Utilization of low-stability variants in protein evolutionary engineering.

Autor: Wakisaka M; Department of Biomolecular Chemistry, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan., Tanaka SI; Department of Biomolecular Chemistry, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan., Takano K; Department of Biomolecular Chemistry, Kyoto Prefectural University, Sakyo-ku, Kyoto 606-8522, Japan. Electronic address: takano@kpu.ac.jp.
Jazyk: angličtina
Zdroj: International journal of biological macromolecules [Int J Biol Macromol] 2024 Jun; Vol. 272 (Pt 2), pp. 132946. Date of Electronic Publication: 2024 Jun 05.
DOI: 10.1016/j.ijbiomac.2024.132946
Abstrakt: Evolutionary engineering involves repeated mutations and screening and is widely used to modify protein functions. However, it is important to diversify evolutionary pathways to eliminate the bias and limitations of the variants by using traditionally unselected variants. In this study, we focused on low-stability variants that are commonly excluded from evolutionary processes and tested a method that included an additional restabilization step. The esterase from the thermophilic bacterium Alicyclobacillus acidocaldarius was used as a model protein, and its activity at its optimum temperature of 65 °C was improved by evolutionary experiments using random mutations by error-prone PCR. After restabilization using low-stability variants with low-temperature (37 °C) activity, several re-stabilizing variants were obtained from a large number of variant libraries. Some of the restabilized variants achieved by removing the destabilizing mutations showed higher activity than that of the wild-type protein. This implies that low-stability variants with low-temperature activity can be re-evolved for future use. This method will enable further diversification of evolutionary pathways.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this study.
(Copyright © 2024 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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