Cell-free protein synthesis is advantageous to structural biology. We have established protein preparation techniques, using the Escherichia coli cell-free protein synthesis system, for the large-scale production of difficult targets suitable for structure determination by X-ray crystallography. The quantity and the quality of the cell-free expressed proteins were generally better than those of the proteins expressed in conventional cell-based systems, because various parameters can be more precisely controlled. Cell-free protein synthesis in the presence of detergents and/or lipids is suitable for the expression of integral membrane proteins. Microbial rhodopsins were prepared by the cell-free methods and crystallized by the lipidic cubic phase method. Their crystal structures have been solved by X-ray crystallography. Furthermore, human GPCRs were prepared by the cell-free method, and their ligand binding activities indicated that these cell-free synthesized GPCRs were functional. Monoclonal antibodies were raised against the cell-free synthesized GPCRs, and some exhibited tertiary structure-dependent binding to the antigens. Another example is the human claudins, which form the tight junction strands to seal intercellular spaces. Many members of this family were prepared by the cell-free synthesis method. Claudin-4 prepared by the cell-free method exhibited tight binding to a bacterial toxin, which disrupts the claudin assembly in the tight junction. The crystal structure of human claudin-4 in complex with the bacterial toxin was determined by X-ray crystallography, and the mechanism of tight-junction disruption was revealed.