Concurrent purification of two defense proteins from French bean seeds: a defensin‐like antifungal peptide and a hemagglutinin

Leung, Edwin H. W.; Wong, Jack H.; Ng, T. B.

A purification protocol is described herein for concurrent isolation of two defense proteins including a 6‐kDa defensin‐like antifungal peptide and a 60‐kDa dimeric hemagglutinin from seeds of the French bean (Phaseolus vulgaris). It involved ion‐exchange chromatography on SP‐Sepharose, affinity chromatography on Affi‐gel blue gel, ion‐exchange chromatography on Q‐Sepharose, and gel filtration on Superdex Peptide (for defensin‐like antifungal peptide) or Superdex 200 (for hemagglutinin). Both antifungal and hemagglutinating activities were adsorbed on SP‐Sepharose and then on Affi‐gel blue gel. Hemagglutinin was subsequently unadsorbed and defensin‐like antifungal peptide adsorbed on Q‐Sepharose. The antifungal activity of the antifungal peptide was stable in the temperature range of 0–90 °C for 20 min, in the pH range of 4–10, and after exposure to trypsin (1 mg/ml) at 37 °C for 1 h. The hemagglutinin was stable from 10 to 80 °C, from pH 1 to 12, and after treatment with trypsin at 37 °C for 2 h. It inhibited [methyl‐(3)H]thymidine incorporation into breast cancer (MCF‐7), leukemia (L1210), hepatoma (HepG2) and human embryonic liver (WRL68) cells with an IC(50) of 6.6, 7, 13 and 15 µm, respectively, and elicited maximal mitogenic response from mouse splenocytes at 1 µm concentration. It curtailed HIV‐1 reverse transcriptase activity with an IC(50) of 1.9 µm, but was devoid of antifungal activity. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.