Recombinant NS2B-NS3 (A1394-L1440)GGGGSGGGG(A1476-K1660), NS3 Protease domain S7 & NS2B Cofactor

The construct design was based on a published structure in the Protein Data Bank (PDB ID: 4M9K): The construct includes the cytoplasmic cofactor region of NS2B spanning residues A1394-L1440, connected via a GGGG-S-GGGG linker to the NS3 protease domain which spans residues A1476-K1660. The NS2B region contains four mutations (T1402A, K1405R, D1407E, D1408E). Additionally, the construct contains an N-terminal overhang of six amino acids (GSHMLE) with the initial glycine remaining from the TEV cleavage site.

Protein Construct: A1394-L1440/GGGGSGGGG/A1476-K1660
Source: Dengue Virus
Expression Host: Escherichia coli
Molecular weight [kDa]: 26.29
pI: 5.03
Extinction Coefficient [M^-1cm^-1]: 41940
Concentration: 16.52 mg/mL
Storage Buffer: 50 mM Tris pH 8.5, 50 mM NaCl, 5% Glycerol
Tag: none

Amino acid sequence:

QC Data: Datasheet
Purity SDS: >95 %
Hydrodynamic Radius (nm): 2.88
Polydispersity Index (PDI): 0.1
T_on (°C): 42.96
T_m (°C): 48.92
Calculated Mass [Da]: 26281.294
Measured Mass [Da]: 26280.3716

Structure and function

The N-terminal residues of the NS3 protease form a chymotrypsin-like serine protease domain which mediates polyprotein cleavage to release individual viral proteins. It requires NS2B as a cofactor for proper folding and enzymatic activity. The N-terminal region of the NS2B cofactor has been shown to facilitate protease folding by contributing a β-strand to the N-terminal β-sheet of the NS3 protease. The C-terminal region of NS2B is required for protease activity, as it completes the substrate-binding pocket of the NS3 protease domain when adopting the "closed conformation". NS2B can adopt distinct conformations and dynamically interconverts between an open (inactive) and closed (active) state.

Ns2B-NS3 as important drug target

The mosquito-borne Dengue virus remains a major global health threat, highlighting the urgent need for innovative drug discovery strategies to develop effective antiviral therapeutics. Disruption of viral polyprotein processing verhindert dengue virus replication and propagation. Consequently, the NS3 protease domain represents an attractive target for drug development. Antiviral therapeutic strategies have so far focused on both orthosteric inhibitors (mimicking the polyprotein cleavage site) and allosteric inhibitors that prevent the transition to the active "closed" state.