Inhibition of SARS-CoV 3CL protease by flavonoids

Jo, Seri; Kim, Suwon; Shin, Dong Hae; Kim, Mi-Sun

The coding sequence of SARS-CoV nsp5-pp1a/pp1ab 3CL (chymotrypsin-like) protease (3CLpro) was obtained at the NCBI database (NP_828863.1). The catalytic domain of 3CLpro (M1$T196) was synthesised chemically by Bioneer (Daejeon, Korea) and cloned into a bacteriophage T7-based expression vector, pBT7. The plasmid DNA was transformed into E. coli BL21 (DE3) for protein expression. E. coli BL21 (DE3) cells were grown on Luria-Bertani (LB) agar plates containing 150 lg ml À1 ampicillin. Several colonies were picked and grown in capped test tubes with 10 ml LB broth containing 150 lg ml À1 ampicillin. A cell stock composed of 0.85 ml culture and 0.15 ml glycerol was prepared and frozen at 193 K for use in a large culture. The frozen cell stock was grown in 5 ml LB medium and diluted into 1000 ml fresh LB medium. The culture was incubated at 310 K with shaking until an OD 600 of 0.6-0.8 was reached. At this point, the expression of the SARS-CoV 3CLpro was induced using isopropyl-b-D-1-thiogalactopyranoside (IPTG) at a final concentration of 1 mM. The culture was further grown at 310 K for 3 h in a shaking incubator. Cells were harvested by centrifugation at 7650g (6500 rev min À1 ) for 10 min in a high-speed refrigerated centrifuge at 277 K. The cultured cell paste was resuspended in 25 ml of a buffer consisting of 50 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM imidazole, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 lg ml À1 DNase I. The cell suspension was disrupted using an ultrasonic cell disruptor (Digital Sonifier 450, Branson, USA). Cell debris was pelleted by centrifugation at 24,900g (15,000 rev min -1 ) for 30 min in a high-speed refrigerated ultra-centrifuge at 277 K.