Panton Rojhae A, Stern Lawrence A
Department of Chemical, Biological, and Materials Engineering, University of South Florida, Tampa, FL, USA.
Methods Mol Biol. 2022;2491:217-233. doi: 10.1007/978-1-0716-2285-8_12.
High-throughput protein selection methods are a cornerstone for protein engineering and pharmaceutical development. Traditional high-throughput selection strategies rely largely on recombinant antigen to generate target specificity. Though effective, this selection strategy can be limited by soluble target quality, particularly in the case of recombinant extracellular domains of transmembrane proteins. Recent advances in cell-based selection techniques provide new opportunities for improving the outcomes of ligand selection campaigns but can introduce technical challenges in maintaining antigen specificity due to the heterogeneity of biomacromolecule expression on the mammalian cell surface. Here, we describe a combination technique using recombinant antigen to "train" library target specificity followed by cell panning selections to ensure that isolated ligands bind cell-expressed target, as well as a facile microscopy technique for assessing target specificity on a clonal basis without the need to produce soluble ligand.
高通量蛋白质筛选方法是蛋白质工程和药物开发的基石。传统的高通量筛选策略在很大程度上依赖重组抗原来产生靶标特异性。尽管这种筛选策略有效，但它可能受到可溶性靶标质量的限制，特别是在跨膜蛋白的重组细胞外结构域的情况下。基于细胞的筛选技术的最新进展为改善配体筛选活动的结果提供了新机会，但由于哺乳动物细胞表面生物大分子表达的异质性，在维持抗原特异性方面可能会带来技术挑战。在这里，我们描述了一种组合技术，即使用重组抗原来“训练”文库靶标特异性，然后进行细胞淘选以确保分离的配体与细胞表达的靶标结合，以及一种简便的显微镜技术，用于在无需产生可溶性配体的情况下基于克隆评估靶标特异性。
