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Exp Parasitol. 2013 Sep;135(1):116-25. doi: 10.1016/j.exppara.2013.06.005. Epub 2013 Jun 28.

Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

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1
Laboratório de Fisiologia e Bioquímica de Microrganismos, Centro de Biociências e Biotecnologia, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes-RJ 28013-600, Brazil.

Abstract

Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 μg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

KEYWORDS:

AMPs; Antimicrobial peptides; CD; EK; His; Human pathogen; LB; LIC; Luria–Bertani; Neglected disease; New antiparasitic substances; Recombinant expression; TB; Terrific Broth; Trx; Vu-Def; Vu-Defr; antimicrobial peptides; circular dichroism; enterokinase; histidine tag; ligation-independent cloning; natural Vigna unguiculata defensin; recombinant Vigna unguiculata defensin; thioredoxin tag

PMID:
23816644
DOI:
10.1016/j.exppara.2013.06.005
[Indexed for MEDLINE]
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