A Novel Technology for Engineering Binders to Membrane Proteins

膜蛋白结合剂工程新技术

• 批准号: 10040547
• 负责人: Zhilei Chen
• 金额: $ 22.71万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10040547
• 关键词: Ankyrin Repeat; Antibodies; Base Sequence; Binding Proteins; Bypass; Cell Surface Receptors; Cell membrane; Cell surface; Cells; Coin; Complementary DNA; Coupled; DNA; Dependence; Drug Targeting; Engineering; Fostering; G-Protein-Coupled Receptors; Goals; Immobilization; In Vitro; Incubated; Ion Channel Protein; Label; Libraries; Magic; Mammalian Cell; Membrane Proteins; Messenger RNA; Methods; Modeling; Nucleotides; Oligonucleotides; Phage Display; Process; Protein Engineering; Proteins; RNA; Recombinants; Refractory; Reporting; Sampling; Single-Stranded DNA; Site; Solid; Structure; Structure of thyroid parafollicular cell; Supporting Cell; System; Technology; Therapeutic Intervention; antibody engineering; design; druggable target; human disease; member; new technology; stem; success; therapeutic protein; therapeutic target; unnatural amino acids

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.

抗体被称为对付许多人类疾病的“灵丹妙药”。然而， 在靶向多通道膜蛋白的抗体的工程化中的重大挑战， 包括大量的治疗靶点，例如细胞表面受体和离子通道蛋白。 工程化膜蛋白结合剂的困难源于体外电流的限制 选择/淘选技术，例如噬菌体展示，其需要高度纯化的靶蛋白。 不幸的是，由于膜蛋白对细胞的依赖性，它们通常难以纯化 膜进行适当的折叠和活动。目前，还没有有效的体外技术用于 发现/工程化多通道膜蛋白的结合剂。本研究的总体目标是开发 一种新的技术- SMURF（简单近端偶联mRNA展示）-用于工程化蛋白结合剂， 蛋白质靶向细胞表面，从而绕过了纯化靶蛋白的需要。SMURF组合 mRNA显示与邻近辅助的DNA组装现象，并不像传统的淘选， 其中固体支持物的所有结合剂都是富集的，SMURF促进结合剂仅富集到所需的浓度。 细胞表面的靶蛋白。在目标1中，我们将使用寡核苷酸证明SMURF原理 并优化引物序列。目的2将建立模型蛋白质在一个细胞中的SMURF富集。 溶液中的非靶蛋白的混合物。最后，在目标3中，模型蛋白质将显示在 将筛选哺乳动物细胞表面和结合物文库，以证明和定量全细胞 SMURF富集效率。这项研究的成功完成将建立一种新的技术， 容易发现/工程化的结合剂，全细胞展示的膜蛋白，并应大大扩大 适合治疗干预的药物靶点库。