Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima.
J Biosci Bioeng
; 138(1): 29-35, 2024 Jul.
Article
en En
| MEDLINE
| ID: mdl-38719683
ABSTRACT
Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), strand-displacing DNA polymerase (Pol), and an ATP-regenerating enzyme. Considering the onsite use of RPA reagents, lyophilized RPA reagents with long storage stability are highly desired. In this study, as one of the approaches to solve this problem, we attempted to use a thermostable pyruvate kinase (PK). PK gene was isolated from a thermophilic bacterium Thermotoga maritima (Tma-PK). Tma-PK was expressed in Escherichia coli and purified from the cells. Tma-PK exhibited higher thermostability than human PK. The purified Tma-PK preparation was applied to RPA as an ATP-regenerating enzyme. Liquid RPA reagent with Tma-PK exhibited the same performance as that with human PK. Lyophilized RPA reagent with Tma-PK exhibited higher performance than that with human PK. Combined with our previous results of RPA reagents of thermostable Pol from a thermophilic bacterium, Aeribacillus pallidus, the results in this study suggest that thermostable enzymes are preferable to mesophilic ones as a component in lyophilized RPA reagents.
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Texto completo:
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Base de datos:
MEDLINE
Asunto principal:
Piruvato Quinasa
/
Estabilidad de Enzimas
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Thermotoga maritima
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Técnicas de Amplificación de Ácido Nucleico
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Liofilización
Idioma:
En
Revista:
J Biosci Bioeng
Asunto de la revista:
ENGENHARIA BIOMEDICA
/
MICROBIOLOGIA
Año:
2024
Tipo del documento:
Article