Fig 1.

Recombinant VACV proteins produced in the baculovirus expression system. Schematic diagrams depict the full length (top) or the truncated, recombinant form (second from top) of VACV A28 (A), F9 (C) and variola virus M1 (D). In C and D, the diagram of soluble L1 (bottom) was also shown for comparison. Grey rectangles indicate putative transmembrane domains (TM), black lollipops represent sites for N-linked glycosylation potentially used in the secreted recombinant proteins, dotted line in (C) depicts the region of L1 which shares no homology with F9 (C), M points to the myristoylation site (D), S symbolizes conserved cysteines or intramolecular disulfide bonds (C and D) and H6 represent the six histidine tail used for affinity purification. Asterisks in (D) point to the two residues in variola virus M1 that are different in VACV L1 (R177 and I248 in M1 versus K177 and M248 in L1). Secreted, recombinant A28 (A), F9 (C) or M1 (D) were purified by nickel chelate affinity chromatography, separated by SDS-PAGE under reducing conditions and stained with silver nitrate (SS), or transferred to a nitrocellulose membrane and then probed with a monoclonal antibody against the His-tag (WB). Migration positions of the recombinant proteins are indicated with arrows. The molecular weights of the markers are shown in kilodaltons. Note that recombinant L1 and M1 are not expected to be myristoylated since the sequence for a melittin signal peptide was appended to the 5’ end of their ORFs (Aldaz-Carroll et al., 2005). (B) Recombinant A28 shares conformational epitopes with the authentic VACV A28. Purified soluble A28 or control A33 (Xiao et al., 2007) was resolved by SDS-PAGE under denaturing or native conditions, transferred to membranes and probed with a rabbit hyper-immune serum raised against VACV. Arrow points to the putative A33 dimer.