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

Protein Expr Purif. 2021 Jul:183:105877. doi: 10.1016/j.pep.2021.105877. Epub 2021 Mar 25.

Abstract

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.

Keywords: Expression in E. coli; IMAC; Kinetic characterization; Leucyl aminopeptidases; pET-15b vector.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminopeptidases* / biosynthesis
  • Aminopeptidases* / chemistry
  • Aminopeptidases* / genetics
  • Aminopeptidases* / isolation & purification
  • Escherichia coli* / genetics
  • Escherichia coli* / metabolism
  • Kinetics
  • Leishmania major* / enzymology
  • Leishmania major* / genetics
  • Protozoan Proteins* / biosynthesis
  • Protozoan Proteins* / chemistry
  • Protozoan Proteins* / genetics
  • Protozoan Proteins* / isolation & purification
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification

Substances

  • Protozoan Proteins
  • Recombinant Proteins
  • Aminopeptidases