Hall Sejal S, Daugherty Patrick S
The Institute for Energy Efficiency, University of California, Santa Barbara, California 93106, USA.
Protein Sci. 2009 Sep;18(9):1926-34. doi: 10.1002/pro.203.
Despite the critical importance of molecular specificity in bimolecular systems, in vitro display technologies have been applied extensively for affinity maturation of peptides and antibodies without explicitly measuring the specificity of the desired interaction. We devised a general strategy to measure, screen, and evolve specificity of protein ligand interactions analogous to widely used affinity maturation strategies. The specificity of binding to target and nontarget antibodies labeled with spectrally distinct fluorophores was measured simultaneously in protein mixtures via multiparameter flow cytometry, thereby enabling screening for high target antibody specificity. Isolated antibody specific ligands exhibited varying specificity, revealing critical amino acid determinants for target recognition and nontarget avoidance in complex mixtures. Molecular specificity in the mixture was further enhanced by quantitative directed evolution, yielding a family of epitopes exhibiting improved specificities equivalent, or superior to, the native peptide antigen to which the antibody was raised. Specificity screening simultaneously favored affinity, yielding ligands with three-fold improved affinity relative to the parent epitope. Quantitative specificity screening will be useful to screen, evolve, and characterize the specificity of protein and peptide interactions for molecular recognition applications.
尽管分子特异性在双分子系统中至关重要，但体外展示技术已被广泛应用于肽和抗体的亲和力成熟，却未明确测量所需相互作用的特异性。我们设计了一种通用策略，用于测量、筛选和进化蛋白质配体相互作用的特异性，类似于广泛使用的亲和力成熟策略。通过多参数流式细胞术在蛋白质混合物中同时测量与用光谱不同的荧光团标记的靶标和非靶标抗体的结合特异性，从而能够筛选出高靶标抗体特异性。分离出的抗体特异性配体表现出不同的特异性，揭示了在复杂混合物中靶标识别和非靶标规避的关键氨基酸决定因素。通过定量定向进化进一步提高了混合物中的分子特异性，产生了一系列表位，其表现出与产生抗体的天然肽抗原相当或更优的改进特异性。特异性筛选同时有利于亲和力，产生的配体相对于亲本表位的亲和力提高了三倍。定量特异性筛选将有助于筛选、进化和表征用于分子识别应用的蛋白质和肽相互作用的特异性。
