Characterization of cell-free protein synthesis system and theapplication for the synthesis of cell membrane protein

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We reconstructed the membrane protein for the mimicking the cell function. During the replication processes, coat proteins of Pf3 phage is synthesized in the bacteria and inserted into bacteria membrane. Since one mutant of coat protein Pf3 phages, 3L-Pf3, having longer hydrophobic region could be inserted into lipid bilayer membrane even in the absence of membrane proteins, studies on the interaction of coat protein of bacterial phages with lipid bilayer membrane are expected to fundamental principles on the formation of membrane protein. Using 3L-Pf3 as a template, we prepared two mutants of 3L-DR and 3L-RD. Modification of the peptide with His-tag enabled the simple and rapid purification of the synthesized peptide, and those with T7and Myc-tags enabled clear determination of their topologies in the liposomal membrane. The two peptides were found to be spontaneously and directionally inserted into negatively charged large unilamellar vesicle, according to the charge distribution in the peptides. Substitution of amino acids in a peptide caused remarkable differences in their immunoreactivities with antidodies. Observed differences in immunoreactivities among the peptides were not due to the differences in efficiencies of their transfer onto nitrocellulose or PVDF membranes. Rather, possible folding of the peptide on the membrane was considered to be the reason for their distinct immunoreactivities with the antibodies.