Abstract
The genomes of higher plants encode many membrane proteins. Some of the membrane proteins play important roles in biosynthesis and accumulation of useful secondary metabolites. They function as enzymes, catalyzing reaction in biosynthetic pathways, or as transporters of metabolites across membranes. Precise biochemical analyses of these membrane proteins are necessary for elucidating molecular mechanism of biosynthesis and accumulation of useful metabolites. Lately, cell-free protein synthesis systems have been applied to preparation of various membrane proteins for structural and functional analyses. We attempted to synthesize and reconstitute membrane-bound enzymes by wheat germ cell-free protein synthesis system. Cell-free syntheses of enzymes with a membrane-binding domain in their N-termini were performed using wheat-germ extracts in the presence or absence of liposomes. In the case of cofactor-dependent enzymes, optimum concentrations of each cofactor in the reaction mixture for cell-free translation were determined. We have succeeded in cell-free reconstitution of several membrane-bound enzymes.
