Bacterial expression of a Trichosanthes kirilowii defensin (TDEF1) and its antifungal activity on Fusarium oxysporum

Abstract

The gene encoding Trichosanthes kirilowii defensin (TDEF1) was cloned by reverse transcriptase-polymerase chain reaction (RT-PCR). The newly discovered TDEF1 cDNA contains 231 bp (Genbank accession number DQ526373) and encodes a 76-amino acid protein, which consists of a 29-amino acid signal peptide and a 47-amino acid mature peptide. The partial cDNA, corresponding to the mature peptide coding region of TDEF1, was inserted into bacterial expression vector pET32a(+). Subsequent expression showed that TDEF1 was produced as a 26-kDa fusion protein in the form of inclusion body in Escherichia coli BL21 (DE3). After protein refolding and purification, the fusion TDEF1 displayed an inhibitive activity against the fungal pathogen, Fusarium oxysporum, with EC(50) of 247 microg/ml by means of fungal growth inhibition method.