Cytolytic toxins are produced by a variety of living organisms, particularly certain bacteria, insects, poisonous reptiles and stinging marine invertebrates. Many of these toxins appear to function simply by forming pores in cell membranes, disrupting the permeability barrier and leading eventually to cell death. Few examples have been studied in plants. Enterolobin was the first plant cytolytic protein described in the literature, and is the most studied one. It is a membrane pore-forming protein extracted from Enterolobium contortisiliquum seeds. The current work aimed at producing E. contortisiliquum callus from seeds as a possible source material for enterolobin purification and comparing protein expression of callus and seeds. For callus production, seeds were randomly selected and scarified, and then incubated in distilled water overnight. Sodium hipochloride 30% was used for seed decontamination. Callus and seed materials were powdered in liquid nitrogen using pestle, followed by precipitation by trichloroacetic acid (TCA) in acetone solution. The resulting pellet was extracted in 1 mL of 2-D electrophoresis (2-DE) buffer containing urea 7 M and thiourea 2 M. The protein concentration was measured using a Plus One 2D QuantKit. The samples were submitted to 2‑DE using immobilized pH gradient gels in the 3‑10 pH range for isoelectric focusing. Comparative analysis of 2-DE maps showed different protein expression between callus and seed. To check the presence of the cytolysin in callus, Western blotting was carried out against enterolobin antibodies raised in rabbit. The results did not show a strong signal of enterolobin in callus protein extracts so far.

 (Supported by CAPES and CNPq)