In vitro scanning saturation mutagenesis of an antibody binding pocket
In vitro scanning saturation mutagenesis of an antibody binding pocket
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DOI：
10.1073/pnas.94.2.412
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发表时间：
1997-01-21
影响因子：
11.1
通讯作者：
Iverson, BL
中科院分区：
文献类型：
--
作者：
Burks, EA;Chen, G;Iverson, BL
我们将PCR诱变与体外转录/翻译和ELISA相结合，用于抗体突变体的快速生成和表征，PCR产物直接用作体外转录/翻译反应的模板，由于不需要克隆步骤，一个残基的体外饱和诱变可由一名研究者在一周内重复完成，体外扫描饱和诱变用于分析六个关键接触残基（H：Tyr-33， H:Asn-35、H:Tyr-50、H:Trp-100、L:Val-94和L:Pro-96)在源自26-10单克隆抗体的单链Fv抗体的结合口袋中，总共产生了114个突变抗体；在 6 个选定位置的每个位置上进行了所有 19 个取代，分析了突变体与地高辛、洋地黄毒苷、地高辛和哇巴因的结合，从而生成了 456 个相对亲和力值的综合数据库。观察到体外合成的突变体抗体获得的相对亲和力值与先前报道的纯化突变体单克隆抗体获得的平衡亲和力数据之间具有极好的一致性，大约 75% 的单氨基酸突变体表现出与一种或多种地高辛类似物的显着结合，确定了改变不同地高辛类似物的特异性，并且在某些情况下反向特异性的突变，体外扫描饱和诱变代表了蛋白质结构功能和工程研究的新工具，并且可以与实验室自动化连接，以便更高的通量可以构建和分析蛋白质突变体。
We have combined PCR mutagenesis with in vitro transcription/translation and ELISA for the rapid generation and characterization of antibody mutants, The PCR products are used directly as the template for the in vitro transcription/translation reactions and because no cloning steps are required, the in vitro saturation mutagenesis of one residue can be completed in duplicate within a week by a single investigator, In vitro scanning saturation mutagenesis was used to analyze the role and plasticity of six key contact residues (H:Tyr-33, H:Asn-35, H:Tyr-50, H:Trp-100, L:Val-94, and L:Pro-96) in the binding pocket of a single chain Fv antibody derived from the 26-10 monoclonal antibody, A total of 114 mutant antibodies were produced; all 19 substitutions at each of the 6 chosen positions, The mutants were analyzed for binding to digoxin, digitoxin, digoxigenin, and ouabain resulting in the generation of a comprehensive data base of 456 relative affinity values. Excellent agreement between the relative affinity values obtained with in vitro synthesized mutant antibodies and equilibrium affinity data obtained with previously reported purified mutant monoclonal antibodies was observed, Approximately 75% of the single amino acid mutants exhibited significant binding to one or more of the digoxin analogs, Mutations that alter and, in some cases, reverse specificity for the different digoxin analogs were identified, In vitro scanning saturation mutagenesis represents a new tool for protein structure-function and engineering studies and can be interfaced with laboratory automation so that an even higher throughput of protein mutants can be constructed and analyzed.