Expression and purification of LASV NP from E. coli Rosetta 2(DE3) cells transformed with construct pMAL-c2x:NP. 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 (lanes 4–10) SEC fractions 4–10. The blot was probed with a rabbit α-MBP polyclonal antibody and then detected with an HRP-conjugated goat α-rabbit IgG antibody. (B) The Western blot in panel A was stripped, reprobed with LASV mAb mix containing NP-specific mAbs, and then detected with an HRP-conjugated goat α-mouse IgG antibody. The identity of each lane is the same as that indicated in Panel A. (C) SDS-PAGE and Coomassie blue stain of proteins in (lane 2) whole bacterial cell lysate, (lane 3) amylose capture eluate, (lane 4) Factor Xa cleavage reaction, (lane 5) SEC-purfied NP generated from pooled NP-containing fractions, and (lane 6) SEC-purified MBP. (Lane 1) SeeBlue® Plus2 pre-stained molecular weight markers, with sizes (kDa) shown to the left of each panel. NP, MBP, and NP-MBP are indicated.