Expression and Purification of Z Protein from Junín Virus
Figure 2
Expression, purification and cleavage of Tio-Z-V5-His. (a) Diagram for the recombinant Tio-Z-V5-His protein expressed in E. coli. The dark gray region represents the thioredoxine (Tio) fusion protein; the striped block indicates the specific protease Enterokinase (Gly-Gly-Gly-Gly-Lys) target sequence; the light gray region corresponds to Z protein; the black line shows the V5 epitope localization; and the white arrow indicates the His-tag. The molecular weight of the complete recombinant protein is 27.3 kDa, the cleavage products being 12.8 kDa (thioredoxine) and 14.5 kDa (Z-V5-His). (b) 12% SDS-PAGE of samples of the whole cell culture at initial time () and after 4 h at C (). The arrow indicates Tio-Z-V5-His recombinant protein. (c) 15% SDS-PAGE of the centrifuged cell lysate of E. coli expressing Tio-Z-V5-His. : supernatant from the whole cell lysate expressing Tio-Z-V5-His (soluble fraction). P: pellet from the whole cell lysate (insoluble fraction). The arrow indicates Tio-Z-V5-His. (d) The chromatography profile (upper part) corresponds to the elution of the bound protein with an imidazole gradient (black line). Fractions 10 to 18 (indicated with a bracket) were loaded on 12% SDS-PAGE. The arrow indicates Tio-Z-V5-His. In the lower part, is shown the 12% SDS-PAGE from Immobilized Ni2+ affinity chromatography (IMAC) His-trap (Amersham) fractions of the soluble fraction of Tio-Z-V5-His (S, from (c) part). (e) 12% SDS-PAGE of the gel filtration (Superdex 200) fractions 13 to 21 (pooled), of the Tio-Z-V5-His purification. This sample was the substrate of the cleavage with the enterokinase. (f) 15% SDS-PAGE of the cleavage of Tio-Z-V5-His with the protease enterokinase. The first lane is the purified Tio-Z-V5-His. The following lanes correspond to an incubation of 2:30 h at C with different dilutions of the enterokinase (indicated with a bracket). The last lane is Tio-Z-V5-His protein without treatment. The arrows indicate the two peptides produced by the cleavage, shown in diagram A. (g) 15% SDS-PAGE of the purification of Z-V5-His with IMAC. (h) Western blot of the cleavage products of Tio-Z-V5-His with Enterokinase. The arrow indicates Z-V5-His.
