OmpG nanopore for single molecule protein sensing

 描述（由申请人提供）：我们的中心目标是创建一个纳米孔传感器，可以调整到专门检测几乎任何这些疾病相关的蛋白质。该传感器是一种工程形式的外膜蛋白G（OmpG）从E。杆菌连接OmpG β-桶链的环要么附加配体，要么用识别序列延长，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，能够区分针对相同半抗原产生的抗体混合物中的变体。为了扩大传感器的实用性，我们将探索控制传感器-目标相互作用的基本机制。此外，OmpG环内新结合位点的掺入将通过两种途径进行。第一个是合理设计，我们将纳入已知的多肽序列，识别既定的目标。第二条途径利用OmpG在E. coli外膜。将使用高通量筛选和富集方法直接从细菌中选择OmpG突变体的随机文库用于新的靶亲和力。

 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.