Protease resistance profile of P22sTsp.

SDS-PAGE analysis of P22sTsp following treatment with A, chicken GI fluid proteases, B, trypsin, C, chymotrypsin and D, pepsin and chymotrypsin in the presence or absence of 10% BSA. A, Lane 1, molecular weight markers; lane 2, untreated P22sTsp; lanes 3–5, P22sTsp incubated with protease solutions at 37°C for 5 min, 20 min and 2 h, respectively. B, C, Lane 1, molecular weight marker; lane 2, untreated P22sTsp; lane 3, trypsin-treated (B) or chymotrypsin-treated (C) P22sTsp (1 h at 37°C). A control protein (single-domain antibody) incubated with the GI fluid (37°C, 2 h), trypsin or chymotrypsin (37°C, 1 h) showed complete digestion (data not shown). D, All reaction samples, including controls (no proteases) were subjected to a purification step with PureProteome™ Nickel Magnetic Beads prior to their analysis by SDS-PAGE (see Materials and Methods). Digestion reactions were carried out for 0 and 60 min. The positions of the molecular weight markers are indicated on the left of the panel D figures.