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Highlights: [•] A protease construct containing full length NS2B and NS3pro of Dengue virus was expressed. [•] Detergent screening was conducted for protein extraction from membrane. [•] Protease was active in the LMPC micelles. [Copyright &y& Elsevier]

Abstract: Dengue virus causes serious diseases affecting people in tropical and sub-tropical regions. The nonstructural (NS) protein 2B is an integral membrane protein and important for the regulation of viral protease NS3, which is significant for virus replication. The NS2B-NS3 complex is an important drug target for treating dengue fever. However, little is known about the structure of NS2B in its entirety. Herein, we describe the expression and purification of this integral membrane protein from cell membrane and inclusion bodies of Escherichia coli cells. The initial nuclear magnetic resonance (NMR) and circular dichroism (CD) results indicate that the purified protein adopts alpha-helical structures in LMPG and TDPC micelles. [Copyright &y& Elsevier]

Abstract The Zika virus (ZIKV) genome encodes a polyprotein that can be post-translationally processed into functional viral proteins. The viral protease is indispensable in the maturation of viral proteins. The Zika protease comprises of two components crucial for catalysis. The N-terminal region of NS3 contains the catalytic triad and approximately 40 amino acids of NS2B are essential for folding and protease activity. NS2B is a membrane protein with transmembrane domains that are critical for the localization of NS3 to the membrane. In this study, we expressed and purified full-length NS2B from ZIKV in E. coli. Purified NS2B was then reconstituted into lyso-myristoyl phosphatidylglycerol (LMPG) micelles. It was found that compared to wild type NS2B, NS2B C11S mutation in LMPG exhibited dispersed cross peaks in the 1H 15N-HSQC spectrum, thereby suggesting the feasibility for structural characterization using solution NMR spectroscopy. Highlights • Full length NS2B of Zika virus was purified. • NS2B in LMPG micelles exhibited dispersed NMR signals. • Structure and dynamics of NS2B were explored. • Free NS2B contains a long unstructured region. [ABSTRACT FROM AUTHOR]

Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease. [ABSTRACT FROM AUTHOR]