AVITHRAPID - Standard Operating Procedure - DENV1-4 protease expression and protein purification

This document describes, in details, the SOP followed for the expression and purification of DENV proteases, from all the four serotypes of Dengue virus, carried out under the Work Package, WP T1.8 of AVITHRAPID project. The DENV herein forth, refers to the Dengue Virus protease (NS2B_NS3) where the NS2B, cofactor, part is fused with the NS3 protease part using a 4GS4G linker sequence, the number refers to the serotype. The plasmids harboring the protease genes are pET28a based where the PreS/3C protease site is inserted for His6-tag removal in case of DENV1, DENV2 and DENV4 except for DENV3 where TEV site has been used for GST tag removal. Henceforth, for simplicity purpose the plasmids are named as followed: pET28a_PreS_DENV1, pET28a_PreS_DENV2, pET28a_TeV_DENV3, and pET28a_PreS_DENV4. Whereas the resultant mutants are named as DENV1_Delta, DENV1_S135A, DENV1_DeltaS135A, and DENV2_S135A. The purified protein is intended to use for biochemical and biophysical evaluation of inhibitors within the AVITHRAPID Framework.