OmpG nanopore for single molecule protein sensing

 描述(申请人提供)：我们的中心目标是创造一种纳米孔传感器，它可以被调节到专门检测几乎任何这些与疾病相关的蛋白质。该传感器是来自大肠杆菌的外膜蛋白G(OmpG)的工程形式。连接OMPG的β-Barrel链的环要么被附加上配体，要么被识别序列加长，以创建特定的传感元件。我们的初步结果表明，OmpG纳米孔传感器非常敏感，能够区分所有针对同一半抗原的抗体混合物中的变体。为了扩大传感器的用途，我们将探索控制传感器与目标相互作用的基本机制。此外，在OmpG的环中加入新的结合位点将通过两条途径进行。第一个是合理的设计，我们将结合识别既定靶点的已知多肽序列。第二条途径是利用OmpG在大肠杆菌外膜中的表达。将使用高通量筛选和浓缩方法直接从细菌中选择OmpG突变体的随机文库，以获得新的靶标亲和力。

 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.