Sharma Preeti, Procko Erik, Kranz David M
Department of Biochemistry, University of Illinois, Urbana, IL, USA.
Cancer Center at Illinois, University of Illinois, Urbana, IL, USA.
Methods Mol Biol. 2022;2491:117-142. doi: 10.1007/978-1-0716-2285-8_7.
Protein engineering using display platforms such as yeast display and phage display has allowed discovery of proteins with therapeutic and industrial applications. Antibodies and T cell receptors developed for therapeutic applications are often engineered by constructing libraries of mutations in loops of five to ten residues called complementarity determining regions that are in proximity to the antigen. In the past decade, deep mutational scanning has become a powerful tool in a protein engineer's toolbox, as it allows one to compare the impact of all 20 amino acids at each position, across the length of the protein. Thus, a single experiment can provide a sequence-activity landscape with information about hotspots or suboptimal binding sites in the original proteins. These residues or regions may be overlooked by engineering methods that are driven solely by structures or directed evolution of error-prone PCR libraries. Here, we describe experimental methods to engineer proteins by combining yeast display and deep mutational scanning mutagenesis, using T cell receptors as an example.
利用酵母展示和噬菌体展示等展示平台进行蛋白质工程，已使得具有治疗和工业应用价值的蛋白质得以发现。用于治疗应用的抗体和T细胞受体通常通过在靠近抗原的由五到十个残基组成的环（称为互补决定区）中构建突变文库来进行工程改造。在过去十年中，深度突变扫描已成为蛋白质工程师工具箱中的一项强大工具，因为它能让人们在蛋白质的整个长度上比较每个位置上所有20种氨基酸的影响。因此，单个实验就能提供一个序列-活性图谱，其中包含有关原始蛋白质中热点或次优结合位点的信息。这些残基或区域可能会被仅由结构驱动的工程方法或易错PCR文库的定向进化所忽视。在此，我们以T细胞受体为例，描述通过结合酵母展示和深度突变扫描诱变来对蛋白质进行工程改造的实验方法。
