Thermostability of recombinant pernisine^co.

<p>Time courses of the residual activity of recombinant pernisine at the indicated temperatures, at pH 8.0.</p

SDS-PAGE analysis and zymography of purified pernisine^co and pernisine^S355Aco.

<p>Representative gels of the purified pernisines, following electrophoresis on standard 12% SDS-PAGE (A) and on 12% SDS-PAGE with casein as substrate (B) for the zymography activity (4 h at 80°C). Staining was with Coomassie blue dye. Lanes 0, protein MW markers (indicated left); lanes 1, recombinant pernisine^co; lanes 2, recombinant pernisine^S355Aco; lanes 3 and 4, heat-activated pernisine^co and pernisine^S355Aco. Selected protein bands of pernisine^co that were analysed by MS/MS are marked as I and II, (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123288#pone.0123288.s003" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123288#pone.0123288.s004" target="_blank">S4</a> Figs). *protein load of pernisine^{<b>S355Aco</b>} is three times higher than pernisine^co.</p

Dependence of the activity of recombinant pernisine on temperature and pH.

<p>Relative activity of recombinant pernisine according to temperature and pH (as corrected based on temperature change). Assays were carried out in triplicates, and means of the relative activity dependence are presented as a function of temperature and corrected pH. Colour legend on the left indicates the relative activities.</p

Residual protease activities of recombinant pernisine in the presence of the reductants, denaturants and detergent.

<p>Residual protease activities of recombinant pernisine in the presence of the reductants, denaturants and detergent.</p

Residual protease activities of recombinant pernisine in the presence of the protease inhibitors.

<p>ND, no data.</p><p>Residual protease activities of recombinant pernisine in the presence of the protease inhibitors.</p

Experimental flowchart and pernisine expression and analysis.

<p>(A) Flowchart of the experimental procedures. (B, C) Gel-exchange chromatography of pernisine^co in pMCSG7 using BL21(DE3) <i>E</i>. <i>coli</i> cells (B), and the corresponding SDS-PAGE analysis (C). The red line represents the selected fractions. (D) Time expression analysis after induction (1, 2, 3, 4 h) of <i>pernisine</i>^<i>wt</i> and <i>pernisine</i>^<i>co</i> for total cell lysates. Proteins were transferred (dot blot) onto nitrocellulose membranes and His₆-tagged pernisine was detected with anti-His₅-tag antibodies. Quantification was done using the ImageJ software (right panel). (E) SDS-PAGE analysis of pernisine^co and pernisine^S355Aco for total cell lysates of BL21(DE3) <i>E</i>. <i>coli</i> containing the pMCSGx series of vectors. 1, 4, Pernisine^co/wt-pMCSG7; 2, 5, Pernisine^co/wt-pMCSG9; 3, 6, Pernisine^co/wt-pMCSG10. (F) Immunodetection of pernisine^wt and pernisine^co for cell lysates containing the pMCSGx series of vectors. Proteins were transferred onto nitrocellulose membranes and His₆-tagged pernisine was detected using anti-His₆-tag antibodies. (G, H) Azocasein assays of the purified pernisine^co, showing effects of CaCl₂ (G) and NaCl (H). Relative proteolytic activities of activated pernisine^co are shown according to the CaCl₂ concentrations, and to the NaCl concentrations in the presence (grey, dot-dash line) and absence (black line) of 1 mM CaCl₂. Non-activated pernisine^co in the absence of 1 mM CaCl₂ is also shown (black, dot line). Abbreviations: co-codon-optimised, wt-wild-type.</p