Specific protein labeling in cells with engineered BirA

使用工程 BirA 标记细胞中的特定蛋白质

基本信息

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
项目类别：
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

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

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项目摘要

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。