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

Author:

Morimoto Kenta,Juma Kevin Maafu,Yamagata Masaya,Takita Teisuke,Kojima Kenji,Suzuki Koichiro,Yanagihara Itaru,Fujiwara Shinsuke,Yasukawa Kiyoshi

Abstract

Abstract 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.

Funder

Japan Society for the Promotion of Science

Japan Agency for Medical Research and Development

Japan Science and Technology Agency

Publisher

Springer Science and Business Media LLC

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