Biotechnical Faculty, University of Ljubljana, Ljubljana, Slovenia.
PLoS One. 2012;7(6):e39548. doi: 10.1371/journal.pone.0039548. Epub 2012 Jun 28.
An R30 fraction from the growth medium of Aeropyrum pernix was analyzed for the protease that can digest the pathological prion protein isoform (PrP(Sc)) from different species (human, bovine, deer and mouse).
METHODOLOGY/PRINCIPAL FINDINGS: Degradation of the PrP(Sc) isoform by the R30 fraction and the purified protease was evaluated using the 6H4 anti-PrP monoclonal antibody. Fragments from the N-terminal and C-terminal of PrP(Sc) were also monitored by Western blotting using the EB8 anti-PrP monoclonal antibody, and by dot blotting using the C7/5 anti-PrP monoclonal antibody, respectively. For detection of smaller peptides from incomplete digestion of PrP(Sc), the EB8 monoclonal antibody was used after precipitation with sodium phosphotungstate. Characterization of the purified active protease from the R30 fraction was achieved, through purification by fast protein liquid chromatography, and identification by tandem mass spectrometry the serine metalloprotease pernisine. SDS-PAGE and zymography show the purified pernisine plus its proregion with a molecular weight of ca. 45 kDa, and the mature purified pernisine as ca. 23 kDa. The purified pernisine was active between 58 °C and 99 °C, and between pH 3.5 and 8.0. The temperature and pH optima of the enzymatic activity of the purified pernisine in the presence of 1 mM CaCl(2) were 105 °C ± 0.5 °C and pH 6.5 ± 0.2, respectively.
CONCLUSIONS/SIGNIFICANCE: Our study has identified and characterized pernisine as a thermostable serine metalloprotease that is secreted from A. pernix and that can digest the pathological prion protein PrP(Sc).
从 Aeropyrum pernix 的生长培养基中分析出一种 R30 片段，该片段中含有一种蛋白酶，能够消化来自不同物种（人类、牛、鹿和鼠）的病理性朊病毒蛋白（PrP(Sc)）。
方法/主要发现：使用 6H4 抗-PrP 单克隆抗体评估 R30 片段和纯化蛋白酶对 PrP(Sc) 同工型的降解作用。使用 EB8 抗-PrP 单克隆抗体通过 Western 印迹法和 C7/5 抗-PrP 单克隆抗体通过斑点印迹法分别监测 PrP(Sc) 的 N 端和 C 端片段。为了检测 PrP(Sc) 不完全消化产生的较小肽，使用 EB8 单克隆抗体进行沉淀后，再进行检测。通过快速蛋白液相色谱法对 R30 片段中的纯化活性蛋白酶进行了鉴定，并通过串联质谱法鉴定出丝氨酸金属蛋白酶 pernisine。SDS-PAGE 和酶谱法显示纯化的 pernisine 及其前体约为 45 kDa，成熟的纯化 pernisine 约为 23 kDa。纯化的 pernisine 在 58°C 至 99°C 之间以及 pH3.5 至 8.0 之间具有活性。在 1 mM CaCl2 存在的情况下，纯化的 pernisine 的酶活性的最佳温度和 pH 值分别为 105°C±0.5°C 和 pH6.5±0.2。
结论/意义：我们的研究鉴定并表征了 pernisine，它是一种耐热的丝氨酸金属蛋白酶，由 A. pernix 分泌，能够消化病理性朊病毒蛋白 PrP(Sc)。
