Details

Autor(en) / Beteiligte

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

Titel

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

Ist Teil von

• Molecular biology reports, 2024-12, Vol.51 (1), p.367-367, Article 367

Ort / Verlag

Dordrecht: Springer Netherlands

Erscheinungsjahr

2024

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.