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

Aguado, M. E., González-Matos, M., Izquierdo, M., Quintana, J. , Field, M. C. and González-Bacerio, J. (2021) Expression in Escherichia coli, purification and kinetic characterization of LAPLm, a Leishmania major M17-aminopeptidase. Protein Expression and Purification, 183, 105877. (doi: 10.1016/j.pep.2021.105877) (PMID:33775769)

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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 (appKM = 30 μM, appkcat = 14.7 s−1). Optimal temperature is 50 °C, and the enzyme is insensitive to 4 mM Co2+, Mg2+, Ca2+ and Ba2+. However, rLAPLm was activated by Zn2+, Mn2+ and Cd2+ 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 Ki value of 994 nM. We suggest that rLAPLm is a suitable target for inhibitor identification.

Item Type:Articles
Additional Information:This work was supported by Wellcome Trust Centre awards 203134/Z/16/Z to M.I. and J.G.-B., and 204697/Z/16/to Paul Wyatt, M.C.F. and others; the International Foundation for Sciences (grant F/4730–2) to J.G.-B.; and the project assigned to J.G.-B. and associated with the Cuban National Program of Basic Sciences. J.Q. is supported by a Sir Henry Wellcome postdoctoral fellowship (221640/Z/20/Z).
Keywords:Expression in E. coli, IMAC, kinetic characterization, leucyl aminopeptidases, pET-15b vector.
Glasgow Author(s) Enlighten ID:Quintana, Dr Juan
Creator Roles:
Quintana, J.Investigation, Methodology, Writing – review and editing
Authors: Aguado, M. E., González-Matos, M., Izquierdo, M., Quintana, J., Field, M. C., and González-Bacerio, J.
College/School:College of Medical Veterinary and Life Sciences > School of Biodiversity, One Health & Veterinary Medicine
Journal Name:Protein Expression and Purification
ISSN (Online):1096-0279
Published Online:25 March 2021

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
310358Molecular basis of pathogen-induced disruption of host circadian rhythmsJuan QuintanaWellcome Trust (WELLCOTR)221640/Z/20/ZMVLS - Polyomics Facility