Figure 3

Expression and purification of LASV GP2 from E. coli Rosetta gami 2 cells transformed with construct pMAL-c2x:GP2. An E. coli lysate was generated from IPTG-induced cells, the clarified supernatant was applied to an amylose resin column, the protein was eluted with 10 mM maltose, cleaved with Factor Xa, and purified by SEC. (A) Western blot of protein in (lane 2) amylose capture eluate, (lane 3) Factor Xa cleavage reaction, and (lane 4) pooled SEC fractions. The blot was probed with LASV mAb mix containing GP2-specific mAbs, then detected with an HRP-conjugated goat α-mouse IgG antibody. (Lane 1) Western blot XP molecular weight markers, with sizes (kDa) shown to the left of the panel. (B) SDS-PAGE and Coomassie blue stain of proteins in (lane 2) amylose capture eluate, (lane 3) Factor Xa cleavage reaction, and (lane 4) SEC-purified GP2 generated from pooled GP2-containing fractions. (Lane 1) SeeBlue^® Plus2 pre-stained molecular weight markers, with sizes (kDa) shown to the left of the panel. GP2, MBP, and GP2-MBP are indicated.