Regulated N-Terminal Modification of Proteins Synthesized Using a Reconstituted Cell-Free Protein Synthesis System

The N-terminal modificationof nascent proteins, suchas acetylationand myristoylation, is one of the most abundant post-translationalmodifications. To analyze the function of the modification, it isimportant to compare the modified and unmodified proteins under definedconditions. However, it is technically difficult to prepare unmodifiedproteins because cell-based systems contain endogenous modificationsystems. In this study, we developed a cell-free method to conductN-terminal acetylation and myristoylation of nascent proteins in vitro using a reconstituted cell-free protein synthesissystem (PURE system). Proteins synthesized using the PURE system weresuccessfully acetylated or myristoylated in a single-cell-free mixturein the presence of modifying enzymes. Furthermore, we performed proteinmyristoylation in giant vesicles, which resulted in their partiallocalization to the membrane. Our PURE-system-based strategy is usefulfor the controlled synthesis of post-translationally modified proteins.