FIG. 2.

Membrane modification in E. coli by JEV NS2B-NS3 proteins. E. coli BL21(DF3)pLysS harboring individual JEV NS genes cloned in pET21 plasmids was incubated without rifampin (no R.) or with rifampin plus IPTG (I.) in the absence or presence of hygromycin B (H.) after various periods as indicated at the top of the gels. Protein samples were labeled with [³⁵S]methionine and analyzed by SDS-PAGE as described in Materials and Methods. (A) Full-length NS2B-NS3 (1 to 750); (B) full-length NS1; (C and D) partial NS2B-NS3 (amino acids 1 to 373 and 127 to 373, respectively). The radioimmunoprecipitation (RIP) of expressed protein is shown in panel D by using antibody against T7 tag (lane 6) or JEV NS3 (lane 7). The major protein bands as predicted for each construct are indicated by arrowheads, and the numbers on the sides of the gels denote the molecular masses of protein standards.