore; functional group; in vivo; interest; irradiation; mutant; photo switch; reaction rate; receptor; 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）：提出了针对具有小分子探针的重组蛋白在体内标记的新方法（例如荧光团，交叉链接器，照片开关和自旋标签）。该方法将采用生物素连接酶（BIRA）的重新设计突变体，这是一种细菌酶，该酶在13-氨基酸共有序列中特定于特异性生物素化赖氨酸侧链（“受体肽”；蓝色所示的修饰的赖氨酸残留物，下面显示为蓝色，下面显示）。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”），该酶（“探针连接酶”）特定于位点结合了各种探针以重新组合受体肽的蛋白质。蛋白质偶联的生物素类似物可以通过生物直接结合酮，叠氮化物或炔烃组，以适当功能化功能化的荧光团或其他生物物理探针，分别通过氢化援助形成，Staudinger结扎和[3+2] Sharpless Cycloadition。 具体目的：1。在体外工作的探针连接酶的发展。 2。开发在活细胞中起作用的探针连接酶。 3。将方法的应用用于鉴定谷氨酸受体的蛋白质相互作用伙伴。