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.

抗体被认为是对抗许多人类疾病的“灵丹妙药”。然而，仍然存在