Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
Proc Natl Acad Sci U S A. 2012 Jul 24;109(30):11993-8. doi: 10.1073/pnas.1205427109. Epub 2012 Jul 9.
Standard genetic approaches allow the production of protein composites by fusion of polypeptides in head-to-tail fashion. Some applications would benefit from constructions that are genetically impossible, such as the site-specific linkage of proteins via their N or C termini, when a remaining free terminus is required for biological activity. We developed a method for the production of N-to-N and C-to-C dimers, with full retention of the biological activity of both fusion partners and without inflicting chemical damage on the proteins to be joined. We use sortase A to install on the N or C terminus of proteins of interest the requisite modifications to execute a strain-promoted copper-free cycloaddition and show that the ensuing ligation proceeds efficiently. Applied here to protein-protein fusions, the method reported can be extended to connecting proteins with any entity of interest.
标准的遗传方法允许通过多肽的头到尾融合来生产蛋白质复合物。有些应用需要通过其 N 或 C 末端进行蛋白质的定点连接等在遗传上不可能的构建，当需要生物活性时，留下一个游离末端。我们开发了一种用于生产 N-N 和 C-C 二聚体的方法，完全保留了融合伙伴的生物活性，并且不会对要连接的蛋白质造成化学损伤。我们使用 sortase A 在感兴趣的蛋白质的 N 或 C 末端安装必要的修饰以执行应变促进的无铜环加成反应，并表明随后的连接过程高效进行。这里应用于蛋白质-蛋白质融合，所报道的方法可以扩展到连接任何感兴趣的蛋白质实体。
