Extraction and purification of the lectin found in the tubers of Eranthis hyemalis (winter aconite)

Abstract

Lectin are proteins which play an important role in the defence mechanisms of plants against the attack of microorganisms and insects: this role has provoked particular interest in the -elds of biotechnology and agriculture. ;is paper describes the extraction and puri-cation of the lectin found in tubers of the winter aconite (Eranthis hyemalis), with the aim of improving and modernising the existing extraction protocol. ;e Eranthis hyemalis lectin (EHL) is a member of the type-2 Ribosome Inactivating Proteins (RIP) family, proteins which have the ability to inhibit in vitro protein synthesis. RIPs have been linked to plant defence by their antiviral, a ntifungal and insecticidal properties, and some have been found to be potent inhibitors of the Human Immunode-ciency Virus-1 (HIV-1) virus. EHL was puri-ed using aQnity column chro matography and ammonium sulphate precipitation; thiourea was used as antioxidant in order to prevent EHL denaturing during the extraction process. ;e presence of EHL in the extract was veri-ed using a blood agglutination test with rabbit erythrocytes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) analysis was employed to determine the lectin size; EHL was found to be formed of two chains with molecular weights of approximately 31 kDa; the size of the whole protein was estimated as approximately 60 kDa. ;e concentration of the EHL in the post-column eluent, determined using the Bradford Assay, was 380.1 μg.cm -3 . ;is improved extraction protocol is the -rst step which will enable future research on the potential use of EHL in crop protection, by studying its insecticidal, fungicidal and bactericidal properties.