A new technology platform for studying protein function

研究蛋白质功能的新技术平台

基本信息

批准号：
7845989
负责人：
MATTHEW P DELISA
金额：
$ 3.98万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-01-09 至 2009-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7845989
关键词：
Acquired Immunodeficiency Syndrome Affinity Antibodies Antigens Antisense RNA Arginine Bacteria Binding Biological Assay Biological Process Categories Cell Nucleus Cells Cytoplasm Degenerative Disorder Development Disease Dreams Drug Delivery Systems Engineering Environment Exhibits Gene Deletion Gene Expression Generations Genes Goals Half-Life Human Immunoglobulins In Vitro Knock-out Knowledge Libraries Life Malignant Neoplasms Mammalian Cell Mediating Methods Molecular Monoclonal Antibodies One-Step dentin bonding system Outcome Pathway interactions Pharmaceutical Preparations Phenotype Play Process Property Proteins Proteome Proteomics Quality Control RNA RNA Interference Reagent Research Role Scheme Specificity Structure System Technology Testing Time Twin Multiple Birth Validation Work bacterial genetics base biological research cellular targeting combinatorial design disulfide bond extracellular functional genomics genetic selection in vivo innovation interest new technology novel protein function research study tool

项目摘要

DESCRIPTION (provided by applicant): Ever since the invention of monoclonal antibodies in 1975 and, more recently, the development of various in vitro antibody display technologies, antibodies have become one of the most powerful tools in biological research and are presently the fastest growing category of new drug entities. One molecular format that shows great promise is the intracellular antibody or intrabody that exploits the specificity and diversity of immunoglobulins to target a wide range of intracellular proteins by expressing the antibody in vivo. In principle, whatever can be achieved by a monoclonal antibody in the extracellular environment can be similarly achieved inside of a cell using an intrabody. Since intrabody synthesis can be constitutive or inducible, the level of inactivation can be toggled which might allow for a wider range of phenotypes than can be observed with gene deletion, antisense or RNAi-based knockdown strategies. Further, since intrabodies are proteins, they possess a much longer half-life compared to RNA and are also more specific to their target molecules. Also, it is possible to design or engineer intrabodies to block certain domains of a particular target protein, thus allowing for the decoupling of multiple protein activities of a single target. This might prove particularly useful for essential targets that have more than one cellular activity. Finally, since intrabodies can be multivalent, simultaneous functional knockout of two or more cellular targets is possible. Based on the above features, intrabodies are expected to play an important and immediate role for target identification and validation in functional genomics and/or proteomics. The long-term objective of this research effort is to develop a proteome-wide repertoire of intrabodies for probing and modulating protein activities inside living cells. The objective of this particular application, which is the first step towards our long-term goal, is to create a novel platform technology based on the bacterial twin-arginine translocation (Tat) pathway that enables rapid, one-step genetic selection of single-chain intrabodies against virtually any intracellular target protein. To accomplish the overall objective of this application, the following specific aims are proposed: (1) develop a genetic selection based on unique mechanistic features of the bacterial Tat system for isolating intrabody-antigen pairings; and (2) engineer intrabodies that specifically inhibit biological processes. Intrabodies are an emerging class of antibody molecules that function (e.g., bind their cognate antigen) intracellularly and, owing to their specificity and diversity, have the potential to block, suppress, alter or even enhance a vast array of biological processes. Therefore, the focus of these studies is to develop a technology platform for rapid, large-scale synthesis of intrabodies that could be used as (i) functional genomics reagents that enable characterization of novel gene products and validation of these gene products as potential drug targets and (ii) drug entities that be used in the treatment of human disorders such as cancer, AIDS or neuro-degenerative disorders.

描述（由申请人提供）：自1975年单克隆抗体发明以来，以及最近的各种体外抗体展示技术的开发，抗体已成为生物学研究中最有力的工具之一，目前是增长最快的新药实体类别。一种表现出巨大希望的一种分子格式是细胞内抗体或内征的特异性和多样性，通过在体内表达抗体，以靶向各种细胞内蛋白。原则上，使用内部体制可以在细胞内类似地实现单克隆抗体在细胞外环境中可以实现的任何方法。由于内部合成可以是组成型的或可诱导的，因此可以切换失活的水平，这可能允许使用基因缺失，反义或基于RNAI的敲低策略观察到的表型更大。此外，由于内构为蛋白质，与RNA相比，它们具有更长的半衰期，并且对目标分子的特异性也更为特异。同样，可以设计或工程内形式来阻止特定靶蛋白的某些结构域，从而使单个靶标的多种蛋白质活性解耦。对于具有多个细胞活性的基本靶标，这可能是特别有用的。最后，由于内置可能是多价的，因此可以同时对两个或多个细胞靶标的功能敲除。基于上述特征，预期内遗嘱将在功能基因组学和/或蛋白质组学中的目标识别和验证中起重要而直接的作用。这项研究工作的长期目的是开发全体生殖的全蛋白质组曲目，用于探测和调节活细胞内的蛋白质活性。该特定应用的目的是朝着我们的长期目标迈出的第一步，是基于细菌双精氨酸易位（TAT）途径创建一种新颖的平台技术，该技术可以快速，一步遗传单链内代属性，几乎可以针对任何细胞内靶蛋白。为了实现此应用程序的总体目标，提出了以下特定目的：（1）基于细菌TAT系统的独特机械特征来开发遗传选择，用于隔离内部抗原配对；（2）专门抑制生物学过程的工程师内生内形式。内遗嘱是一类新兴的抗体分子（例如，结合其同源抗原）的抗体分子，由于它们的特异性和多样性，具有阻断，抑制，改变甚至增强一系列生物学过程的潜力。因此，这些研究的重点是开发一个技术平台，以快速，大规模合成生殖的合成，可以用作（i）功能基因组学试剂，该试剂能够表征新型基因产物并将这些基因产物作为潜在药物靶标和（ii）药物的验证，这些药物用于治疗诸如癌症，艾滋病，艾滋病，艾滋病，艾滋病，艾滋病，神经疾病或神经性差异。

项目成果

期刊论文数量（4）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Efficient isolation of soluble intracellular single-chain antibodies using the twin-arginine translocation machinery.

DOI：
10.1016/j.jmb.2008.10.051
发表时间：
2009-01-09
期刊：
JOURNAL OF MOLECULAR BIOLOGY
影响因子：
5.6
作者：
Fisher, Adam C.;DeLisa, Matthew P.
通讯作者：
DeLisa, Matthew P.

Exploration of twin-arginine translocation for expression and purification of correctly folded proteins in Escherichia coli.

DOI：
10.1111/j.1751-7915.2008.00041.x
发表时间：
2008-09
期刊：
Microbial biotechnology
影响因子：
5.7
作者：
Fisher AC;Kim JY;Perez-Rodriguez R;Tullman-Ercek D;Fish WR;Henderson LA;DeLisa MP
通讯作者：
DeLisa MP

Design and Functional Characterization of Synthetic E3 Ubiquitin Ligases for Targeted Protein Depletion.