Centro de Estudio de Proteínas, Facultad de Biología, Universidad de La Habana, calle 25 #455 entre I y J, Vedado, 10400, Havana, Cuba.
Wellcome Centre for Anti-Infectives Research, School of Life Sciences, University of Dundee, Dow Street, Dundee, Scotland, DD1 5EH, UK.
Protein J. 2019 Apr;38(2):167-180. doi: 10.1007/s10930-019-09823-w.
The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appK = 74 µM, appk = 4.4 s). The optimal temperature is 50 °C, and the cations Mg, Cd, Ba, Ca and Zn at 4 mM inhibited the activity by 60% or more, while Mn inhibited by only 15% and addition of Co activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a K value of 881 nM. The enzyme is a good target for inhibitor identification.
克氏锥虫 M17 亮氨酰氨基肽酶（LAPTc）是一种治疗恰加斯病的新型药物靶点。本工作旨在大肠杆菌中获得重组 LAPTc（rLAPTc）。设计了一个 LAPTc 基因，对其在大肠杆菌中的表达进行了优化，合成并克隆到 pET-19b 载体中。在 25°C 用 1mM IPTG 诱导 20 小时，可在大肠杆菌 BL21(DE3)pLysS 中生产可溶性 rLAPTC，该酶具有催化活性。rLAPTc 酶通过 IMAC 一步纯化。重组蛋白的纯度为 90%，每升培养物的体积产率为 90mg。该酶的最适 pH 为 9.0，对 Leu-p-硝基苯胺（appK=74µM，appk=4.4s）具有偏好性。最适温度为 50°C，4mM 的 Mg、Cd、Ba、Ca 和 Zn 使活性抑制 60%或以上，而 Mn 仅抑制 15%，Co 则使活性增加 40%。重组酶对蛋白酶抑制剂 PMSF、TLCK、E-64 和胃蛋白酶抑制剂 A 不敏感，但对 EDTA 和最佳抑制剂敏感。最佳抑制剂是该酶的非竞争性抑制剂，K 值为 881nM。该酶是抑制剂鉴定的良好靶标。
