Figure 4.

PCS1 has proteolytic activity. (A) PCS1 is a glycosylated protein. The western blot containing protein extracts with or without EndoH digestion from the 35S:PCS1–FAST transgenic plants was probed with the anti-FLAG-M2 antibody. (B) SDS–polyacrylamide gel electrophoresis (SDS–PAGE) analysis of purified PCS1–FAST. Lanes 2 and 3 contain proteins eluted after single-step purification with the anti-FLAG-M2 affinity gel and after second-step purification with a Strep-tactin column, respectively. Lane 1 is the protein elute from nontransgenic plants subjected to M2 affinity purification. (C) SDS–PAGE analysis of fragmented casein catalysed by the purified PCS1–FAST at the two pH conditions. Lanes 1, 3: casein only; lanes 2, 4: casein+PCS1–FAST. The elute from transgenic plants expressing only the FAST tag (lane 5) did not have protease activity. Arrows 1 and 2 point to casein and PCS1–FAST, respectively. (D) Semiquantitative assay on proteolytic activity of PCS1–FAST. Reaction was carried out at pH 5.5.