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

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 re...

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Bibliographic Details
Published in:Molecular biology reports 2024-12, Vol.51 (1), p.367-367, Article 367
Main Authors: Morimoto, Kenta, Juma, Kevin Maafu, Yamagata, Masaya, Takita, Teisuke, Kojima, Kenji, Suzuki, Koichiro, Yanagihara, Itaru, Fujiwara, Shinsuke, Yasukawa, Kiyoshi
Format: Article
Language:English
Subjects:
Amino Acids
Animal Anatomy
Animal Biochemistry
Biomedical and Life Sciences
DNA polymerase
E coli
Enzymes
Freeze drying
Gene Library
Histology
Kinases
Life Sciences
Molecular biology
Morphology
Mutagenesis
Nucleotide sequence
Original
Original Article
Phages
Proteins
Reagents
Recombinase
Recombinases - genetics
Saturation mutagenesis
Solubility
Thermal cycling
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Summary: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.
ISSN:0301-4851
1573-4978
DOI:10.1007/s11033-024-09367-y