An antifungal defensin from Phaseolus vulgaris cv. 'Cloud Bean'

Abstract

An antifungal peptide with a defensin-like sequence and exhibiting a molecular mass of 7.3kDa was purified from dried seeds of Phaseolus vulgaris 'Cloud Bean'. The isolation procedure entailed anion exchange chromatography on DEAE-cellulose, affinity chromatography an Affi-gel blue gel, cation exchange chromatography on SP-Sepharose, and gel filtration by fast protein liquid chromatography on Superdex 75. Although the antifungal peptide was unadsorbed on DEAE-cellulose, it was adsorbed on both Affi-gel blue gel and SP-Sepharose. The antifungal peptide exerted antifungal activity against Mycosphaerella arachidicola with an IC(50) value of 1.8 μM. It was also active against Fusarium oxysporum with an IC(50) value of 2.2 μM. It had no inhibitory effect on HIV-1 reverse transcriptase when tested up to 100 μM. Proliferation of L1210 mouse leukemia cells and MBL2 lymphoma cells was inhibited by the antifungal peptide with an IC(50) of 10 μM and 40 μM, respectively.

Fig. 1

Ion exchange chromatography of fraction B2 (from Affi-gel blue gel column) on SP-Sepharose. Column dimensions: 2.5 cm × 20 cm. Starting buffer: 10 mM NH₄OAc buffer (pH 5.0). Slanting line across right half of chromatogram represents the linear 0–1 M NaCl gradient included in the 10 mM NH₄OAc buffer used to elute adsorbed proteins.

Fig. 2

Gel filtration by FPLC on a Superdex 75 HR 10/30 column. Buffer: 0.2 M NH₄HCO₃ (pH 8.5). Flow rate: 0.4 ml/min. Fraction size: 0.8 ml. Sample: Fraction S2 from SP-Sepharose column chromatography.

Fig. 3

SDS-polyacrylamide gel electrophoresis of fraction SU3 (from Superdex 75 column chromatography). Left lane: molecular mass marker from GE Healthcare. Right lane: fraction SU3 (purified cloud bean antifungal peptide).

Fig. 4

Antifungal activity of purified antifungal peptide toward Mycosphaerella arachidicola. (A) Control, 15 μl 50 mM MES buffer (pH 6.0). (B) 12 μg peptide in 15 μl MES buffer. (C) 2.4 μg peptide in 15 μl MES buffer.

Fig. 5

Antifungal activity of purified antifungal peptide toward Fusarium oxysporum. (A) Control, 15 μl 50 mM MES buffer (pH 6.0). (B) 12 μg peptide in 15 μl MES buffer. (C) 2.4 μg peptide in 15 μl MES buffer.

Fig. 6

Determination of IC₅₀ value of antifungal activity of purified antifungal peptide toward Mycosphaerella arachidicola. (A) 0 μM antifungal peptide. (B) 0.6 μM antifungal peptide. (C) 3 μM antifungal peptide. (D) 15 μM antifungal peptide. The IC₅₀ value was determined to be 1.8 μM.