Protease resistance profile of P22sTsp.

<p>SDS-PAGE analysis of P22sTsp following treatment with <b>A</b>, chicken GI fluid proteases, <b>B</b>, trypsin, <b>C</b>, chymotrypsin and <b>D,</b> pepsin and chymotrypsin in the presence or absence of 10% BSA. <b>A,</b> 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>B</b>, <b>C</b>, Lane 1, molecular weight marker; lane 2, untreated P22sTsp; lane 3, trypsin-treated (<b>B</b>) or chymotrypsin-treated (<b>C</b>) 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). <b>D</b>, 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013904#s4" target="_blank">Materials and Methods</a>). 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.</p>