Semisynthetic tRNA Complement Mediates in Vitro Protein Synthesis

Abstract

Genetic code expansion is a key objective of synthetic biology and protein engineering. Most efforts in this direction are focused on reassigning termination or decoding quadruplet codons. While the redundancy of genetic code provides a large number of potentially reassignable codons, their utility is diminished by the inevitable interaction with cognate aminoacyl-tRNAs. To address this problem, we sought to establish an in vitro protein synthesis system with a simplified synthetic tRNA complement, thereby orthogonalizing some of the sense codons. This quantitative in vitro peptide synthesis assay allowed us to analyze the ability of synthetic tRNAs to decode all of 61 sense codons. We observed that, with the exception of isoacceptors for Asn, Glu, and Ile, the majority of 48 synthetic Escherichia coli tRNAs could support protein translation in the cell-free system. We purified to homogeneity functional Asn, Glu, and Ile tRNAs from the native E. coli tRNA mixture, and by combining them with synthetic tRNAs, we formulated a semisynthetic tRNA complement for all 20 amino acids. We further demonstrated that this tRNA complement could restore the protein translation activity of tRNA-depleted E. coli lysate to a level comparable to that of total native tRNA. To confirm that the developed system could efficiently synthesize long polypeptides, we expressed three different sequences coding for superfolder GFP. This novel semisynthetic translation system is a powerful tool for tRNA engineering and potentially enables the reassignment of at least 9 sense codons coding for Ser, Arg, Leu, Pro, Thr, and Gly.

Repurposing the translation apparatus for synthetic biology

“…For instance, translation systems reconstituted from purified components [56] or crude cell extracts depleted of native tRNAs [57] can be selectively supplied with purified tRNAs to essentially create a custom genetic code with select sense codons left ‘blank’ for nsAA reassignment [58,59]. Alternatively, in a recent effort a 50S ribosomal subunit was developed with mutations at the peptidyl transferase center that abolish its ability to use several native tRNAs for translation [60].…”

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

“…A 60 min RNase A treatment resulted in an average of 99.3% of tRNA degraded for all tRNA assessed, with >99.5% degradation for Ala(GCC), Arg(CGG), Glu(GAA), and Val(GTA) tRNA. Previous methods to remove tRNA involving ethanol–Sepharose matrix achieved lower depletion efficiencies that varied widely for different tRNAs, with an average of 62% depletion and standard deviation of 28% . In addition, the reported depletion efficiencies were measured indirectly via protein synthesis activity.…”

“…Such efficient tRNA depletion is essential to reduce the competition between the native and synthetic coding elements for high fidelity codon reassignment. The presented method is compatible with various approaches of synthetic tRNA production, including in vitro transcription of T7‐promoted tRNA, T7‐promoted hammerhead Ribozyme–tRNA, and flexizyme . Additionally, for the first time, we measured residual tRNA directly using quantitative real‐time PCR, and report the inaccuracy of assessing tRNA removal by protein expression levels only.…”

“…An impressive recent study illustrated the potential of a cell‐free protein synthesis system as a platform to reengineer the codon–tRNA–amino acid relationship by demonstrating the decoding of 61 sense codons by synthetic tRNAs at various efficiencies . The emancipation of multiple sense codons, however, was limited due to low removal efficiency of native tRNA by chromatography, at an average of 62% of a specific tRNA removed . The PURE system, where individually purified transcription/translation components are combined, also has the potential to emancipate codons, but the monetary high cost severely limits its utility .…”

Creating a completely “cell‐free” system for protein synthesis

“…[2][3][4][5][6] The most robust and highest yielding CFPS systems are based on the soluble portion of cell lysates from Escherichia coli, outperforming the expensive systems with individually purified components by >50% in protein production. [7][8][9] To produce these systems, E. coli is grown, harvested by centrifugation, lysed, and finally centrifuged to remove superfluous cellular debris. The resulting supernatant is collected as the final cell-extract.…”