"221640/Z/20/Z" Dotaz Zobrazit nápovědu
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.
- MeSH
- aminopeptidasy * biosyntéza chemie genetika izolace a purifikace MeSH
- Escherichia coli * genetika metabolismus MeSH
- kinetika MeSH
- Leishmania major * enzymologie genetika MeSH
- protozoální proteiny * biosyntéza chemie genetika izolace a purifikace MeSH
- rekombinantní proteiny biosyntéza chemie genetika izolace a purifikace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Aquaglyceroporins (AQPs) are membrane proteins that function in osmoregulation and the uptake of low molecular weight solutes, in particular glycerol and urea. The AQP family is highly conserved, with two major subfamilies having arisen very early in prokaryote evolution and retained by eukaryotes. A complex evolutionary history indicates multiple lineage-specific expansions, losses and not uncommonly a complete loss. Consequently, the AQP family is highly evolvable and has been associated with significant events in life on Earth. In the African trypanosomes, a role for the AQP2 paralogue, in sensitivity to two chemotherapeutic agents, pentamidine and melarsoprol, is well established, albeit with the mechanisms for cell entry and resistance unclear until very recently. Here, we discuss AQP evolution, structure and mechanisms by which AQPs impact drug sensitivity, suggesting that AQP2 stability is highly sensitive to mutation while serving as the major uptake pathway for pentamidine.
