We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al, 2019). During the study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr, a minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 was pH 7.0 and 40°C, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity further (117%) when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.

中文翻译：

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编码来自枯草芽孢杆菌 SJ4 的次要纤维蛋白溶解酶的新 vpr 基因的克隆和 Vpr 的特性。

我们之前已经表征了 AprESJ4，它是枯草芽孢杆菌SJ4 中的主要纤维蛋白溶解酶（Yao等人，2019 年）。在研究过程中，我们观察到一种具有纤溶活性的 68 kDa 蛋白质。在这项研究中，我们克隆了编码 68 kDa 蛋白质的基因 ( vprSJ4 )，这是一种成熟的 Vpr，一种由芽孢杆菌分泌的次要蛋白酶。vprSJ4编码由 810 个氨基酸 (aa) 组成的前酶原，包括信号序列 (28 aa) 和原序列 (132 aa)。成熟酶 (650 aa) 的预测分子量为 68,467.35。与其他枯草芽孢杆菌菌株的 Vprs 不同，VprSJ4 在 C 端有 4 个额外的氨基酸 (DEFA)。vprSJ4在大肠杆菌中过表达。PreproVprSJ4 定位于包涵体中，并通过亲和柱进行体外复性和纯化。SDS-PAGE 和蛋白质印迹显示发生了 preproVprSJ4 的自动加工，并产生了 68 kDa 和更小的蛋白质。重组 VprSJ4 的最适 pH 和温度分别为 pH 7.0 和 40°C。重组 VprSJ4 的动力学参数通过使用人工底物N-琥珀酰-ala-ala-pro-phe- p-硝基苯胺测量。使用 pHY300PLK共表达vprSJ4和aprESJ4与在枯草芽孢杆菌WB600。