Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase.

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 _M  = 30 μM, appk _cat  = 14.7 s ^-1 ). Optimal temperature is 50 °C, and the enzyme is insensitive to 4 mM Co ²⁺ , Mg ²⁺ , Ca ²⁺ and Ba ²⁺ . However, rLAPLm was activated by Zn ²⁺ , Mn ²⁺ and Cd ²⁺ 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 _i value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification.
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