The Application of Cell-Free Protein Synthesis in Genetic Code Expansion for Post-translational Modifications

Abstract

The translation system is a sophisticated machinery that synthesizes proteins from 20 canonical amino acids. Recently, the repertoire of such composition has been expanded by the introduction of non-canonical amino acids (ncAAs) with the genetic code expansion strategy, which provides proteins with designed properties and structures for protein studies and engineering. Although the genetic code expansion strategy has been mostly implemented by using living cells as the host, a number of limits such as poor cellular uptake or solubility of specific ncAA substrates and the toxicity of target proteins have hindered the production of certain ncAA-modified proteins. To overcome those challenges, cell-free protein synthesis (CFPS) has been applied as it allows the precise control of reaction components. Several approaches have been recently developed to increase the purity and efficiency of ncAA incorporation in CFPS. Here, we summarized recent development of CFPS with an emphasis on its applications in generating site-specific protein post-translational modifications by the genetic code expansion strategy.

Automated Cell-Free Multiprotein Synthesis Facilitates the Identification of a Secretory, Oligopeptide Elicitor-Like, Immunoreactive Protein of the Oomycete Pythium insidiosum

“…(i) A plasmid DNA, instead of a PCR product, should be used to express a longer protein-coding sequence (longer than 2 kb). (ii) The posttranslational modification of a synthesized protein can be achieved with a eukaryotic cell extract-based CFPS system prepared from yeast, insect, or rabbit reticulocyte (18,(46)(47)(48)(49). (iii) The baseline protein contamination from the cell extract-based CFPS can be minimized by using the PURE (protein synthesis using recombinant elements) CFPS system, which relies on purified recombinant transcription and translation components (14,50,51).…”

Cell-Free Protein Synthesis: A Promising Option for Future Drug Development

“…Although the naturally occurring PTM machinery is not available in the E. coli lysates, recently proteins with N-glycosylation were synthesized by using E. coli extracts enriched with glycosylation components, including oligosaccharyltransferases (OSTs) and lipid-linked oligosaccharides (LLOs) [41]. Using release factor (RF1) deficient E. coli lysates, proteins were phosphorylated by incorporation of non-canonical amino acids, which will be addressed in a later part of this review [42].…”

“…When non-canonical amino acids (ncAAs) are incorporated into proteins, novel functional, structural, and imaging properties can be generated. This synthetic biology application is fast emerging and has wide applications such as incorporating precise PTMs and adding novel functions to proteins [23,24,42,45]. By taking advantage of the openness of the CFPS, one can add the machinery responsible for the co-translational incorporation of ncAAs directly to the standard reaction components.…”

Cell‐free protein synthesis: The transition from batch reactions to minimal cells and microfluidic devices

“…Notably, genomic material withdrawal from chassis organism bestows circumstances in which desired synthesis reactions are achievable at high rates (Hodgman & Jewett, 2012). Thus, such features enable prevailing utilization of CFPS systems, which are also complementary to in vivo expression for various applications such as biocatalyst development (Rolf, Rosenthal, & Lütz, 2019), complex product fabrication (Chi, Wang, Li, Ren, & Huang, 2015), disease detection (Soltani, Davis, Ford, Nelson, & Bundy, 2018), glycoprotein synthesis (Jaroentomeechai et al, 2018), prototyping minimal cells (Yue, Zhu, & Kai, 2019), synthetic gene networks (Dubuc et al, 2019), as well as protein engineering (Hong, Kwon, & Jewett, 2014; Venkat, Chen, Gan, & Fan, 2019), among others (Table 1 and Figure 1).…”