Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan.
J Pept Sci. 2011 Aug;17(8):569-75. doi: 10.1002/psc.1375. Epub 2011 May 13.
HTLV-I is a debilitating and/or lethal retrovirus that causes HTLV-I-associated myelopathy/tropical spastic paraparesis, adult T-cell leukemia and several inflammatory diseases. HTLV-I protease is an aspartic retropepsin involved in HTLV-I replication and its inhibition could treatHTLV-I infection. A recombinant L40I mutant HTLV-I protease was designed and obtained from Escherichia coli, self-processingand purification by ion-exchange chromatography. The protease was refolded by a one-step dialysis and recovered activity. The cleavage efficiency of the [Ile⁴⁰]HTLV-I protease was at least 300 times higher for a fluorescent substratethan that of our previously reported recombinant His-tagged non-mutated HTLV-I protease. In addition, we designed and synthesized a substrate containing a highly fluorescent Mca moiety in the fragment before the scissile bond, and a chromogenic p-nitrophenylalanine moiety after the scissile bond that greatly amplified spectrometry detection and improved the HTLV-I protease inhibition potency assay. The HTLV-I protease inhibition assay with the [Ile⁴⁰]HTLV-I protease and fluorogenic substrate requires distinctively less protease, substrate, inhibitor and assay time than our previous methods. This means our new assay is more cost-effective and more time-efficient while being reproducible and less labor-intensive.
人类嗜 T 细胞病毒 I 型（HTLV-I）是一种使人虚弱和/或致命的逆转录病毒，可引起 HTLV-I 相关性脊髓病/热带痉挛性截瘫、成人 T 细胞白血病和几种炎症性疾病。HTLV-I 蛋白酶是一种天冬氨酸反转肽酶，参与 HTLV-I 的复制，其抑制作用可治疗 HTLV-I 感染。本研究从大肠杆菌中设计并获得了重组 L40I 突变 HTLV-I 蛋白酶，通过离子交换色谱法进行自身加工和纯化。通过一步透析对蛋白酶进行重折叠并恢复其活性。与我们之前报道的重组 His 标记未突变 HTLV-I 蛋白酶相比，[Ile⁴⁰]HTLV-I 蛋白酶对荧光底物的切割效率至少高 300 倍。此外，我们设计并合成了一种含有高度荧光 Mca 部分的底物，在切割键之前的片段中，以及在切割键之后的生色对硝基苯丙氨酸部分，大大放大了光谱检测并提高了 HTLV-I 蛋白酶抑制效力测定的灵敏度。与我们之前的方法相比，使用 [Ile⁴⁰]HTLV-I 蛋白酶和荧光底物的 HTLV-I 蛋白酶抑制测定需要明显更少的蛋白酶、底物、抑制剂和测定时间。这意味着我们的新测定方法更具成本效益和时间效率，同时具有可重复性且劳动强度更低。
