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

Mol Biol Rep. 2024 Feb 27;51(1):367. doi: 10.1007/s11033-024-09367-y.

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.

Keywords: Isothermal DNA amplification; Recombinase polymerase amplification (RPA); Site saturation mutagenesis library; uvsY.

MeSH terms

  • Amino Acids*
  • Gene Library
  • Mutagenesis
  • Recombinases* / genetics
  • Solubility

Substances

  • Recombinases
  • Amino Acids