Production of enzymatically active recombinant N23SsAP.

<p>A. N23SsAP was expressed in <i>E. coli</i> and purified over Ni-NTA. The expressed and purified protein was analysed by SDS-PAGE stained with coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, <i>E. coli</i> lysate before induction of the protein expression; lane 3, <i>E. coli</i> lysate after induction of the protein expression; lane 4, flow through from the Ni-NTA column; lane 5, purified N23SsAP eluted from the Ni-NTA column. B. N23SsAP (lane 2) was incubated at pH 3.5 and samples taken after 15 (lane 3) and 30 minutes (lane 4). Activation of the zymogen was visualised by silver staining after SDS-PAGE. Downward pointing arrow- zymogen; upward pointing arrow- activated enzyme. C. The activity of the activated enzyme at different pH conditions was measured using a fluorogenic substrate. The highest initial enzyme velocity was designated as 100% and enzyme velocities at other pHs were calculated as a percentage of this maximum value.</p

Digestion of human serum albumin by the recombinant human scabies mite aspartic protease N23SsAP.

<p>Recombinant N23SsAP was tested for its ability to digest human serum albumin. Reactions were run on a 15% non-reducing SDS-PAGE gel and stained with coomassie brilliant blue R-250. Lane 1, molecular weight marker; lanes 2–6, serum albumin plus N23SsAP, incubated for 15 minutes (lane 2), 1 hour (lane 3), 2 hours (lane 4), 4 hours (lane 5) or 12 hours (lane 6).</p

Detection of aspartic protease activity in scabies mite extract using a fluorescent substrate.

<p>The fluorescence signal was recorded at 60 second intervals, commencing after 10 minutes.</p

Digestion of human fibrinogen by the recombinant human scabies mite aspartic protease N23SsAP.

<p>Recombinant N23SsAP was tested for its ability to digest human fibrinogen. Reactions were run on a 15% non-reducing SDS-PAGE gel and stained with coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, fibrinogen alone; lanes 3 and 5, fibrinogen plus N23SsAP; lanes 4 and 6, fibrinogen plus N23SsAP and pepstatin A, after 4 hours incubation (lanes 3 and 4) and 13 hours incubation (lanes 5 and 6).</p

Digestion of acid denatured haemoglobin by scabies mite extract at pH–8.5.

<p>Haemoglobinolytic activity in whole human scabies mite extract was detected using acid denatured human haemoglobin (AD-Hb) as substrate at pH 3.5–8.5. Reactions were run on a 15% non-reducing SDS-PAGE gel and stained with coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, AD-Hb; lanes 3–8, AD-Hb plus scabies mite extract at pH 3.5, 4.5, 5.5, 6.5, 7.5 and 8.5 respectively.</p

Digestion of human collagen III by the recombinant human scabies mite aspartic protease N23SsAP.

<p>Recombinant N23SsAP was tested for its ability to digest human collagen III. Reactions were run on a 15% non-reducing SDS-PAGE gel and stained with coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, collagen III alone; lanes 3 and 5, collagen III plus N23SsAP; lanes 4 and 6, collagen III plus N23SsAP and pepstatin A, after 4 hours incubation (lanes 3 and 4) and 13 hours incubation (lanes 5 and 6).</p

Digestion of human fibronectin by the recombinant human scabies mite aspartic protease N23SsAP.

<p>Recombinant N23SsAP was tested for its ability to digest human fibronectin. Reactions were run on a 15% non-reducing SDS-PAGE gel and stained with coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, fibrinonectin alone; lanes 3 and 5, fibrinonectin plus N23SsAP; lanes 4 and 6, fibrinonectin plus N23SsAP and pepstatin A, after 4 hours incubation (lanes 3 and 4) and 13 hours incubation (lanes 5 and 6).</p

Digestion of human haemoglobin by the recombinant human scabies mite aspartic protease N23SsAP.

<p>The haemoglobinolytic activity of N23SsAP was detected using native human haemoglobin as substrate at pH% non-reducing SDS-PAGE gel and stained with coomassie brilliant blue R-250. Lane 1, molecular weight marker; lane 2, haemoglobin alone; lanes 3–6, haemoglobin plus N23SsAP, incubated for 1 hour (lane 3), 2 hours (lane 4), 4 hours (lane 5) or 12 hours (lane 6).</p