Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries
Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries
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DOI：
10.1073/pnas.95.24.14130
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发表时间：
1998-11-24
影响因子：
11.1
通讯作者：
Plückthun, A
中科院分区：
文献类型：
--
作者：
Hanes, J;Jermutus, L;Plückthun, A
应用核糖体展示从用转录因子GCN 4二聚化结构域的变体肽免疫的小鼠产生的不同文库中亲和选择抗体单链片段（scFv）。三轮核糖体展示后，分离并表征阳性scFv。选择了几种不同的scFv，但最大组中的那些彼此密切相关，并且相对于它们的共有序列（可能的共同祖先）在0至5个氨基酸残基上不同。在溶液中测量，最佳scFv的解离常数为（4 +/- 1）× 10（-11）M。发现互补决定区L1中的一个氨基酸残基负责比可能的祖细胞高65倍的亲和力。似乎该高亲和力scFv选自体外核糖体展示期间发生的突变，并且这构成了该方法中固有的亲和力成熟。体外筛选的单链抗体可以在大肠杆菌周质中以良好的产量进行功能性表达或通过体外复性制备。因此，核糖体展示可以是体外文库筛选和同时序列进化的有力方法。
Ribosome display was applied for affinity selection of antibody single-chain fragments (scFv) from a diverse library generated from mice immunized with a variant peptide of the transcription factor GCN4 dimerization domain. After three rounds of ribosome display, positive scFvs were isolated and characterized. Several different scFvs were selected, but those in the largest group were closely related to each other and differed in 0 to 5 amino acid residues with respect to their consensus sequence, the likely common progenitor. The best scFv had a dissociation constant of (4 +/- 1) x 10(-11) M, measured in solution. One amino acid residue in complementarity determining region L1 was found to be responsible for a 65-fold higher affinity than the likely progenitor. It appears that this high-affinity scFv was selected from the mutations occurring during ribosome display in vitro, and that this constitutes an affinity maturation inherent in this method. The in vitro-selected scFvs could be functionally expressed in the Escherichia coli periplasm with good yields or prepared by in vitro refolding. Thus, ribosome display can be a powerful methodology for in vitro library screening and simultaneous sequence evolution.