Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation

We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9–1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50–88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNAopt) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.

[1]  Y. Li,et al.  A modified procedure for fast purification of T7 RNA polymerase. , 1999, Protein expression and purification.

[2]  Kedar G. Patel,et al.  Surface functionalization of virus-like particles by direct conjugation using azide-alkyne click chemistry. , 2011, Bioconjugate chemistry.

[3]  C. Hilty,et al.  Genetic incorporation of twelve meta-substituted phenylalanine derivatives using a single pyrrolysyl-tRNA synthetase mutant. , 2013, ACS chemical biology.

[4]  J. Swartz,et al.  Total amino acid stabilization during cell-free protein synthesis reactions. , 2006, Journal of biotechnology.

[5]  Cem Albayrak,et al.  Using E. coli-based cell-free protein synthesis to evaluate the kinetic performance of an orthogonal tRNA and aminoacyl-tRNA synthetase pair. , 2013, Biochemical and biophysical research communications.

[6]  G. Hong,et al.  Nucleic Acids Research , 2015, Nucleic Acids Research.

[7]  Yane-Shih Wang,et al.  A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum. , 2012, Journal of the American Chemical Society.

[8]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[9]  T. Terwilliger,et al.  Engineering and characterization of a superfolder green fluorescent protein , 2006, Nature Biotechnology.

[10]  Shigeyuki Yokoyama,et al.  Codon reassignment in the Escherichia coli genetic code , 2010, Nucleic acids research.

[11]  Thomas Huber,et al.  Multiple-site labeling of proteins with unnatural amino acids. , 2012, Angewandte Chemie.

[12]  P G Schultz,et al.  Expanding the Genetic Code of Escherichia coli , 2001, Science.

[13]  J. Swartz,et al.  Development of cell‐free protein synthesis platforms for disulfide bonded proteins , 2008, Biotechnology and bioengineering.

[14]  Ryohei Ishii,et al.  Multistep engineering of pyrrolysyl-tRNA synthetase to genetically encode N(epsilon)-(o-azidobenzyloxycarbonyl) lysine for site-specific protein modification. , 2008, Chemistry & biology.

[15]  Peter G Schultz,et al.  Efficient incorporation of unnatural amino acids into proteins in Escherichia coli , 2006, Nature Methods.

[16]  J. Cooke,et al.  Cell-free production of transducible transcription factors for nuclear reprogramming. , 2009, Biotechnology and bioengineering.

[17]  Probing Protein Structure and Function with an Expanded Genetic Code , 1995 .

[18]  Dominic Esposito,et al.  A novel cell-free protein synthesis system. , 2004, Journal of biotechnology.

[19]  P. Schultz,et al.  The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination , 2004, Nature Biotechnology.

[20]  J. Stapleton,et al.  Cell‐free synthesis and maturation of [FeFe] hydrogenases , 2008, Biotechnology and bioengineering.

[21]  Farren J. Isaacs,et al.  Precise Manipulation of Chromosomes in Vivo Enables Genome-Wide Codon Replacement , 2011, Science.

[22]  Nediljko Budisa,et al.  Recent advances in genetic code engineering in Escherichia coli. , 2012, Current opinion in biotechnology.

[23]  M. Yarus,et al.  The context effect does not require a fourth base pair. , 1986, Science.

[24]  Peter G Schultz,et al.  Adding new chemistries to the genetic code. , 2010, Annual review of biochemistry.

[25]  P G Schultz,et al.  Biosynthetic method for introducing unnatural amino acids site-specifically into proteins. , 1991, Methods in enzymology.

[26]  C. J. Murray,et al.  Microscale to Manufacturing Scale-up of Cell-Free Cytokine Production—A New Approach for Shortening Protein Production Development Timelines , 2011, Biotechnology and bioengineering.

[27]  J. Swartz,et al.  An Economical Method for Cell‐Free Protein Synthesis using Glucose and Nucleoside Monophosphates , 2008, Biotechnology progress.

[28]  M. Jewett,et al.  Mimicking the Escherichia coli cytoplasmic environment activates long‐lived and efficient cell‐free protein synthesis , 2004, Biotechnology and bioengineering.

[29]  C. Kurland,et al.  The concentration of polypeptide chain release factors 1 and 2 at different growth rates of Escherichia coli. , 1994, Journal of molecular biology.

[30]  J. Swartz,et al.  High yield cell-free production of integral membrane proteins without refolding or detergents. , 2008, Biochimica et biophysica acta.

[31]  Nediljko Budisa,et al.  Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. , 2004, Angewandte Chemie.

[32]  E. Lemke,et al.  Genetically Encoded Copper-Free Click Chemistry , 2011, Angewandte Chemie.

[33]  Karthish Manthiram,et al.  Multiply mutated Gaussia luciferases provide prolonged and intense bioluminescence. , 2009, Biochemical and biophysical research communications.

[34]  S. Levy,et al.  Cell-free production of Gaussia princeps luciferase--antibody fragment bioconjugates for ex vivo detection of tumor cells. , 2009, Biochemical and biophysical research communications.

[35]  J. Kur,et al.  Cloning and expression in Escherichia coli of the recombinant his-tagged DNA polymerases from Pyrococcus furiosus and Pyrococcus woesei. , 1998, Protein expression and purification.

[36]  Peter G Schultz,et al.  An enhanced system for unnatural amino acid mutagenesis in E. coli. , 2010, Journal of molecular biology.

[37]  Peter G Schultz,et al.  Unnatural amino acid mutagenesis of green fluorescent protein. , 2003, The Journal of organic chemistry.

[38]  H. Toh,et al.  Organization and nucleotide sequence of the DNA polymerase gene from the archaeon Pyrococcus furiosus. , 1993, Nucleic acids research.

[39]  James R. Swartz,et al.  Site-specific incorporation of p-propargyloxyphenylalanine in a cell-free environment for direct protein-protein click conjugation. , 2010, Bioconjugate chemistry.

[40]  P. Schultz,et al.  In vivo incorporation of an alkyne into proteins in Escherichia coli. , 2005, Bioorganic & medicinal chemistry letters.

[41]  J. Chin,et al.  Genetic encoding and labeling of aliphatic azides and alkynes in recombinant proteins via a pyrrolysyl-tRNA Synthetase/tRNA(CUA) pair and click chemistry. , 2009, Journal of the American Chemical Society.

[42]  G. F. Short,et al.  Effects of release factor 1 on in vitro protein translation and the elaboration of proteins containing unnatural amino acids. , 1999, Biochemistry.

[43]  Andrew B. Martin,et al.  Addition of a photocrosslinking amino acid to the genetic code of Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[44]  R. Giegé,et al.  Evolution of the tRNA(Tyr)/TyrRS aminoacylation systems. , 2005, Biochimie.

[45]  P. Schultz,et al.  Evolution of amber suppressor tRNAs for efficient bacterial production of proteins containing nonnatural amino acids. , 2009, Angewandte Chemie.

[46]  Andrew B. Martin,et al.  Addition of p-azido-L-phenylalanine to the genetic code of Escherichia coli. , 2002, Journal of the American Chemical Society.

[47]  Michael C Jewett,et al.  An integrated cell-free metabolic platform for protein production and synthetic biology , 2008, Molecular systems biology.

[48]  P. Schultz,et al.  Addition of the keto functional group to the genetic code of Escherichia coli , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[49]  J. Miller,et al.  Effects of surrounding sequence on the suppression of nonsense codons. , 1983, Journal of molecular biology.

[50]  W. Mcallister,et al.  Studies of promoter recognition and start site selection by T7 RNA polymerase using a comprehensive collection of promoter variants. , 2000, Biochemistry.

[51]  Bradley C. Bundy,et al.  Escherichia coli‐based cell‐free synthesis of virus‐like particles , 2008, Biotechnology and bioengineering.

[52]  R. Giegé,et al.  Ribozyme processed tRNA transcripts with unfriendly internal promoter for T7 RNA polymerase: production and activity , 1998, FEBS letters.

[53]  T. D. Schneider,et al.  Quantitative analysis of the relationship between nucleotide sequence and functional activity. , 1986, Nucleic acids research.

[54]  S. Mottagui-Tabar,et al.  The second to last amino acid in the nascent peptide as a codon context determinant. , 1994, The EMBO journal.

[55]  Matthew D. Schultz,et al.  RF1 Knockout Allows Ribosomal Incorporation of Unnatural Amino Acids at Multiple Sites , 2011, Nature chemical biology.

[56]  J. F. Curran,et al.  Effects of the nucleotide 3' to an amber codon on ribosomal selection rates of suppressor tRNA and release factor-1. , 1991, Journal of molecular biology.

[57]  Jennifer R Cochran,et al.  Discovery of improved EGF agonists using a novel in vitro screening platform. , 2011, Journal of molecular biology.

[58]  J. Swartz,et al.  Cell‐free synthesis of functional aquaporin Z in synthetic liposomes , 2009, Biotechnology and bioengineering.

[59]  Chia-Wei Wang,et al.  Simultaneous expression and maturation of the iron‐sulfur protein ferredoxin in a cell‐free system , 2006, Biotechnology and bioengineering.

[60]  P. Schultz,et al.  An efficient system for the evolution of aminoacyl-tRNA synthetase specificity , 2002, Nature Biotechnology.

[61]  Brian A. Smith,et al.  A new strategy for the site-specific modification of proteins in vivo. , 2003, Biochemistry.

[62]  P. Schultz,et al.  Adding amino acids with novel reactivity to the genetic code of Saccharomyces cerevisiae. , 2003, Journal of the American Chemical Society.

[63]  P. Schultz,et al.  Beyond the Canonical 20 Amino Acids: Expanding the Genetic Lexicon* , 2010, The Journal of Biological Chemistry.

[64]  T. Terwilliger,et al.  Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein , 2005, Nature Biotechnology.

[65]  James R. Swartz,et al.  High‐level cell‐free synthesis yields of proteins containing site‐specific non‐natural amino acids , 2009, Biotechnology and bioengineering.

[66]  James Swartz,et al.  Amino acid stabilization for cell-free protein synthesis by modification of the Escherichia coli genome. , 2004, Metabolic engineering.

[67]  L. Isaksson,et al.  Only the last amino acids in the nascent peptide influence translation termination in Escherichia coli genes , 1997, FEBS letters.

[68]  W. Scott,et al.  Tertiary Contacts Distant from the Active Site Prime a Ribozyme for Catalysis , 2006, Cell.

[69]  T. Muir,et al.  Genetically encoded 1,2-aminothiols facilitate rapid and site-specific protein labeling via a bio-orthogonal cyanobenzothiazole condensation. , 2011, Journal of the American Chemical Society.

[70]  David H Russell,et al.  A facile system for genetic incorporation of two different noncanonical amino acids into one protein in Escherichia coli. , 2010, Angewandte Chemie.

[71]  Y. Aoyama,et al.  In vitro selection of RNA aptamer against Escherichia coli release factor 1. , 2007, Bioorganic & medicinal chemistry letters.

[72]  P. Schultz,et al.  A general approach for the generation of orthogonal tRNAs. , 2001, Chemistry & biology.

[73]  Shigeyuki Yokoyama,et al.  Structural basis for orthogonal tRNA specificities of tyrosyl-tRNA synthetases for genetic code expansion , 2003, Nature Structural Biology.

[74]  M. Phillips-Jones,et al.  The 3' codon context effect on UAG suppressor tRNA is different in Escherichia coli and human cells. , 1993, Journal of molecular biology.

[75]  J. Kur,et al.  Cloning and Expression inEscherichia coliof the Recombinant His-Tagged DNA Polymerases fromPyrococcus furiosusandPyrococcus woesei , 1998 .