A Novel Technology for Engineering Binders to Membrane Proteins

抗体被认为是对抗多种人类疾病的“灵丹妙药”。然而，仍有遗迹 针对多通道膜蛋白的抗体工程中的重大挑战 包括大量的治疗靶点，如细胞表面受体和离子通道蛋白。 膜蛋白工程结合的困难源于体外电流的限制。 选择/平移技术，如噬菌体展示，这需要高度纯化的目的蛋白。 不幸的是，由于膜蛋白对细胞的依赖，膜蛋白往往难以纯化。 用于适当折叠和活动的膜。目前，还没有有效的体外技术来治疗 多通道膜蛋白结合剂的发现/工程。这项研究的总体目标是开发 一种用于工程蛋白质结合的新技术--SmURF(简单邻近耦合mRNA显示) 蛋白质定位于细胞表面，从而绕过了提纯目标蛋白质的需要。蓝精灵组合 与传统的平移不同，mRNA的显示具有邻近辅助DNA组装现象 其中所有粘合剂对固体支撑物都是浓缩的，蓝精灵培养的粘合剂只能浓缩到所需的程度 细胞表面的靶蛋白。在目标1中，我们将使用寡核苷酸来演示蓝精灵原理 并对引物序列进行了优化。目标2将在一个模型蛋白中建立蓝精灵浓缩 非靶蛋白在溶液中的混合物。最后，在目标3中，模型蛋白质将显示在 将对哺乳动物细胞表面和粘合剂文库进行筛选，以展示和量化整个细胞 蓝精灵浓缩效率。这项研究的成功完成将为 方便地发现/工程全细胞展示的膜蛋白的结合体，并应大大扩展 可用于治疗干预的药物靶点的曲目。

Antibodies have been coined the ‘magic bullets’ against many human diseases. However, there remains significant challenges in the engineering of antibodies targeting multi-pass membrane proteins, which encompass a large number of therapeutic targets such as cell surface receptors and the ion channel proteins. The difficulty in engineering binders to membrane proteins stems from the limitation of the current in vitro selection/panning technologies, such as phage display, which require highly purified target protein. Unfortunately, membrane proteins are often refractory to purification due to their dependence on the cell membrane for proper folding and activity. Currently, there is no effective in vitro technology for the discovery/engineering of binders to multi-pass membrane proteins. The overall goal of this study is to develop a novel technology – SMURF (Simple proxiMity coUpled mRNA display) – for engineering protein binders to protein targets on the cell surface, thus bypassing the need to purify the target protein. SMURF combines mRNA display with the proximity-assisted-DNA-assembly phenomenon and, unlike conventional panning in which all binders to a solid support are enriched, SMURF fosters the enrichment of binders only to a desired target protein on the cell surface. In Aim 1, we will demonstrate the SMURF principle using oligonucleotides and optimize the primer sequences. Aim 2 will establish the SMURF enrichment of a model protein in a mixture of non-target proteins in solution. Finally, in Aim 3, a model protein will be displayed on the mammalian cell surface and a library of binders will be screened to demonstrate and quantify the whole-cell SMURF enrichment efficiency. The successful completion of this study will establish a novel technology for facile discovery/engineering of binders to whole-cell-displayed membrane proteins and should greatly expand the repertoire of drug targets amenable to therapeutic intervention.