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

Authors :: Morimoto K
Juma KM
Yamagata M
Takita T
Kojima K
Suzuki K
Yanagihara I
Fujiwara S
Yasukawa K
Source :: Molecular biology reports [Mol Biol Rep] 2024 Feb 27; Vol. 51 (1), pp. 367. Date of Electronic Publication: 2024 Feb 27.
Publication Year :: 2024
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.<br />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) <subscript>6</subscript> -tagged uvsY.<br />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.<br />Conclusions: The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.<br /> (© 2024. The Author(s).)

Subjects :: Solubility
Gene Library
Mutagenesis
Recombinases genetics
Amino Acids

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