Expression and Purification of Z Protein from Junín Virus
Figure 4
Z Protein Expression in Sf9 Insect Cell Line. (a) Diagram of the recombinant His-Z protein expressed on Sf9 Insect cell line. The white region represents the His-tag; the squared block indicates the specific protease TEV (GluAsnLeuTyrPheGlnGly) target sequence; and the gray arrow shows the Z protein. The molecular weight of the complete recombinant protein is 14 kDa, the cleavage products being 11 kDa (Z) and 3 kDa (His-TEV). (b) 16% SDS-PAGE of Sf9 cells infected with v_Ac-Z. Lane 1 shows the Sf9 cells without infection. Lanes 2 and 3 correspond to the Sf9 cells infected with v_Ac-Z at different moi's (1 and 5 resp.) after an incubation of 36 h. The arrows have shown the possible Z protein expression. (c) Western blotting with -Tio-Z-V5-His. Total protein extracts from Sf9 cells infected with v_Ac-Z (recombinant AcMNPV expressing Junín Z protein; lanes 1 and 2) and AcMNPV (wild type baculovirus; lane 4) were loaded in the indicated lanes. Lane 3 corresponds to the control of uninfected cells (ui). Tio-Z-V5-His purified sample was used as a positive control (indicated with a red arrow). (d) Western blotting with α-His Tag polyclonal serum (Santa Cruz Biotechnology). Ultracentrifugated supernatant and cellular extract of Sf9 cells infected with recombinant v_Ac-Z was loaded in the indicated lanes. At the bottom of each lane the origin of the sample is shown; Cell: Sf9 protein extract, UC pellet: ultracentrifugation pellet. Tio-Z-V5-His positive control is indicated with a red arrow.
