SDS-PAGE analysis of purified proteases NS2B(H)-NS3pro. Proteins were obtained by a two-step purification procedure using immobilized metal affinity chromatography and size-exclusion chromatography on a Superdex 75 HR10/300 GL column as described under Methods. Proteins shown were loaded in the presence of 8 M urea under denaturing conditions prior to refolding. Lane M, molecular weight marker proteins with molecular weight indicated; lane 1, NS2B(H)-NS3pro protease wild-type; lane 2, inactive S135A mutant of NS3pro; lanes 3-11, purified protein samples of nine active site mutants L115A, D129A, G133A, T134A, Y150A, G151A, N152A, S163A and I165A in NS2B(H)-NS3pro, respectively. The arrow indicates the band of NS2B(H)-NS3pro migrating at an apparent molecular weight of 37 kDa. Samples were run on 15% SDS-PAGE gels and stained with Coomassie Brillant Blue.