Cell-free protein synthesis systems for post-translational modifications

Post-translational modification (PTM) is an important event of the cell. It aids function and behavior to the proteins. It is highly regulated and reported to govern various phenomenon of cells. Moreover, dysfunctionality in PTM also drives diseases development. Approximately 650 types of PTM have been reported so far, of which phosphorylation, methylation, acetylation, ubiquitination, glycosylation, lipidation, citrullination, and SUMOylation are among the most studied till date. Many pharmaceutical valued proteins are also post-translationally modified. In order to investigate or use such polypeptide as therapeutics, homogeneous population of targeted protein with authentic PTM is required. Conventional in vivo approaches pose challenges to produce a population of proteins with correct PTM. However, with the gain of interest to examine the PTM among the researchers across the globe, cell-free protein synthesis (CFPS) platforms emerged as a tool of choice to quench valuable insights. As found in vivo, CFPS mediated PTM of proteins enables the user to acquire the structural and functional properties of protein with ease. Several strategies such as GlycoPRIME for glycosylation, soluble expression strategies such as SIMPLEx, orthogonal translation systems for the site specific phosphoserine insertion, continuous exchange CFPS system for hydroxylation, PURE based systems for acetylation, and cell engineering for robust CFPS mediated disulfide bond formation has been proposed over the years. Current chapter describes such CFPS mediated strategy to produce proteins with PTM. Overall, CFPS mediated approaches provide a controlled and efficient means to produce proteins with defined PTMs, facilitating mechanistic studies and therapeutic applications.