Purification and molecular mass determination of a lipid transfer protein exuded from Vigna unguiculata seeds

Abstract

Plants exude a variety of substances through their surface especially of roots and germinating seeds. Some of these released compounds seem to have an inhibitory action against certain pathogens. Lipid transfer proteins (LTPs) are 9 kDa cysteine-rich cationic peptides and are thought to play a role in the protection of plants against microbial infections. The aim of this work was to isolate and determine the molecular mass of a LTP present in the exudates of imbibed cowpea seeds. For exudation induction, 50 seeds were submerged in 50 mL sterile 100 mmol.L-1 Na-acetate buffer, pH 4.5 and shaken at 30 ºC for 24 h. The resulting exudate was subjected to ammonium sulphate fractionation and the pellet formed between 0 and 70 % saturation was dialysed and recovered by freeze drying. Further purification steps were carried out using chromatographic methods and the molecular mass of the LTP determined. All of these steps were monitored by SDS-Tricine gel electrophoresis and Western blotting using an anti-LTP serum. The purified LTP showed a relative molecular mass of 9 kDa.