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

为了模拟细胞功能，我们重构了膜蛋白。在复制过程中，Pf 3噬菌体的外壳蛋白在细菌中合成并插入细菌膜。由于细菌外壳蛋白Pf 3的一个突变体3L-Pf 3具有更长的疏水区，即使在没有膜蛋白的情况下也能插入到脂双层膜中，因此研究细菌外壳蛋白与脂双层膜的相互作用有望为膜蛋白的形成奠定基础。以3L-Pf 3为模板，制备了3L-DR和3L-RD两种突变体。用His标签修饰的肽能够简单快速地纯化合成的肽，而用T7和Myc标签修饰的肽能够清楚地确定它们在脂质体膜中的拓扑结构。根据肽中的电荷分布，发现这两个肽自发地定向插入带负电荷的大单层囊泡中。肽中氨基酸的取代引起它们与解毒剂的免疫反应性的显着差异。观察到的肽之间的免疫反应性的差异不是由于其转移到硝酸纤维素或PVDF膜的效率的差异。相反，肽在膜上的可能折叠被认为是它们与抗体的不同免疫反应性的原因。

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.