Purification of Aster Yellows Agent from Diseased Lettuce Using Affinity Chromatography

Abstract

An affinity chromatographic procedure was developed for purification of the mycoplasmalike organism (MLO) from lettuce with aster yellows (AY) disease. The affinity column consisted of Staphylococcus protein A covalently linked to a 6MB Sepharose matrix and coupled with monoclonal antibody specific against AY-MLO. AY-MLO was concentrated from clarified crude sap of diseased lettuce by differential centrifugation. After a low-speed centrifugation, supernatants containing AY-MLO were loaded on the affinity column. Extraneous unbound plant materials from the crude sap were washed from the column after an incubation period. The aster yellows agent retained in the column was released by mechanical shaking and then eluted. The entire procedure took 3 hr to complete. Intact and undamaged cells were observed by electron microscopy, and the high purity of this preparation was revealed by electrophoretic protein profiles and the production of polyclonal AY-antiserum. In addition, the specificity of the monoclonal antibodies was confirmed in Western blots.