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, DD1 5EH, Dundee, Scotland, UK.
Protein Expr Purif. 2021 Jul;183:105877. doi: 10.1016/j.pep.2021.105877. Epub 2021 Mar 25.
The Leishmania major leucyl-aminopeptidase (LAPLm), a member of the M17 family of proteases, is a potential drug target for treatment of leishmaniasis. To better characterize enzyme properties, recombinant LAPLm (rLAPLm) was expressed in Escherichia coli. A LAPLm gene was designed, codon-optimized for expression in E. coli, synthesized and cloned into the pET-15b vector. Production of rLAPLm in E. coli Lemo21(DE3), induced for 4 h at 37 °C with 400 μM IPTG and 250 μM l-rhamnose, yielded insoluble enzyme with a low proportion of soluble and active protein, only detected by an anti-His antibody-based western-blot. rLAPLm was purified in a single step by immobilized metal ion affinity chromatography. rLAPLm was obtained with a purity of ~10% and a volumetric yield of 2.5 mg per liter, sufficient for further characterization. The aminopeptidase exhibits optimal activity at pH 7.0 and a substrate preference for Leu-p-nitroanilide (appK = 30 μM, appk = 14.7 s). Optimal temperature is 50 °C, and the enzyme is insensitive to 4 mM Co, Mg, Ca and Ba. However, rLAPLm was activated by Zn, Mn and Cd but is insensitive towards the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, being inhibited by EDTA and bestatin. Bestatin is a potent, non-competitive inhibitor of the enzyme with a K value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification.
利什曼原虫亮氨酰氨基肽酶（LAPLm）是 M17 家族蛋白酶的成员，是治疗利什曼病的潜在药物靶点。为了更好地描述酶的特性，在大肠杆菌中表达了重组 LAPLm（rLAPLm）。设计了 LAPLm 基因，针对大肠杆菌中的表达进行了密码子优化，合成并克隆到 pET-15b 载体中。在 37°C 下用 400 μM IPTG 和 250 μM l-鼠李糖诱导 4 小时，在大肠杆菌 Lemo21(DE3)中生产 rLAPLm ，产生不溶性酶，只有可溶性和活性蛋白用抗 His 抗体的 western blot 检测到。rLAPLm 通过固定化金属离子亲和层析一步纯化。rLAPLm 的纯度约为 10%，体积产率为每升 2.5 毫克，足以进行进一步的表征。该氨肽酶在 pH 7.0 下表现出最佳活性，对 Leu-p-硝基苯胺（appK=30 μM，appk=14.7 s）有偏好。最佳温度为 50°C，该酶对 4 mM Co、Mg、Ca 和 Ba 不敏感。然而，rLAPLm 被 Zn、Mn 和 Cd 激活，但对蛋白酶抑制剂 PMSF、TLCK、E-64 和胃蛋白酶抑制剂 A 不敏感，被 EDTA 和 bestatin 抑制。Bestatin 是该酶的一种有效、非竞争性抑制剂，K 值为 994 nM。我们建议 rLAPLm 是鉴定抑制剂的合适靶标。
