Mechanistic insights into Zika virus NS3 helicase inhibition by Epigallocatechin-3-gallate: Supplementary Files

Deepak Kumar; Nitin Sharma; Murali Aarthy; Sanjeev Singh; Rajanish Giri

ZIKV NS3 helicase coding region (1342 bp) corresponding to PDB ID: 5GJC was synthesized by GeneArt Gene synthesis services provided by Invitrogen (USA). This gene was further ligated into pET 151/D-TOPO vector purchased from Thermo Fisher Scientific (USA). The final construct containing N-terminal 6X-His tag with TEV protease cleavage site was transformed into BL21 (Sigma) E.coli cells and thereafter positive clones were expressed in LB broth media (inducing with 1mM IPTG at 20 0 C overnight). Cells containing recombinant protein were harvested by centrifugation at 6000g at 4 0 C and re-suspended in binding buffer (50mM Tris, 300mM NaCl, 40mM imidazole, 5%glycerol pH8.0). Protease inhibitor cocktail (Thermo Fisher Scientific, USA) was added before cell lysis. Cells were lysed by sonication and adding 50% B-PER (Thermo scientific) reagent. After centrifugation at 16000 rpm for 30 min at 4 0 c, the supernatant containing recombinant protein was filtered and loaded on HisTrap FF 5ml column (GE healthcare). Recombinant protein was eluted by using a linear imidazole gradient from 0 to 100 %. Eluted fractions were analyzed on 10% SDS-PAGE for the purity. Buffer exchange was carried out to remove the Imidazole by using Amicon 10 kDa (Merck Millipore) centrifugal filters. Recombinant protein was kept in storage buffer (50mM Tris, 100mM NaCl, 5% glycerol) stored in aliquots at -80 0 C.