Advances and Challenges in Cell-Free Incorporation of Unnatural Amino Acids Into Proteins

Abstract

Incorporation of unnatural amino acids (UNAAs) into proteins currently is an active biological research area for various fundamental and applied science. In this context, cell-free synthetic biology (CFSB) has been developed and recognized as a robust testing and biomanufacturing platform for highly efficient UNAA incorporation. It enables the orchestration of unnatural biological machinery toward an exclusive user-defined objective of unnatural protein synthesis. This review aims to overview the principles of cell-free unnatural protein synthesis (CFUPS) systems, their advantages, different UNAA incorporation approaches, and recent achievements. These have catalyzed cutting-edge research and diverse emerging applications. Especially, present challenges and future trends are focused and discussed. With the development of CFSB and the fusion with other advanced next-generation technologies, CFUPS systems would explicitly deliver their values for biopharmaceutical applications.

A bead-based method for the removal of the amino acid lysine from cell-free transcription-translation systems

“…(Pure) expression systems contain only the strictly necessary compounds for gene expression. These compounds are individually expressed, purified and mixed at a later stage (Gao et al, 2019;Singh-Blom et al, 2014). This avoids the presence of residual matter from the cell lysate (in particular amino acids such as lysine).…”

“…Moreover results cannot be transferred to in vivo situations because of the minimalistic constitution of the pure system (Gao et al, 2019;Singh-Blom et al, 2014). The system depends on the bacteriophage T7 RNA polymerase, which leads to high protein yield.…”

“…They include the entire endogenous transcription-translation machinery (Garenne et al, 2019;Schenkelberger et al, 2017;Worst et al, 2015). In principle lysine-free expression systems could be derived from lysine auxotrophic E. coli strains (Gao et al, 2019;Singh-Blom et al, 2014). However, these strains require addition of the depleted amino acid for growth, leading again to the presence of residual amino acids in the final extract.…”

“…There are different methods to remove particular substances from an expression system. Filtering processes separate as a function of different properties, like hydrophobicity, size, charge, however, confounding lysine with other compounds with similar properties (Brödel et al, 2014;Gao et al, 2019). Antibodies or aptamers enable the separation of compounds in a much more specific way.…”

Selenomethionine as an Expressible Handle for Bioconjugations

“…(21,22) To address the issues surrounding natural amino acid specificity, unnatural amino acids can be introduced through auxotrophic expression or amber codon suppression for bioorthogonal conjugations. (23)(24)(25) For example, methionine can be substituted in auxotrophic systems with homologs such as azidohomoalanine and propargyl glycine for copper-catalyzed alkyne-azide cycloadditions (CuAAC, "click" chemistry). In precedent to our current work, Palmer and coworkers labelled selenomethione with a variety of alkyl halides at high concentration but this method has yet to evolve as a protein conjugation strategy.…”