ISOLATION AND PURITY DETERMINATION OF A GLYCOPROTEIN ELICITOR FROM WHEAT-STEM RUST BY MEDIUM-PRESSURE LIQUID-CHROMATOGRAPHY

Previously the isolation of a single glycoprotein (pgt-elicitor) from cell walls of the phytopathogenic fungus Puccinia graminis f.sp. tritici Erics. & Henn., which elicits defence reactions in wheat leaves was described. The apparent molecular mass of this compound, isolated via concanavalin A affinity chromatography and anion-exchange chromatography, was 67 000 as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis. A scaled-up procedure for purifying larger amounts of fungal elicitor based on anion-exchange fast protein liquid chromatography (FPLC) with Q-Sepharose Fast Flow medium has now been developed. The yield of pure elicitor was further increased by the use of the volatile buffer ammonium carbonate for eluting the bound glycoproteins. Analytical size-exclusion FPLC supplemented gel electrophoretic results by quantifying small amounts of inactive components contaminating the most active elicitor fraction. Further, purity determination by FPLC provided evidence of well defined complex formation leading to co-migration of elicitor-associated glycoproteins during anion-exchange chromatography. The glycoprotein complexes were separated during size-exclusion FPLC at lowered pH (100 mM acetic acid). Thus, size exclusion FPLC resulted in final purification of the pgt-elicitor. © 1990.