Leung Edwin H W, Wong Jack H, Ng T B
Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, New Territories, Hong Kong, China.
J Pept Sci. 2008 Mar;14(3):349-53. doi: 10.1002/psc.946.
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.
本文描述了一种纯化方案，用于从菜豆（Phaseolus vulgaris）种子中同时分离两种防御蛋白，包括一种6 kDa的防御素样抗真菌肽和一种60 kDa的二聚体血凝素。该方案包括在SP-琼脂糖上进行离子交换色谱、在Affi-凝胶蓝胶上进行亲和色谱、在Q-琼脂糖上进行离子交换色谱，以及在Superdex Peptide（用于防御素样抗真菌肽）或Superdex 200（用于血凝素）上进行凝胶过滤。抗真菌和血凝活性均吸附在SP-琼脂糖上，然后吸附在Affi-凝胶蓝胶上。随后，血凝素未被吸附，而防御素样抗真菌肽吸附在Q-琼脂糖上。抗真菌肽的抗真菌活性在0-90℃温度范围内20分钟、pH值4-10范围内以及在37℃下用胰蛋白酶（1 mg/ml）处理1小时后保持稳定。血凝素在10-80℃、pH值1-12范围内以及在3,7℃下用胰蛋白酶处理2小时后保持稳定。它抑制[甲基-(3)H]胸苷掺入乳腺癌（MCF-7）、白血病（L1210）、肝癌（HepG2）和人胚胎肝（WRL68）细胞，IC50分别为6.6、7、13和15 microM，并在1 microM浓度下从小鼠脾细胞中引发最大的促有丝分裂反应。它以1.9 microM的IC50抑制HIV-1逆转录酶活性，但没有抗真菌活性。
