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Updated: Jul 2, 2025
In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity
Published on: March 25, 2020
Increase in the solubility of uvsY using a site saturation mutagenesis library for application in a lyophilized
Kenta Morimoto1, Kevin Maafu Juma1, Masaya Yamagata1
1Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Kyoto, 606-8502, Japan.
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.
