Burks E A, Chen G, Georgiou G, Iverson B L
Department of Chemistry and Biochemistry, University of Texas, Austin 78712, USA.
Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):412-7. doi: 10.1073/pnas.94.2.412.
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.
我们将聚合酶链反应（PCR）诱变与体外转录/翻译及酶联免疫吸附测定（ELISA）相结合，用于快速生成和表征抗体突变体。PCR产物直接用作体外转录/翻译反应的模板，由于无需克隆步骤，一名研究人员在一周内可分两次完成一个残基的体外饱和诱变。体外扫描饱和诱变用于分析源自26 - 10单克隆抗体的单链Fv抗体结合口袋中六个关键接触残基（重链：酪氨酸-33、重链：天冬酰胺-35、重链：酪氨酸-50、重链：色氨酸-100、轻链：缬氨酸-94和轻链：脯氨酸-96）的作用和可塑性。共产生了114种突变抗体；在所选的6个位置上，每个位置都有19种取代。分析了这些突变体与地高辛、洋地黄毒苷、地高辛配基和哇巴因的结合情况，从而生成了一个包含456个相对亲和力值的综合数据库。观察到体外合成的突变抗体获得的相对亲和力值与先前报道的纯化突变单克隆抗体获得的平衡亲和力数据之间具有良好的一致性。约75%的单氨基酸突变体对一种或多种地高辛类似物表现出显著结合。鉴定出了改变甚至在某些情况下逆转对不同地高辛类似物特异性的突变。体外扫描饱和诱变代表了一种用于蛋白质结构-功能和工程研究的新工具，并且可以与实验室自动化相结合，从而能够构建和分析更高通量的蛋白质突变体。
