Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized reagent for recombinase polymerase amplification.

Autor: Morimoto K; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan., Juma KM; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan., Yamagata M; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan., Takita T; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan., Kojima K; Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo, 670-8524, Japan., Suzuki K; The Research Foundation for Microbial Diseases of Osaka University, Suita, Osaka, 565-0871, Japan., Yanagihara I; Department of Developmental Medicine, Research Institute, Osaka Women's and Children's Hospital, Izumi-Shi, Osaka, 594-1101, Japan., Fujiwara S; Department of Biosciences, School of Biological and Environmental Sciences, Kwansei-Gakuin University, Sanda, Hyogo, 669-1330, Japan., Yasukawa K; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan. yasukawa.kiyoshi.7v@kyoto-u.ac.jp.
Jazyk: angličtina
Zdroj: Molecular biology reports [Mol Biol Rep] 2024 Feb 27; Vol. 51 (1), pp. 367. Date of Electronic Publication: 2024 Feb 27.
DOI: 10.1007/s11033-024-09367-y
Abstrakt: Background: Recombinase uvsY from bacteriophage T4, along with uvsX, is a key enzyme for recombinase polymerase amplification (RPA), which is used to amplify a target DNA sequence at a constant temperature. uvsY, though essential, poses solubility challenges, complicating the lyophilization of RPA reagents. This study aimed to enhance uvsY solubility.
Methods: Our hypothesis centered on the C-terminal region of uvsY influencing solubility. To test this, we generated a site-saturation mutagenesis library for amino acid residues Lys91-Glu134 of the N-terminal (His) 6 -tagged uvsY.
Results: Screening 480 clones identified A116H as the variant with superior solubility. Lyophilized RPA reagents featuring the uvsY variant A116H demonstrated enhanced performance compared to those with wild-type uvsY.
Conclusions: The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.
(© 2024. The Author(s).)
Databáze: MEDLINE
Externí odkaz: