Genetically expanded cell‐free protein synthesis using endogenous pyrrolysyl orthogonal translation system

Abstract

Cell-free protein synthesis offers a facile and rapid method for synthesizing, monitoring, analyzing, and purifying proteins from a DNA template. At the same time, genetic code expansion methods are gaining attention due to their ability to site-specifically incorporate unnatural amino acids (UAAs) into proteins via ribosomal translation. These systems are based on the exogenous addition of an orthogonal translation system (OTS), comprising an orthogonal tRNA, and orthogonal aminoacyl tRNA synthetase (aaRS), to the cell-free reaction mixture. However, these components are unstable and their preparation is labor-intensive, hence introducing a major challenge to the system. Here, we report on an approach that significantly reduces the complexity, effort and time needed to express UAA-containing proteins while increasing stability and realizing maximal suppression efficiency. We demonstrate an endogenously introduced orthogonal pair that enables the use of the valuable yet insoluble pyrrolysyl-tRNA synthetase in a cell-free system, thereby expanding the genetic repertoire that can be utilized in vitro and enabling new possibilities for bioengineering. With the high stability and efficiency of our system, we offer an improved and accessible platform for UAA incorporation into proteins.

Genetic Code Expansion by Degeneracy Reprogramming of Arginyl Codons

“…Chemla et al. reported a similar yield (∼200 μg mL −1 ) in their synthesis of a GFP mutant with propargyllysine at a single position by reprogramming the amber codon in a CFPS system deficient in RF1 . And a similar yield was reported by Hong et al.…”

Cell-sized mechanosensitive and biosensing compartment programmed with DNA

“…G-GECO is dim in the absence of Ca 2+ and bright when bound to Ca 2+ with a Ca 2+ -dependent fluorescence increase of ~23–26 fold (Fig. 4A) 7 . We cloned G-GECO under the P70a promoter and verified that it can sense Ca 2+ in a plate reader assay (Fig.…”

“…Modern TXTL platforms are used for medicine and biomolecular manufacturing, such as the production of vaccine and therapeutics 5, 6 . By performing non-natural chemistries 3, 7, 8 , the TXTL technology has been improved to expand the molecular repertoire of biological systems. Because the time for design-build-test cycle is dramatically reduced, TXTL has become a powerful platform to rapidly prototype genetic programs in vitro , from testing single regulatory elements to recapitulating metabolic pathways 9 .…”

Residue-specific Incorporation of Noncanonical Amino Acids into Model Proteins Using an <em>Escherichia coli</em> Cell-free Transcription-translation System

“…A slight modification of the same system enabled site-specific incorporation of different pyrrolysine analogs into a model protein via stop codon suppression 33 . The employed cell-free system [31][32][33] is based on an all E. coli transcription-translation system.…”

“…A slight modification of the same system enabled site-specific incorporation of different pyrrolysine analogs into a model protein via stop codon suppression 33 . The employed cell-free system [31][32][33] is based on an all E. coli transcription-translation system. Nevertheless, it enables protein expression as efficiently as in current bacteriophage systems (0.5 -1 mg/ml of recombinant protein) 32 , while retaining much of the original transcription-translation modularity.…”