Figure 4.

Expression of Stisa Proteins in E. coli.

Stisa proteins were expressed as S-tagged fusion proteins in E. coli.

(A) Separation of soluble extracts of E. coli expressing Stisa1, Stisa2, or Stisa3 by SDS-PAGE. Proteins were blotted onto nitrocellulose and visualized by developing the blots with biotinylated S-protein (Invitrogen, Carlsbad, CA). Molecular mass is indicated in kD.

(B) Stisa1, Stisa2, and Stisa3 were purified by binding to S-agarose. The bound proteins were released by thrombin cleavage, separated by SDS-PAGE, and stained with Coomassie blue. The Stisa proteins appear pure by this method. Stisa2 was cleaved by thrombin within the isoamylase peptide as well as at the point of fusion to the S-TAG, so it was reduced in size compared with the mature protein.