Antibody affinity maturation using bacterial surface display
Antibody affinity maturation using bacterial surface display
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DOI：
10.1093/protein/11.9.825
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发表时间：
1998-09-01
期刊：
影响因子：
--
通讯作者：
Georgiou, G
中科院分区：
文献类型：
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作者：
Daugherty, PS;Chen, G;Georgiou, G
利用大肠杆菌表面展示技术和荧光激活细胞分选技术，建立了一种筛选单链抗体（scFv）组合库的定量系统。该系统用于从通过随机化抗地高辛26-10衍生抗体scFv（dig）中的重链和轻链残基构建的文库中分离对荧光标记的半抗原具有高亲和力的克隆。流式细胞术的使用使得能够直接在异质群体中检测稀有文库成员并在分选之前优化选择条件。采用越来越严格的选择条件，在三轮后选择具有野生型亲和力（KD = 0.91 +/- 0.22 nM）和预期代表频率小于1 x 10（6）的重链突变体至同质。分离的克隆相对于野生型DNA序列具有两个不同的点突变，但仍编码野生型氨基酸序列，表明野生型残基在随机化位置可能是最佳的。在单个分选步骤中，从较小的轻链文库中分离亲和力改善的克隆（K-D = 0.30 +/-0.05 nM），其解离常数比野生型抗体低约三倍。流式细胞术被证明是一种简单而快速的方法，用于测定所选克隆的相对半抗原解离速率常数，而不需要亚克隆。通过FAGS估计的相对速率常数通过产生可溶形式的scFv抗体并通过表面等离子体共振（SPR）测量半抗原结合动力学来证实。这些结果表明，大肠杆菌表面展示，加上使用流式细胞仪的定量选择和分析，有可能成为从大型多肽库中快速分离和表征所需突变体的强大工具，
A quantitative system for screening combinatorial single-chain Fv (scFv) antibody libraries was developed utilizing surface display on Escherichia coli and fluorescence-activated cell sorting (FACS). This system was employed to isolate clones with high-affinity to a fluorescently-labeled hapten from libraries constructed by randomizing heavy and light-chain residues in the anti-digoxin 26-10 derived antibody, scFv(dig). The use of flow cytometry enabled the detection of rare library members directly in heterogeneous populations and the optimization of selection conditions prior to sorting. A heavy-chain mutant having wild-type affinity (K-D = 0.91 +/- 0.22 nM) and an expected representation frequency of less than 1 x 10(6), was selected to homogeneity after three rounds utilizing increasingly stringent selection conditions. The isolated clone possessed two distinct point mutations relative to the wild-type DNA sequence, yet still coded for the wild-type amino acid sequence, suggesting that the wild-type residues may be optimal at the randomized positions. An affinity improved clone (K-D = 0.30 +/- 0.05 nM), having a dissociation constant approximately threefold lower than the wild-type antibody, was isolated from a smaller light-chain library in a single sorting step. Flow cytometry was shown to be a simple and rapid method for the determination of the relative hapten dissociation rate constants of selected clones without requiring subcloning, The relative rate constants estimated by FAGS were confirmed by producing the scFv antibodies in soluble form and measuring hapten binding kinetics by surface plasmon resonance (SPR). These results demonstrate that E.coli surface display, coupled with quantitative selection and analysis using FACS, has the potential to become a powerful tool for rapid isolation and characterization of desirable mutants from large polypeptide libraries,