Concurrent purification of two defense proteins from French bean seeds: a defensin-like antifungal peptide and a hemagglutinin.
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 degrees C for 20 min, in the pH range of 4-10, and after exposure to trypsin (1 mg/ml) at 37 degrees C for 1 h. The hemagglutinin was stable from 10 to 80 degrees C, from pH 1 to 12, and after treatment with trypsin at 37 degrees 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 IC50 of 6.6, 7, 13 and 15 microM, respectively, and elicited maximal mitogenic response from mouse splenocytes at 1 microM concentration. It curtailed HIV-1 reverse transcriptase activity with an IC50 of 1.9 microM, but was devoid of antifungal activity.
Leung
Edwin H W
EH
Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, New Territories, Hong Kong, China.
Wong
Jack H
JH
Ng
T B
TB
D000595:Amino Acid Sequence; D000818:Animals; D000935:Antifungal Agents / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D023082:Defensins / Q000737:chemistry* / Q000302:isolation & purification; D006388:Hemagglutinins / Q000302:isolation & purification*; D006801:Humans; D051379:Mice; D027805:Phaseolus / Q000737:chemistry*; D037121:Plant Lectins / Q000302:isolation & purification; D012639:Seeds / Q000737:chemistry
D000935:Antifungal Agents; D023082:Defensins; D006388:Hemagglutinins; D037121:Plant Lectins