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.

抗体被称为对抗许多人类疾病的“灵丹妙药”，但抗体仍然存在。 针对多通道膜蛋白的抗体工程面临重大挑战， 涵盖大量治疗靶点，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目前体外技术的限制 选择/淘选技术，例如噬菌体展示，需要高度纯化的目标蛋白。 不幸的是，膜蛋白由于对细胞的依赖性而通常难以纯化 目前，还没有有效的体外技术来实现正确的折叠和活性。 多通道膜蛋白结合剂的发现/工程化本研究的总体目标是开发。 一项新技术 – SMURF（简单邻近耦合 mRNA 显示） – 用于工程蛋白质结合剂 蛋白质靶向细胞表面，从而无需纯化 SMURF 组合。 mRNA 展示具有邻近辅助 DNA 组装现象，与传统的淘选不同 固相支持物上的所有结合物均被富集，SMURF 仅促进结合物的富集至所需的值 在目标 1 中，我们将使用寡核苷酸演示 SMURF 原理。 目标 2 将建立模型蛋白的 SMURF 富集。 最后，在目标 3 中，模型蛋白质将显示在溶液中。 将筛选哺乳动物细胞表面和粘合剂库以展示和量化全细胞 SMURF 富集效率这项研究的成功完成将建立一种新技术。 轻松发现/设计全细胞展示膜蛋白的结合物，并且应该大大扩展 适合治疗干预的药物靶点清单。