Integrated Bioscience Section, Graduate School of Science and Technology, National University Corporation, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka, 422-8529, Japan.
Biological Science Course, Graduate School of Science, National University Corporation, Shizuoka University, Oya 836, Suruga-ku, Shizuoka, 422-8529, Japan.
Biochem Biophys Res Commun. 2021 Jun 30;560:32-36. doi: 10.1016/j.bbrc.2021.04.118. Epub 2021 May 6.
Eleven genes, including prss59.1, were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. To address the role of prss59.1, the prss59.1 gene knock-out zebrafish strain is currently being established by genome editing. In this study, for further phenotypic analysis of prss59.1, biochemical characterization of Prss59.1 was conducted using recombinant protein. A C-terminal histidine-tagged version of zebrafish Prss 59.1 was constructed. Although E. coli-produced recombinant Prss59.1 showed almost no activity, peptidase activities appeared after denaturation and renaturation. Zebrafish Prss59.1 showed the highest activity against Lys-MCA. The optimal temperature and pH of the activity toward Lys-MCA were 37 °C and pH 8.0, respectively. The Km value was 0.17 mM. Thus, zebrafish Prss59.1 possesses the closed character of trypsin, as expected from the DNA sequence.
基于我们之前的微阵列分析和 RNA 测序研究，选择了包括 prss59.1 在内的 11 个基因作为候选排卵诱导基因。为了研究 prss59.1 的作用，目前正在通过基因组编辑建立 prss59.1 基因敲除斑马鱼品系。在这项研究中，为了进一步对 prss59.1 进行表型分析，使用重组蛋白对 Prss59.1 进行了生化特性分析。构建了带有 C 端组氨酸标签的斑马鱼 Prss 59.1。尽管大肠杆菌产生的重组 Prss59.1 几乎没有活性，但变性和复性后出现了肽酶活性。斑马鱼 Prss59.1 对 Lys-MCA 的活性最高。活性的最适温度和 pH 值分别为 37°C 和 pH 8.0。Km 值为 0.17 mM。因此，斑马鱼 Prss59.1 具有预期的 DNA 序列所具有的胰蛋白酶的封闭特征。
