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

摘要

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 20h at 25 degrees C with 1mM 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 90mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appK(M)=74 mu M, appk(cat)=4.4s(-1)). The optimal temperature is 50 degrees C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ 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 881nM. The enzyme is a good target for inhibitor identification.