Utilização da galactomanana de sementes de algaroba (Prosopis juliflora) como matriz cromatográfica de afinidade para purificação de proteínas galactose-ligantes.

Abstract

Carbohydrates are complex biopolymers found in nature with various functions and applications. One of those applications is due to its ability to be specifically recognized by a protein class. Therefore, several carbohydrates may be used as a source to produce chromatographic matrices to purify specific classes of carbohydrate-binding proteins, as some classes of RIPs (Ribosome Inactivating Proteins) and CBM (Carbohydrate-Binding Modules). Knowing the properties of galactomannan, a polysaccharide very common in leguminous seeds, the present work aimed to evaluate the technical viability of galactose binding protein purification using a galactomannan affinity chromatographic matrix of mesquite seeds (Prosopis juliflora), a widespread species in the Brazilian northeastern semiarid. The pods were immersed in water and mildly ground to facilitate capsule separation in order to produce a powder. The carbohydrate was isolated by ethanol precipitation and then centrifuged and lyophilized. Powder containing refined galactomannan was used for reticulation with epichlorohydrin, resulting in a chromatographic matrix production. In order to prove its functionality the chromatographic matrix was tested using total crude extract of Artocarpus incisa, Artocarpus integrifolia, Abrus precatorius and Ricinus communis, through purification of Frutalin, Jacalin, Abrin and Ricin, respectively. To certify purification efficiency and functional integrity of these proteins, they were all evaluated by polyacrylamide gel electrophoresis and haemagglutination assay. Visualization of electrophoretic profile confirmed the purification of the cited molecules, using Comassie Brilliant Blue R-250 dying. For the functional assay, it was seen that the all purified proteins presented expected haemagglutination activity. Even though the matrix showed to be efficient for purification of all proposed molecules, other assays comparing yield, purity and specific activity of the proteins compared to commercial methodologies are necessary, aiming the development of a more refined biotechnological product.