High-Level Expression in Escherichia coli, Purification and Kinetic Characterization of LAPTc, a Trypanosoma cruzi M17-Aminopeptidase

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 _M = 74 µM, appk _cat = 4.4 s ⁻¹ ). The optimal temperature is 50 °C, and the cations Mg ²⁺ , Cd ²⁺ , Ba ²⁺ , Ca ²⁺ and Zn ²⁺ at 4 mM inhibited the activity by 60% or more, while Mn ²⁺ inhibited by only 15% and addition of Co ²⁺ 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 _i value of 881 nM. The enzyme is a good target for inhibitor identification.