Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs
Rapid identification of reactive cysteine residues for site-specific labeling of antibody-Fabs
复制标题
DOI：
10.1016/j.jim.2007.12.011
复制
发表时间：
2008-03-20
影响因子：
2.2
通讯作者：
Lowman, Henry B.
中科院分区：
文献类型：
--
作者：
Junutula, Jagath R.;Bhakta, Sunil;Lowman, Henry B.
具有反应性巯基的半胱氨酸是用于蛋白质的位点特异性标记的有吸引力的工具。将反应性半胱氨酸残基工程化到具有多个二硫键的蛋白质中通常是一项具有挑战性的任务，因为它可能干扰蛋白质的结构和功能特性。在这里，我们开发了一种基于噬菌体展示的生物化学测定，PHESelector（用于选择反应性硫醇的噬菌体ELISA），以快速筛选抗体片段上的反应性硫醇基团，而不干扰其抗原结合，使用曲妥珠单抗-Fab（hu 4D 5 Fab）作为模型系统。使用可用的晶体结构信息计算hu 4D 5 Fab中所有氨基酸残基的溶剂可及性值。选择具有最高溶剂可及性值的丝氨酸、丙氨酸和缬氨酸残基并进行测试，以将基于结构的设计与PHESelector生化方法进行比较。在部分溶剂可及的丙氨酸或缬氨酸残基处的半胱氨酸取代比在丝氨酸残基处的取代表现出更好的硫醇反应性值。工程化的hu 4D 5 Fab变体的溶剂可及性分数与硫醇反应性之间的相关性较差，表明PHESELECTOR生物化学测定法鉴定抗体-Fab表面上的反应性硫醇基团的价值。生物素化的ThioFab（具有工程化半胱氨酸的Fab）变体的质谱分析证实，缀合仅发生在轻链或重链的工程化半胱氨酸硫醇处。在恒定结构域（CL和CH 1）中具有工程化半胱氨酸残基的ThioFab应允许抗体-Fab的位点特异性缀合的普遍应用。(C)2007 Elsevier B. V.保留所有权利。
Cysteines with reactive thiol groups are attractive tools for site-specific labeling of proteins. Engineering a reactive cysteine residue into proteins with multiple disulfide bonds is often a challenging task as it may interfere with structural and functional properties of the protein. Here we developed a phage display-based biochemical assay, PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) to rapidly screen reactive thiol groups on antibody fragments without interfering with their antigen binding, using trastuzumab-Fab (hu4D5Fab) as a model system. The solvent accessibility values for all the amino acid residues in the hu4D5Fab were calculated using available crystal structure information. Serine, alanine and valine residues with highest solvent accessibility values were selected and tested to compare structure-based design with the PHESELECTOR biochemical method. Cysteine substitutions at partially solvent-accessible alanine or valine residues exhibited better thiol reactivity values than substitutions at serine residues. The poor correlation between fractional solvent accessibility and thiol reactivity of the engineered hu4D5Fab variants indicated the value of PHESELECTOR biochemical assay to identify reactive thiol groups on the antibody-Fab surface. Mass spectrometric analysis of biotinylated ThioFab (Fab with engineered cysteine) variants confirmed that conjugation occurred only at the engineered cysteine thiols of either light or heavy chains. ThioFabs with engineered cysteine residues in the constant domains (CL and CH1) should allow universal application for site-specific conjugation of antibody-Fabs. (C) 2007 Elsevier B.V. All rights reserved.