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

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2024年2月27日

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

摘要

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.