Computer-based Engineering of Thermostabilized Antibody Fragments.
Computer-based Engineering of Thermostabilized Antibody Fragments.
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DOI：
10.1002/aic.16864
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发表时间：
2019-11
期刊：
影响因子：
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通讯作者：
Jiwon Lee;Bryan S. Der;C. Karamitros;Wenzong Li;Nicholas M. Marshall;Oana I. Lungu;Aleksandr E. Miklos;Jianqing Xu;T. Kang;Chang-Han Lee;Bing Tan;R. Hughes;S. Jung;G. Ippolito;Jeffrey J. Gray;Yan Zhang;B. Kuhlman;G. Georgiou;A. Ellington
中科院分区：
文献类型：
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作者：
Jiwon Lee;Bryan S. Der;C. Karamitros;Wenzong Li;Nicholas M. Marshall;Oana I. Lungu;Aleksandr E. Miklos;Jianqing Xu;T. Kang;Chang-Han Lee;Bing Tan;R. Hughes;S. Jung;G. Ippolito;Jeffrey J. Gray;Yan Zhang;B. Kuhlman;G. Georgiou;A. Ellington
我们使用分子建模程序Rosetta来确定抗体片段(scFv和scabs)中的氨基酸替换簇，这些替换可以提高全球蛋白质的稳定性和抗热失活能力。使用这种方法，我们提高了与肉毒梭菌血凝素蛋白(抗HA33)结合的抗体片段的熔融温度(Tm)和抗热处理能力。设计的两个带有两个氨基酸替换簇的抗体片段变体，旨在稳定局部区域，与亲本scFv相比，具有更高的Tm，更重要的是，在70°C孵育2小时后仍保持完全的抗原结合活性。一个热稳定的ScFv变体的晶体结构在1.6？下求解，并与Rosetta抗体模型预测非常一致。
We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (Tm) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher Tm compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.