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

Abstract

The N-terminal modification of nascent proteins, such as acetylation and myristoylation, is one of the most abundant post-translational modifications. To analyze the function of the modification, it is important to compare the modified and unmodified proteins under defined conditions. However, it is technically difficult to prepare unmodified proteins because cellbased systems contain endogenous modification systems. In this study, we developed a cell-free method to conduct N-terminal acetylation and myristoylation of nascent proteins in vitro using a reconstituted cell-free protein synthesis system (PURE system). Proteins synthesized using the PURE system were successfully acetylated or myristoylated in a single-cell-free mixture in the presence of modifying enzymes. Furthermore, we performed protein myristoylation in giant vesicles, which resulted in their partial localization to the membrane. Our PURE-system-based strategy is useful for the controlled synthesis of post-translationally modified proteins.

N-Terminal Amino Acid Affects the Translation Efficiency at Lower Temperatures in a Reconstituted Protein Synthesis System

“…The major advantage of the system is that it consists only of purified factors that are required for translation, and it is devoid of nucleases, proteases, and other unrelated factors, such as metabolic enzymes [1]. The PURE system is currently used not only for studying the mechanism of translation and protein folding [2][3][4][5][6][7][8], but also in synthetic biology and protein engineering applications [9][10][11][12][13][14][15][16]. The yield of the proteins synthesized using the PURE system was initially lower than that of the E. coli S30 extract system, but it has now increased to 1 mg/mL following optimization of the reaction composition [17].…”