Biochemical characterization of zebrafish Prss59.1
Biochemical characterization of zebrafish Prss59.1
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斑马鱼 Prss59.1 的生化特征
DOI：
10.1016/j.bbrc.2021.04.118
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发表时间：
2021
影响因子：
3.1
通讯作者：
Md. Maisum Sarwar Jyoti and Toshinobu Tokumoto
中科院分区：
文献类型：
--
作者：
Md. Rubel Rana;Md. Forhad Hossain;Md. Hasan Ali;Md. Maisum Sarwar Jyoti and Toshinobu Tokumoto
在前期研究的基础上，通过基因芯片分析和RNA测序，筛选出包括prss 59.1在内的11个基因作为候选促排卵基因。为了解决prss 59.1的作用，目前正在通过基因组编辑建立prss 59.1基因敲除斑马鱼品系。在本研究中，为了进一步分析Prss 59.1的表型，利用重组蛋白对Prss 59.1进行了生化特性分析，构建了C-末端组氨酸标记的斑马鱼Prss 59.1。虽然大肠杆菌表达的重组Prss 59.1几乎没有活性，但变性和复性后出现了肽酶活性。斑马鱼Prss 59.1对Lys-MCA的活性最高。对Lys-MCA的最适反应温度为37℃，最适pH为8.0。Km值为0.17 mm。因此，斑马鱼Prss 59.1具有胰酶的封闭特性，正如DNA序列所预期的那样。
Eleven genes, includingprss59.1,were selected as candidate ovulation-inducing genes on the basis of microarray analysis and RNA sequencing in our previous study. To address the role ofprss59.1, the prss59.1 gene knock-out zebrafish strain is currently being established by genome editing. In this study, for further phenotypic analysis ofprss59.1, biochemical characterization of Prss59.1 was conducted using recombinant protein.A C-terminal histidine-tagged version of zebrafish Prss 59.1 was constructed. AlthoughE. 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.