Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

doi:10.1186/1475-2859-7-7

Microbial Cell Factories

Volume 7

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Campos MA
Silva MS
Magalhães CP
Ribeiro SG
Sarto RP
Vieira EA
Grossi de Sá MF

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Silva MS
Magalhães CP
Ribeiro SG
Sarto RP
Vieira EA
Grossi de Sá MF
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Research

Expression in Escherichia coli, purification, refolding and antifungal activity of an osmotin from Solanum nigrum

Magnólia de A Campos¹^,2^,3^,4 , Marilia S Silva³^,4 , Cláudio P Magalhães¹^,4 , Simone G Ribeiro¹^,4 , Rafael PD Sarto¹^,2^,4 , Eduardo A Vieira³^,4 and Maria F Grossi de Sá¹^,4

¹EMBRAPA Recursos Genéticos e Biotecnologia, PO Box 02372, 70770-900, Brasília-DF, Brazil

²Departamento de Biologia Celular, Universidade de Brasília, PO Box 90710-900, Brasília-DF, Brazil

³EMBRAPA Cerrados, BR 020 Km 18, PO Box 08223, 73310-970, Planaltina-DF, Brazil

⁴Departamento de Biologia, Universidade Federal de Lavras, P.O.Box 3037, 37200-000, Lavras-MG, Brazil

author email corresponding author email

Microbial Cell Factories 2008, 7:7doi:10.1186/1475-2859-7-7


Published:	11 March 2008

Abstract

Background

Heterologous protein expression in microorganisms may contribute to identify and demonstrate antifungal activity of novel proteins. The Solanum nigrum osmotin-like protein (SnOLP) gene encodes a member of pathogenesis-related (PR) proteins, from the PR-5 sub-group, the last comprising several proteins with different functions, including antifungal activity. Based on deduced amino acid sequence of SnOLP, computer modeling produced a tertiary structure which is indicative of antifungal activity.

Results

To validate the potential antifungal activity of SnOLP, a hexahistidine-tagged mature SnOLP form was overexpressed in Escherichia coli M15 strain carried out by a pQE30 vector construction. The urea solubilized His₆-tagged mature SnOLP protein was affinity-purified by immobilized-metal (Ni²⁺) affinity column chromatography. As SnOLP requires the correct formation of eight disulfide bonds, not correctly formed in bacterial cells, we adapted an in vitro method to refold the E. coli expressed SnOLP by using reduced:oxidized gluthatione redox buffer. This method generated biologically active conformations of the recombinant mature SnOLP, which exerted antifungal action towards plant pathogenic fungi (Fusarium solani f. sp.glycines, Colletotrichum spp., Macrophomina phaseolina) and oomycete (Phytophthora nicotiana var. parasitica) under in vitro conditions.

Conclusion

Since SnOLP displays activity against economically important plant pathogenic fungi and oomycete, it represents a novel PR-5 protein with promising utility for biotechnological applications.