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

Development of a High-Throughput Screening Assay to Identify Inhibitors of the Major M17-Leucyl Aminopeptidase from Trypanosoma cruzi Using RapidFire Mass Spectrometry

“…M17-LAPs are usually assayed at alkaline pH and elevated temperature, 29,[31][32][33][34] and the T. cruzi enzyme displays maximal activity at pH 9.0 and 50 C. 24 Although these conditions are incompatible with HTS, LAPTc retained sufficient activity at pH 7.5 and room temperature for HTS assay development (Figs. 1A, 2A, and 2C).…”

“…LAPTc was produced in recombinant form in Escherichia coli according to Izquierdo et al 24 As a second purification step, gel filtration chromatography was performed using a Superose 6 HR10/30 column with 50 mM Tris-HCl buffer, pH 8.0, 300 mM NaCl. The flow rate was 1 mL/min, the fraction size was 2 mL, and runs were monitored at 280 nm.…”

Characterization of a Trichinella spiralis aminopeptidase and its participation in invasion, development and fecundity

“…The results of the enzymatic activity assay showed that rTsAP has the enzymatic activity of natural aminopeptidase to hydrolyze the substrate Leu-pNA with an optimal temperature of 50 °C and optimal pH of 8.0. The rTsAP enzymatic activity was significantly enhanced by three metal ions (Mn 2+ , Co 2+ and Ni 2+ ), indicating that TsAP is a leucyl-aminopeptidase that requires divalent metal cations and a basic optimal pH for catalysis [45].…”

“…To determine the enzymatic activity of rTsAP, the serially diluted rTsAP (0, 0.004, 0.008, 0.016, 0.032, 0.064 and 0.128 μg/μL) was pre-incubated at 37 °C for 10 min in various pH buffers (pH 4.0-5.0 sodium acetate, pH 6.0-7.0 sodium phosphate, pH 8.0-9.0 Tris-HCl, and pH 10.0-11 sodium bicarbonate) [44]. Subsequently, the aminopeptidase substrate 1 mM Leu-P-nitroaniline (Leu-pNA, Sigma-Aldrich, USA) was added into the reaction mixture and incubated at different temperatures (20-100 °C) for 15 min [45], and the absorbance at 405 nm was measured with a spectrophotometer. In order to verify whether the rTsAP enzymatic activity is metal ion-dependent, four common auxiliary metal ions (Co 2+ , Zn 2+ , Mn 2+ and Ni 2+ ) were selected and added into the reaction system at the same concentration (0.5 mM) to analyze their ability to affect rTsAP activity [46].…”

“…In order to verify whether the rTsAP enzymatic activity is metal ion-dependent, four common auxiliary metal ions (Co 2+ , Zn 2+ , Mn 2+ and Ni 2+ ) were selected and added into the reaction system at the same concentration (0.5 mM) to analyze their ability to affect rTsAP activity [46]. Different enzymatic inhibitors (2 mM 1, 10-Phenanthroline, 1 mM AEBSF and 5 μM E-64) were used to determine the effects of various inhibitors on the rTsAP enzyme activity [45]. The reaction rates of rTsAP to hydrolyze the substrate Leu-pNA at different concentrations (0.5, 1, 2, 3, 4, 5, 6, 7 and 8 mM) were calculated according to the product standard curve, and the Michaelis-Menten plot and Lineweaver-Burk plot were performed to obtain the K m value [39,47].…”