A Novel Technology for Engineering Binders to Membrane Proteins

膜蛋白结合剂工程新技术

基本信息

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
项目类别：
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

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

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项目摘要

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.

抗体已被创造出对许多人类疾病的“魔力”。但是，仍然存在针对多通膜蛋白的抗体工程中的重大挑战，包括大量的治疗靶标，例如细胞表面受体和离子通道蛋白。工程粘合剂对膜蛋白的困难源于体外电流的局限性选择/平移技术，例如噬菌体显示，它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，膜蛋白通常对纯化是难治性的膜进行适当的折叠和活动。目前，没有有效的体外技术对多通膜蛋白的粘合剂的发现/工程。这项研究的总体目标是发展一种新型技术 - 蓝精（简单接近耦合mRNA显示） - 用于工程蛋白质粘合剂细胞表面的蛋白质靶标，因此绕开了净化靶蛋白的需求。蓝精灵结合 mRNA显示具有接近辅助的DNA组装现象，并且与常规平移不同所有对固体支撑的粘合剂都富集，蓝精灵仅将粘合剂富集到所需的靶蛋白在细胞表面。在AIM 1中，我们将使用寡核苷酸演示蓝精子原理并优化引物序列。 AIM 2将在A中建立模型蛋白的蓝精灵富集溶液中非目标蛋白的混合物。最后，在AIM 3中，模型蛋白将显示在将筛选哺乳动物细胞表面和粘合剂库，以证明和量化整个细胞蓝精灵的效率。这项研究的成功完成将为轻松发现/工程对全细胞脱落的膜蛋白的粘合剂，应大大扩展药物靶标的曲目可应对治疗干预。