Application of recombinant human pyruvate kinase in recombinase polymerase amplification

Kenji Kojima; Kenta Morimoto; Kevin Maafu Juma; Teisuke Takita; Kazuki Saito; Itaru Yanagihara; Shinsuke Fujiwara; Kiyoshi Yasukawa

doi:10.1016/j.jbiosc.2023.08.005

Application of recombinant human pyruvate kinase in recombinase polymerase amplification

J Biosci Bioeng. 2023 Nov;136(5):341-346. doi: 10.1016/j.jbiosc.2023.08.005. Epub 2023 Sep 16.

Authors

Kenji Kojima¹, Kenta Morimoto², Kevin Maafu Juma², Teisuke Takita², Kazuki Saito¹, Itaru Yanagihara³, Shinsuke Fujiwara⁴, Kiyoshi Yasukawa⁵

Affiliations

¹ Division of Bioanalytical Chemistry, Faculty of Pharmaceutical Sciences, Himeji Dokkyo University, Himeji, Hyogo 670-8524, Japan.
² Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
³ Department of Developmental Medicine, Research Institute, Osaka Women's and Children's Hospital, Izumi-shi, Osaka 594-1101, Japan.
⁴ Department of Biosciences, School of Biological and Environmental Sciences, Kwansei-Gakuin University, Sanda, Hyogo 669-1330, Japan.
⁵ 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.

PMID: 37718149
DOI: 10.1016/j.jbiosc.2023.08.005

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. In this study, we attempted to use pyruvate kinase instead of creatine kinase (CK) that has been consistently used as an ATP-regenerating enzyme in RPA. Human pyruvate kinase M1 (PKM) was expressed in Escherichia coli and purified from the cells. RPA with PKM was performed at 41°C with the in vitro synthesized urease subunit β (ureB) DNA from Ureaplasma parvum serovar 3 as a standard DNA. The optimal concentrations of PKM and phosphoenolpyruvate were 20 ng/μL and 10 mM, respectively. The RPA reaction with PKM was more sensitive than that with CK. PKM exhibited higher thermostability than CK, suggesting that the RPA reagents with PKM are preferable to those with CK for onsite use.

Keywords: Creatine kinase; Isothermal DNA amplification; Pyruvate kinase; Recombinase polymerase amplification.