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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公開日
2024-02-27
資源種別
journal article
権利情報
  • https://creativecommons.org/licenses/by/4.0
  • https://creativecommons.org/licenses/by/4.0
DOI
  • 10.1007/s11033-024-09367-y
公開者
Springer Science and Business Media LLC

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<jats:title>Abstract</jats:title> <jats:sec> <jats:title>Background</jats:title> <jats:p>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.</jats:p> </jats:sec> <jats:sec> <jats:title>Methods</jats:title> <jats:p>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)<jats:sub>6</jats:sub>-tagged uvsY.</jats:p> </jats:sec> <jats:sec> <jats:title>Results</jats:title> <jats:p>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.</jats:p> </jats:sec> <jats:sec> <jats:title>Conclusions</jats:title> <jats:p>The uvsY variant A116H emerges as an appealing choice for RPA applications, offering improved solubility and heightened lyophilization feasibility.</jats:p> </jats:sec>

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