M. JunaidC. AngsuthanasombatJ. E.S. WikbergN. AliG. KatzenmeierUniversity of MalakandMahidol UniversityUppsala UniversitetKhyber Medical University2018-10-192018-10-192013-08-01Biochemistry (Moscow). Vol.78, No.8 (2013), 920-92416083040000629792-s2.0-84882672686https://repository.li.mahidol.ac.th/handle/20.500.14594/31260Dengue virus threatens around 2.5 billion people worldwide; about 50 million become infected every year, and yet no vaccine or drug is available for prevention and/or treatment. The flaviviral NS2B-NS3pro complex is indispensable for flaviviral replication and is considered to be an important drug target. The aim of this study was to develop a simple and generally applicable experimental strategy to construct, purify, and assay a highly active recombinant NS2B(H)-NS3pro complex that would be useful for high-throughput screening of potential inhibitors. The sequence of NS2B(H)-NS3pro was generated by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro complex was expressed in E. coli predominantly as insoluble protein and purified to >95% purity by single-step immobilized metal affinity chromatography. SDS-PAGE followed by immunoblotting of the purified enzyme demonstrated the presence of the NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 37, 21, and 10 kDa bands, respectively. Kinetic parameters, Km, kcat, and kcat/Kmfor the fluorophore-linked protease model substrate Ac-nKRR-amc were obtained using inner-filter effect correction. The kinetic parameters Km, kcat, and kcat/Kmfor Ac-nKRR-amc substrate were 100 μM, 0.112 s-1, and 1120 M-1·s-1, respectively. A simplified procedure for the cloning, overexpression, and purification of the NS2B(H)-NS3pro complex was applied, and a highly active recombinant NS2B(H)-NS3pro complex was obtained that could be useful for the design of high-throughput assays aimed at flaviviral inhibitor discovery. © 2013 Pleiades Publishing, Ltd.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1134/S0006297913080099