OmpG nanopore for single molecule protein sensing

用于单分子蛋白质传感的 OmpG 纳米孔

基本信息

批准号：
9901553
负责人：
Min Chen
金额：
$ 30.65万
依托单位：
UNIVERSITY OF MASSACHUSETTS AMHERST
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9901553
关键词：
Achievement Affect Affinity Amino Acid Sequence Antibodies Area Bacteria Binding Proteins Binding Sites Biological Biological Markers Biomedical Research Biotin Charge Chemicals Data Detection Development Devices Diagnosis Disease Disease Outbreaks Docking Elements Engineering Escherichia coli Fingerprint Fluorescence-Activated Cell Sorting Future G-substrate GTP-Binding Proteins Goals Hand Haptens Immunoassay Length Libraries Ligands Mass Spectrum Analysis Measures Membrane Membrane Proteins Methods Modification Molecular Molecular Conformation Nature Pattern Peptides Process Protein Array Proteins Randomized Route Site Streptavidin Structure Surface Technology Testing Time Variant Viral Virus Diseases base cancer diagnosis cancer therapy cost effective design extracellular flexibility high throughput screening insight interest mutant nanopore novel novel strategies pathogen peptide G polypeptide portability protein aminoacid sequence protein expression protein protein interaction protein purification public health relevance screening sensor single molecule virtual voltage weapons

项目摘要

DESCRIPTION (provided by applicant): Our central goal is to create a nanopore sensor that can be tuned to specifically detect virtually any of these disease-related protein. The sensor is an engineered form of outer membrane protein G (OmpG) from E. coli. The loops that connect the strands of OmpG's β-barrel are either appended with a ligand or lengthened with a recognition sequence to create the specific sensing elements. Our preliminary results demonstrate that the OmpG nanopore sensor is remarkably sensitive, able to distinguish variants within a mixture of antibodies that were all raised against the same hapten. To expand the utility of the sensor, we will explore the fundamental mechanisms that govern sensor-target interactions. Furthermore, the incorporation of new binding sites within OmpG's loops will proceed by two routes. The first is rational design, where we will incorporate known polypeptide sequences that recognize established targets. The second route takes advantage of OmpG's expression in the E. coli outer membrane. A randomized library of OmpG mutants will be selected for novel target affinity directly from the bacteria using a high- throughput screening and enrichment approach.

描述（由适用提供）：我们的核心目标是创建一个可以调整的纳米孔传感器，该传感器几乎可以特异性检测这些疾病相关的蛋白质中的任何一种。该传感器是大肠杆菌的外膜蛋白G（OMPG）的工程形式。连接OMPGβ-桶的链的环要么用配体附加，要么以识别序列加长以创建特定的传感器元件。我们的初步结果表明，OMPG纳米孔传感器非常敏感，能够区分抗体混合物中的变体，这些变体都针对同一触觉。为了扩展传感器的效用，我们将探索控制传感器目标相互作用的基本机制。此外，OMPG循环中的新绑定站点的使用将通过两条路线进行。第一个是理性设计，我们将结合认识到已建立目标的已知多肽序列。第二个路线利用了OMPG在大肠杆菌外膜中的表达。使用高通量筛选和富集方法，将选择一个直接从细菌中的新型目标亲和力选择的OMPG突变体的随机库。