DNA洗牌
噬菌体展示
突变
突变体
大肠杆菌
选择(遗传算法)
折叠(DSP实现)
定向进化
化学
蛋白质折叠
蛋白质工程
寡核苷酸
噬菌体
计算生物学
分子生物学
DNA
生物
生物化学
噬菌体
基因
计算机科学
酶
肽
人工智能
电气工程
工程类
作者
Sora Jung,Annemarie Honegger,Andreas Plückthun
标识
DOI:10.1006/jmbi.1999.3196
摘要
A library of mutants of a single-chain Fv fragment (scFv) was generated by a combination of directed and random mutagenesis, using oligonucleotides randomized at defined positions and two rounds of DNA shuffling. The library was based on the already well folding and stable scFv fragment 4D5Flu. In order to further improve this framework and test the efficiency of various selection strategies, phage display selection was carried out under different selective pressures for higher thermodynamic stability. Incubation of the display phages at elevated temperatures was compared to exposure of the phages to high concentrations of guanidinium chloride. Temperature stress-guided selection yielded the most stable scFv mutant after two rounds of mutagenesis and selection, due to the irreversibility of the unfolding process. It possessed only two mutations (His(L27d)Asn and Phe(L55)Val) and showed a thermodynamic stability improved by roughly 4 kcal/mol, threefold better expression yields in Escherichia coli as well as a 20-fold better binding constant than the 4D5Flu wild-type. The selection results obtained in this study delineate the advantages, disadvantages and limitations of different stability stress selection methods in phage display.
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