Acquisition of Protein Purification Instrumentation for Isoflavonoid Research

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This award supports the acquisition of a protein chromatography system and associated protein purification and analysis equipment to be used in research activities and undergraduate education at Southeastern Oklahoma State University. The instrumentation will be used to study isoflavonoids and isoflavonoid conjugates which are natural products produced in many legume plant species. Isoflavonoids have been proposed to play several roles in plant survival, including defense against fungal pathogens and recruitment of beneficial nitrogen-fixing bacteria. Specific enzymatic hydrolysis of isoflavonoid conjugates may be essential during these processes. Isoflavonoids are also potentially important constituents of human foods, in that diets rich in isoflavonoids are thought to decrease rates of certain cancers, osteoporosis, and other diseases. Hydrolysis of isoflavonoid conjugates before or during digestion may greatly affect the uptake, bioavailability and stability of isoflavonoids. Despite their potential importance, little has been published on beta-glucosidases which cleave isoflavonoid beta-glucosides. The instrumentation will facilitate the biochemical analysis of plant beta-glucosidases with high specificity towards isoflavonoid conjugates, for which clones were recently isolated from Medicago truncatula. Conditions will be investigated for expression and purification of large amounts of recombinant enzymes for use in animal diet studies designed to determine the relative importance of isoflavonoid conjugate hydrolysis in nutrition and disease prevention. The instrumentation will also allow the purification and eventual cloning of the enzyme 7,2'-dihydroxy-4'-methoxy-isoflavanol dehydratase (DMID). This enzyme catalyzes the final step in the biosynthesis of the antifungal isoflavonoid medicarpin, and may be involved in the biosynthesis of other important isoflavonoid derivatives such as coumestans. Acquisition of this instrumentation will also facilitate the training of students in current biochemical and molecular biology techniques, and will directly involve undergraduates in functional genomics research. The instrumentation will be extremely beneficial to revisions of the existing Biochemistry laboratory courses and the initiation of a new Advanced Protein Techniques half-course, for biology, chemistry and biotechnology majors. Southeastern Oklahoma State University serves a diverse student body, including Native American students, students from rural areas, and first generation college students. The instrumentation will aid in preparing these students for biochemistry, biotechnology and interdisciplinary research careers.