Achieving unprecedented stability in lyophilized recombinase polymerase amplification with thermostable pyruvate kinase from Thermotoga maritima.

Juma, Kevin Maafu; Murakami, Yuto; Morimoto, Kenta; Takita, Teisuke; Kojima, Kenji; Suzuki, Koichiro; Yanagihara, Itaru; Ikuta, Soichiro; Fujiwara, Shinsuke; Yasukawa, Kiyoshi

Juma, Kevin Maafu; Murakami, Yuto; Morimoto, Kenta; Takita, Teisuke; Kojima, Kenji; Suzuki, Koichiro; Yanagihara, Itaru; Ikuta, Soichiro; Fujiwara, Shinsuke; Yasukawa, Kiyoshi.

Afiliación

Juma KM; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Murakami Y; Department of Biosciences, School of Biological and Environmental Sciences, Kwansei-Gakuin University, Sanda, Hyogo 669-1330, Japan.
Morimoto K; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Takita T; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Kojima K; Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo 670-8524, Japan.
Suzuki K; The Research Foundation for Microbial Diseases of Osaka University, Suita, Osaka 565-0871, Japan.
Yanagihara I; Department of Developmental Medicine, Research Institute, Osaka Women's and Children's Hospital, Izumi, Osaka 594-1101, Japan.
Ikuta S; Department of Biosciences, School of Biological and Environmental Sciences, Kwansei-Gakuin University, Sanda, Hyogo 669-1330, Japan.
Fujiwara S; Department of Biosciences, School of Biological and Environmental Sciences, Kwansei-Gakuin University, Sanda, Hyogo 669-1330, Japan.
Yasukawa K; Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Electronic address: yasukawa.kiyoshi.7v@kyoto-u.ac.jp.

J Biosci Bioeng ; 138(1): 29-35, 2024 Jul.

Article en En | MEDLINE | ID: mdl-38719683

ABSTRACT

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.

Asunto(s)

Estabilidad de Enzimas; Liofilización; Técnicas de Amplificación de Ácido Nucleico; Piruvato Quinasa; Thermotoga maritima; Thermotoga maritima/enzimología; Thermotoga maritima/genética; Piruvato Quinasa/metabolismo; Piruvato Quinasa/genética; Piruvato Quinasa/química; Técnicas de Amplificación de Ácido Nucleico/métodos; Humanos; Recombinasas/metabolismo; Recombinasas/química; Recombinasas/genética; Escherichia coli/genética; Escherichia coli/metabolismo; ADN Polimerasa Dirigida por ADN/metabolismo; ADN Polimerasa Dirigida por ADN/química; ADN Polimerasa Dirigida por ADN/genética; Proteínas Bacterianas/genética; Proteínas Bacterianas/metabolismo; Proteínas Bacterianas/química

Palabras clave

Isothermal DNA amplification; Pyruvate kinase; Recombinase polymerase amplification; Thermostable enzyme

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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Piruvato Quinasa / Estabilidad de Enzimas / Thermotoga maritima / Técnicas de Amplificación de Ácido Nucleico / Liofilización Límite: Humans Idioma: En Revista: J Biosci Bioeng Asunto de la revista: ENGENHARIA BIOMEDICA / MICROBIOLOGIA Año: 2024 Tipo del documento: Article País de afiliación: Japón Pais de publicación: Japón

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