Liposome‐Based in Vitro Evolution of Aminoacyl‐tRNA Synthetase for Enhanced Pyrrolysine Derivative Incorporation

Methanosarcina species pyrrolysyl‐tRNA synthetase (PylRS) attaches Pyl to its cognate amber suppressor tRNA. The introduction of two mutations (Y384F and Y306A) into PylRS was previously shown to generate a mutant, designated LysZ‐RS, that was able to attach N‐benzyloxycarbonyl‐L‐lysine (LysZ) to its cognate tRNA. Despite the potential of LysZ derivatives, further LysZ‐RS engineering has not been performed; consequently, we aimed to generate LysZ‐RS mutants with improved LysZ incorporation activity through in vitro directed evolution. Using a liposome‐based in vitro compartmentalization (IVC) approach, we screened a randomly mutagenized gene library of LysZ‐RS and obtained a mutant that showed increased LysZ incorporation activity both in vitro and in vivo. The ease and high flexibility of liposome‐based IVC should enable the evolution of not only LysZ‐RS that can attach various LysZ derivatives but also of other enzymes involved in protein translation.

[1]  T. Yomo,et al.  Synthesis of milligram quantities of proteins using a reconstituted in vitro protein synthesis system. , 2014, Journal of bioscience and bioengineering.

[2]  G. Roelfes,et al.  Novel artificial metalloenzymes by in vivo incorporation of metal-binding unnatural amino acids , 2017 .

[3]  T. Yomo,et al.  Identification of giant unilamellar vesicles with permeability to small charged molecules , 2014 .

[4]  Tetsuya Yomo,et al.  Liposome display for in vitro selection and evolution of membrane proteins , 2014, Nature Protocols.

[5]  Jeffery M. Tharp,et al.  Pyrrolysyl-tRNA synthetase: an ordinary enzyme but an outstanding genetic code expansion tool. , 2014, Biochimica et biophysica acta.

[6]  Martin Fischlechner,et al.  One in a Million: Flow Cytometric Sorting of Single Cell-Lysate Assays in Monodisperse Picolitre Double Emulsion Droplets for Directed Evolution , 2014, Analytical chemistry.

[7]  Andrew D. Ellington,et al.  Directed evolution of genetic parts and circuits by compartmentalized partnered replication , 2013, Nature Biotechnology.

[8]  Adam R Abate,et al.  Ultrahigh-throughput sorting of microfluidic drops with flow cytometry. , 2013, Lab on a chip.

[9]  Tetsuya Yomo,et al.  In vitro evolution of α-hemolysin using a liposome display , 2013, Proceedings of the National Academy of Sciences.

[10]  Peter G Schultz,et al.  Protein conjugation with genetically encoded unnatural amino acids. , 2013, Current opinion in chemical biology.

[11]  J. Krzycki,et al.  PylSn and the Homologous N-terminal Domain of Pyrrolysyl-tRNA Synthetase Bind the tRNA That Is Essential for the Genetic Encoding of Pyrrolysine* , 2012, The Journal of Biological Chemistry.

[12]  Tetsuya Yomo,et al.  Cell-free protein synthesis inside giant unilamellar vesicles analyzed by flow cytometry. , 2012, Langmuir : the ACS journal of surfaces and colloids.

[13]  T. Yomo,et al.  Construction of a gene screening system using giant unilamellar liposomes and a fluorescence-activated cell sorter. , 2012, Analytical chemistry.

[14]  S. Yokoyama,et al.  Wide-range protein photo-crosslinking achieved by a genetically encoded N(ε)-(benzyloxycarbonyl)lysine derivative with a diazirinyl moiety. , 2012, Molecular bioSystems.

[15]  J. Noel,et al.  Stereochemical Basis for Engineered Pyrrolysyl-tRNA Synthetase and the Efficient in Vivo Incorporation of Structurally Divergent Non-native Amino Acids , 2011, ACS chemical biology.

[16]  Christoph A. Merten,et al.  Miniaturization and parallelization of biological and chemical assays in microfluidic devices. , 2010, Chemistry & biology.

[17]  B. M. Paegel.  Microfluidic landscapes for evolution. , 2010, Current opinion in chemical biology.

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

[19]  Masanori Fujinami,et al.  Population analysis of structural properties of giant liposomes by flow cytometry. , 2009, Langmuir : the ACS journal of surfaces and colloids.

[20]  T. Hohsaka,et al.  Position‐Specific Incorporation of Fluorescent Non‐natural Amino Acids into Maltose‐Binding Protein for Detection of Ligand Binding by FRET and Fluorescence Quenching , 2009, Chembiochem : a European journal of chemical biology.

[21]  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.

[22]  S. Yokoyama,et al.  Crystallographic studies on multiple conformational states of active-site loops in pyrrolysyl-tRNA synthetase. , 2008, Journal of molecular biology.

[23]  D. Söll,et al.  The amino‐terminal domain of pyrrolysyl‐tRNA synthetase is dispensable in vitro but required for in vivo activity , 2007, FEBS letters.

[24]  M. Sisido,et al.  FRET analysis of protein conformational change through position-specific incorporation of fluorescent amino acids , 2006, Nature Methods.

[25]  Yutetsu Kuruma,et al.  Cell‐free translation systems for protein engineering , 2006, The FEBS journal.

[26]  Tetsuya Yomo,et al.  In vitro evolution of proteins. , 2006, Journal of bioscience and bioengineering.

[27]  J. Krzycki.  The direct genetic encoding of pyrrolysine. , 2005, Current opinion in microbiology.

[28]  Fabrizia Negri,et al.  Rational design of helical columnar packing in single crystals. , 2004, Angewandte Chemie.

[29]  David A. Weitz,et al.  Production of Unilamellar Vesicles Using an Inverted Emulsion , 2003 .

[30]  Takuya Ueda,et al.  Cell-free translation reconstituted with purified components , 2001, Nature Biotechnology.

[31]  A. Plückthun,et al.  In vitro display technologies: novel developments and applications. , 2001, Current opinion in biotechnology.