共识序列
序列(生物学)
蛋白质工程
蛋白质稳定性
突变体
定向进化
热稳定性
计算生物学
热稳定性
蛋白质测序
生物
化学
酶
生物化学
肽序列
基因
有机化学
作者
Diego S. Val,Luisina Di Nardo,Fiorela Marchisio,Salvador Peirú,María Eugenia Castelli,Luciano A. Abriata,Hugo G. Menzella,Rodolfo M. Rasia
出处
期刊:Biochemistry
[American Chemical Society]
日期:2024-01-11
标识
DOI:10.1021/acs.biochem.3c00509
摘要
Proteins' extraordinary performance in recognition and catalysis has led to their use in a range of applications. However, proteins obtained from natural sources are oftentimes not suitable for direct use in industrial or diagnostic setups. Natural proteins, evolved to optimally perform a task in physiological conditions, usually lack the stability required to be used in harsher conditions. Therefore, the alteration of the stability of proteins is commonly pursued in protein engineering studies. Here, we achieved a substantial thermal stabilization of a bacterial Zn(II)-dependent phospholipase C by consensus sequence design. We retrieved and analyzed sequenced homologues from different sources, selecting a subset of examples for expression and characterization. A non-natural consensus sequence showed the highest stability and activity among those tested. Comparison of the stability parameters of this stabilized mutant and other natural variants bearing similar mutations allows us to pinpoint the sites most likely to be responsible for the enhancement. Point mutations in these sites alter the unfolding process of the consensus sequence. We show that the stabilized version of the protein retains full activity even in harsh oil degumming conditions, making it suitable for industrial applications.
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