Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis

• Published in: Biochimica et biophysica acta Vol. 1861; no. 11; pp. 3047 - 3052
• Main Authors: Gan, Qinglei, Fan, Chenguang
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.11.2017
• Subjects: amino acids; Amino Acids - metabolism; Amino Acyl-tRNA Synthetases - metabolism; Cell-free protein synthesis; Cell-Free System; Codon; Escherichia coli; Genetic Code; Genetic code expansion; mass spectrometry; Near cognate suppression; Noncanonical amino acid; Phosphoserine; Protein Biosynthesis; Protein Engineering - methods; proteins; Ribosomes - metabolism; RNA, Transfer - metabolism; stop codon; synthetic biology; transfer RNA; tRNA isoacceptor; Genetic code expansion; tRNA isoacceptor; Cell-free protein synthesis; Noncanonical amino acid; Phosphoserine; Near cognate suppression
• ISSN: 0304-4165; 0006-3002; 1872-8006; 1878-2434
• DOI: 10.1016/j.bbagen.2016.12.002

Cell-free protein synthesis provides a robust platform for co-translational incorporation of noncanonical amino acid (ncAA) into proteins to facilitate biological studies and biotechnological applications. Recently, eliminating the activity of release factor 1 has been shown to increase ncAA incorporation in response to amber codons. However, this approach could promote mis-incorporation of canonical amino acids by near cognate suppression. We performed a facile protocol to remove near cognate tRNA isoacceptors of the amber codon from total tRNAs, and used the phosphoserine (Sep) incorporation system as validation. By manipulating codon usage of target genes and tRNA species introduced into the cell-free protein synthesis system, we increased the fidelity of Sep incorporation at a specific position. By removing three near cognate tRNA isoacceptors of the amber stop codon [tRNALys, tRNATyr, and tRNAGln(CUG)] from the total tRNA, the near cognate suppression decreased by 5-fold without impairing normal protein synthesis in the cell-free protein synthesis system. Mass spectrometry analyses indicated that the fidelity of ncAA incorporation was improved. Removal of near cognate tRNA isoacceptors of the amber codon could increase ncAA incorporation fidelity towards the amber stop codon in release factor deficiency systems. We provide a general strategy to improve fidelity of ncAA incorporation towards stop, quadruplet and sense codons in cell-free protein synthesis systems. This article is part of a Special Issue entitled “Biochemistry of Synthetic Biology - Recent Developments" Guest Editor: Dr. Ilka Heinemann and Dr. Patrick O’Donoghue. •Near cognate suppression of amber, opal, and ochre stop codons were examined in the PURE system.•A facile protocol of inhibiting RF1 activity in PURE systems was performed.•A facile protocol of removing target tRNA isoacceptors from total tRNAs was performed.•The purity of phosphoserine incorporation was increased by changing codon usage and tRNA species input.