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.

Automated summary

The study developed a cell-free method using a reconstituted cell-free protein synthesis system (PURE system) to perform N-terminal acetylation and myristoylation of nascent proteins in vitro. The proteins synthesized using the PURE system were successfully modified in the presence of modifying enzymes. The study also demonstrated protein myristoylation in giant vesicles, leading to partial localization to the membrane. The PURE-system-based strategy is useful for controlled synthesis of post-translationally modified proteins.