Over-expression and purification of recombinant Arabidopsis VTE3 and VTE4 involved in tocopherol biosynthesis
Vitamin E is the collective term for a group of eight lipid soluble antioxidants, the tocochromanols, synthesized exclusively in photosynthetic organisms. Tocochromanols, comprised of tocopherols and tocotrienols, are essential dietary nutrients for humans and other mammals. Studies have shown that tocochromanols act as antioxidants and free radical scavengers. Tocopherol biosynthetic enzymes and their corresponding genes have been characterized. However, little is known about the biochemical regulation and the organization of the tocopherol biosynthetic pathway. Tocopherol biosynthetic enzymes, the methyltransferases VTE3 and VTE4, are thought to localize to the inner envelope membrane of chloroplasts. Presented here is a successful strategy for expression of highly hydrophobic 'Arabidopsis' VTE3 and VTE4 methyltransferases, using an 'Escherichia coli' cell-free expression system. Additionally, cell-free expression of VTE3 and VTE4 in the presence of liposomes promoted an association of recombinant proteins with the liposomes, providing an excellent tool for future protein-protein interaction studies with chloroplast lysates.