Development of a rapid purification method for Ricin A chain from Ricinus communis seeds

摘要

Ricin is a highly toxic, naturally occurring lectin (a carbohydrate binding protein) produced in the seeds of the castor oil plant Ricinus communis, and composed of two chains( A and B)(8). Ricin A chain inactivates 60S ribosomal subunit by disrupting the binding site for elongation factor (EF-2), and thus prevents the formation of the initiation complex (7). This toxin specifically influences on eukaryotic cells and affects protein synthesis.Ricin A chain (RTA), has been investigated as a potential candidate for cancer chemotherapy in the form of immunotoxins. Immunotoxins made with RTA have been used in clinical trials for the treatment of patients with bone-marrow transplantation, lymphoma, lupus nephritis, metastatic tumors, and leukemia (11). Ricin B chain (RTB) is a lectin that is able to bind terminal galactose residues on cell surfaces .This chain has no catalytic activity, but can mediate transport of the A-B protein complex from the cell surface to the lumen of the endoplasmic reticulum (ER). In this research we tried to design a simple and rapid method for RTA purification from castor bean. The crude ricin was loaded on column containing acid treated sepharose-4B gel.The column was washed with PBS for several hours (till the optical density reached below 0.05) to remove the unbound proteins. Due to RTB binding to the galactose moiety on sepharose-4B, whole toxin was attached to the sepharose. Then 2ME [Mercapto ethanol] containing buffer (0.2 molar 2ME in PBS) was added to the column for disulfide bound disruption. Fractions were collected manually, and absorbance was recorded at 280 nm. Protein containing fractions were pooled and concentrated. The purity of RTA was evaluated by the sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Purified RTA gives a single band in SDS-PAGE, under reduced condition. The molecular weight of purified RTA was estimated to be 30.1 KDa. The western-blot analysis with anti-RTA monoclonal antibody revealed one strong bound in 30 KDa regions.