A novel approach for separate expression of dengue virus NS3 protease and its NS2B cofactor domain is described in this paper. The two proteins are expressed in E.coli and purified separately and subsequently efficiently co-refolded to form a stable complex. This straightforward and robust method allows for separate isotope labeling of the two proteins, facilitating analysis by nuclear magnetic resonance (NMR) spectroscopy. Unlinked NS2B-NS3pro behaves better in NMR spectroscopy than linked NS2B-NS3pro, which has resulted in the backbone resonance assignment of the unlinked NS2B-NS3 complex bound to a peptidic boronic acid inhibitor. (C) 2017 Elsevier Inc. All rights reserved.