Expression and Purification of Z Protein from Junín Virus
Figure 3
Expression, Purification, and Cleavage of GST-Z. (a) Diagram of the recombinant GST-Z protein expressed in E. coli. The dark gray region represents the glutathione-S-Transferase (GST) fusion protein; the striped block indicates the specific protease Factor Xa. (IleGluGlyArg) target sequence; and the light gray arrow shows the Z protein. The molecular weight of the complete recombinant protein is approx. 37 kDa, the cleavage products being ca. 25 kDa (GST) and ca. 11 kDa (Z). (b) 12% SDS-PAGE of a sample of the whole cell culture at initial time () and after 20 h at C (). (c) 15% SDS-PAGE of the purification of GST-Z with glutathione Sepharose 4B (Amersham). S: Supernatant from the whole cell lysate expressing GST-Z; : Lysate that ran through the column upon loading; W: Wash with PBS; E1 and E2: elution with 1 X PBS plus 25 mM reduced glutathione (Sigma). The arrow indicates GST-Z (37 kDa). (d) Gel Filtration (Superdex 200 GL, Amersham) and 12% SDS-PAGE. The first peak on the chromatogram includes fractions 6 to 10, analyzed in the SDS-PAGE, while the second peak corresponds to fractions 11 to 16. (e) 16% SDS-PAGE GST-Z cleavaged with the protease Factor Xa. The arrow indicates the cleavage product corresponding to (11.56 kDa). : 1 h; : 2 h; : 4 h; : 6 h; and : 16 h, at C. (f) Western-blot of the cleavage products of GST-Z with Factor Xa. The arrow indicates the protein. (g) 15% SDS-PAGE of the limited proteolysis of GST-Z with Chymotrypsin (Roche). The protein products indicated with the symbols I, II, and III were sequenced at the N-terminus. The obtained sequence is indicated adjacent to the diagram of GST-Z.
