Ribosome display: a perspective.

Ribosome display is an in vitro evolution technology for proteins. It is based on in vitro translation, but prevents the newly synthesized protein and the mRNA encoding it from leaving the ribosome. It thereby couples phenotype and genotype. Since no cells need to be transformed, very large libraries can be used directly in selections, and the in vitro amplification provides a very convenient integration of random mutagenesis that can be incorporated into the procedure. This review highlights concepts, mechanisms, and different variations of ribosome display and compares it to related methods. Applications of ribosome display are summarized, e.g., the directed evolution of proteins for higher binding affinity, for higher stability or other improved biophysical parameters and enzymatic properties. Ribosome display has developed into a robust technology used in academia and industry alike, and it has made the cell-free Darwinian evolution of proteins over multiple generations a reality.

[1]  Andreas Plückthun,et al.  A designed ankyrin repeat protein evolved to picomolar affinity to Her2. , 2007, Journal of molecular biology.

[2]  D. M. Brown,et al.  An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. , 1996, Journal of molecular biology.

[3]  H. Bremer.  Modulation of Chemical Composition and Other Parameters of the Cell by Growth Rate , 1999 .

[4]  Richard W Roberts,et al.  In vitro selection of protein and peptide libraries using mRNA display. , 2009, Methods in molecular biology.

[5]  G. P. Smith,et al.  Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. , 1985, Science.

[6]  M. Ehrenberg,et al.  Complementary roles of initiation factor 1 and ribosome recycling factor in 70S ribosome splitting , 2008, The EMBO journal.

[7]  Andreas Plückthun,et al.  Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target , 2007, Nature Methods.

[8]  R. Tsien,et al.  Evolution of new nonantibody proteins via iterative somatic hypermutation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Fields,et al.  A novel genetic system to detect protein–protein interactions , 1989, Nature.

[10]  G. Winter,et al.  Selection of phage antibodies by binding affinity. Mimicking affinity maturation. , 1992, Journal of molecular biology.

[11]  G. Weiss,et al.  Optimizing the affinity and specificity of proteins with molecular display. , 2006, Molecular bioSystems.

[12]  Thomas J Magliery,et al.  Detecting protein-protein interactions with GFP-fragment reassembly , 2004, Nature Methods.

[13]  A. Plückthun,et al.  High-affinity binders selected from designed ankyrin repeat protein libraries , 2004, Nature Biotechnology.

[14]  D. Koshland.  Application of a Theory of Enzyme Specificity to Protein Synthesis. , 1958, Proceedings of the National Academy of Sciences of the United States of America.

[15]  J. Kraus,et al.  Purification of low-abundance messenger RNAs from rat liver by polysome immunoadsorption. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Marinus,et al.  Escherichia coli mutator genes. , 1999, Trends in microbiology.

[17]  J. Wells,et al.  Hormone phage: An enrichment method for variant proteins with altered binding properties , 1990, Proteins.

[18]  T. Yomo,et al.  Nascent chain, mRNA, and ribosome complexes generated by a pure translation system. , 2007, Biochemical and biophysical research communications.

[19]  Klaus Schulten,et al.  Structural Insight into Nascent Polypeptide Chain–Mediated Translational Stalling , 2009, Science.

[20]  J. Szostak,et al.  Directed evolution of ATP binding proteins from a zinc finger domain by using mRNA display. , 2006, Chemistry & biology.

[21]  Andreas Plückthun,et al.  In-vitro protein evolution by ribosome display and mRNA display. , 2004, Journal of immunological methods.

[22]  T. Bratkovič,et al.  Progress in phage display: evolution of the technique and its applications , 2010, Cellular and Molecular Life Sciences.

[23]  J. Douthwaite,et al.  Highly efficient ribosome display selection by use of purified components for in vitro translation. , 2006, Journal of immunological methods.

[24]  G. Winter,et al.  Phage antibodies: filamentous phage displaying antibody variable domains , 1990, Nature.

[25]  N. Doi,et al.  DNA display of biologically active proteins for in vitro protein selection. , 2004, Journal of biochemistry.

[26]  N. Doi,et al.  STABLE: protein‐DNA fusion system for screening of combinatorial protein libraries in vitro , 1999, FEBS letters.

[27]  P. Schatz.  Use of Peptide Libraries to Map the Substrate Specificity of a Peptide-Modifying Enzyme: A 13 Residue Consensus Peptide Specifies Biotinylation in Escherichia coli , 1993, Bio/Technology.

[28]  Shenghua Li,et al.  Affinity maturation of a V(H)H by mutational hotspot randomization. , 2005, Journal of immunological methods.

[29]  A. Plückthun,et al.  Selection and characterization of DARPins specific for the neurotensin receptor 1. , 2009, Protein engineering, design & selection : PEDS.

[30]  P. Daugherty.  Protein engineering with bacterial display. , 2007, Current opinion in structural biology.

[31]  E. Hajnsdorf,et al.  Poly(A)-assisted RNA decay and modulators of RNA stability. , 2009, Progress in molecular biology and translational science.

[32]  M. Taussig,et al.  Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display. , 1999, Journal of immunological methods.

[33]  A. J. Carpousis.  The RNA degradosome of Escherichia coli: an mRNA-degrading machine assembled on RNase E. , 2007, Annual review of microbiology.

[34]  Gideon Schreiber,et al.  Rational design of faster associating and tighter binding protein complexes , 2000, Nature Structural Biology.

[35]  J. Devlin,et al.  Random peptide libraries: a source of specific protein binding molecules. , 1990, Science.

[36]  Andreas Plückthun,et al.  Crystal Structure and Function of a DARPin Neutralizing Inhibitor of Lactococcal Phage TP901-1 , 2009, The Journal of Biological Chemistry.

[37]  M. Ehrenberg,et al.  Class-1 release factor eRF1 promotes GTP binding by class-2 release factor eRF3. , 2006, Biochimie.

[38]  Andreas Plückthun,et al.  Selection and Characterization of Her2 Binding-designed Ankyrin Repeat Proteins* , 2006, Journal of Biological Chemistry.

[39]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[40]  Adrian A. Nickson,et al.  Affinity maturation of phage display antibody populations using ribosome display. , 2012, Methods in molecular biology.

[41]  Y. Aoyama,et al.  Termination‐Free Prokaryotic Protein Translation by Using Anticodon‐Adjusted E. coli tRNASer as Unified Suppressors of the UAA/UGA/UAG Stop Codons. Read‐Through Ribosome Display of Full‐Length DHFR with Translated UTR as a Buried Spacer Arm , 2006, Chembiochem : a European journal of chemical biology.

[42]  Anthony D. Keefe,et al.  Functional proteins from a random-sequence library , 2001, Nature.

[43]  A. Plückthun,et al.  Directed in Vitro Evolution and Crystallographic Analysis of a Peptide-binding Single Chain Antibody Fragment (scFv) with Low Picomolar Affinity* , 2004, Journal of Biological Chemistry.

[44]  M. Kozak.  Initiation of translation in prokaryotes and eukaryotes. , 1999, Gene.

[45]  C. Milstein,et al.  Conformational isomerism and the diversity of antibodies. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[46]  N. Doi,et al.  Bicistronic DNA display for in vitro selection of Fab fragments , 2009, Nucleic acids research.

[47]  L. Jermutus,et al.  An improved method for an efficient and easily accessible eukaryotic ribosome display technology. , 2006, Protein engineering, design & selection : PEDS.

[48]  Hiroshi Yanagawa,et al.  DNA display for in vitro selection of diverse peptide libraries. , 2003, Nucleic acids research.

[49]  R. Roberts,et al.  Design, expression, and stability of a diverse protein library based on the human fibronectin type III domain , 2007, Protein science : a publication of the Protein Society.

[50]  A. Plückthun,et al.  Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[51]  M. Taussig,et al.  Eukaryotic ribosome display with in situ DNA recovery , 2007, Nature Methods.

[52]  A. Plückthun,et al.  Computational analysis of off-rate selection experiments to optimize affinity maturation by directed evolution. , 2010, Protein engineering, design & selection : PEDS.

[53]  J. Shine,et al.  Terminal-sequence analysis of bacterial ribosomal RNA. Correlation between the 3'-terminal-polypyrimidine sequence of 16-S RNA and translational specificity of the ribosome. , 1975, European journal of biochemistry.

[54]  A. Plückthun,et al.  Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems , 1999, FEBS letters.

[55]  J. Scott,et al.  Searching for peptide ligands with an epitope library. , 1990, Science.

[56]  W. Dower,et al.  An in vitro polysome display system for identifying ligands from very large peptide libraries. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[57]  Andrew D Griffiths,et al.  Directed evolution by in vitro compartmentalization , 2006, Nature Methods.

[58]  A. Plückthun,et al.  Beyond binding: using phage display to select for structure, folding and enzymatic activity in proteins. , 1999, Current opinion in structural biology.

[59]  Andreas Plückthun,et al.  Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. , 2003, Journal of molecular biology.

[60]  T. Terwilliger,et al.  Recent Advances in GFP Folding Reporter and Split-GFP Solubility Reporter Technologies. Application to Improving the Folding and Solubility of Recalcitrant Proteins from Mycobacterium tuberculosis , 2005, Journal of Structural and Functional Genomics.

[61]  A. Plückthun,et al.  Rapid selection of specific MAP kinase-binders from designed ankyrin repeat protein libraries. , 2006, Protein engineering, design & selection : PEDS.

[62]  B. Bukau,et al.  Structure and function of the molecular chaperone Trigger Factor. , 2010, Biochimica et biophysica acta.

[63]  R. Glockshuber,et al.  A comparison of strategies to stabilize immunoglobulin Fv-fragments. , 1990, Biochemistry.

[64]  Andreas Plückthun,et al.  Intracellular Kinase Inhibitors Selected from Combinatorial Libraries of Designed Ankyrin Repeat Proteins* , 2005, Journal of Biological Chemistry.

[65]  Dan S. Tawfik,et al.  Directed evolution of an extremely fast phosphotriesterase by in vitro compartmentalization , 2003, The EMBO journal.

[66]  Eric T. Boder,et al.  Yeast surface display for screening combinatorial polypeptide libraries , 1997, Nature Biotechnology.

[67]  U. Rinas,et al.  Roles of heat-shock chaperones in the production of recombinant proteins in Escherichia coli. , 2004, Advances in biochemical engineering/biotechnology.

[68]  W. Stemmer.  Rapid evolution of a protein in vitro by DNA shuffling , 1994, Nature.

[69]  A. Plückthun,et al.  Ribosome display of mammalian receptor domains. , 2005, Protein engineering, design & selection : PEDS.

[70]  A. Plückthun,et al.  In vitro selection and evolution of functional proteins by using ribosome display. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[71]  A. Plückthun,et al.  Residue-resolved stability of full-consensus ankyrin repeat proteins probed by NMR. , 2010, Journal of molecular biology.

[72]  Bei-Wen Ying,et al.  Efficient protein selection based on ribosome display system with purified components. , 2007, Biochemical and biophysical research communications.

[73]  Andreas Plückthun,et al.  Inhibition of caspase-2 by a designed ankyrin repeat protein: specificity, structure, and inhibition mechanism. , 2007, Structure.

[74]  Viktor Stein,et al.  New genotype-phenotype linkages for directed evolution of functional proteins. , 2005, Current opinion in structural biology.

[75]  H. Erickson,et al.  Kinetics of protein-protein association explained by Brownian dynamics computer simulation. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[76]  Katja M. Arndt,et al.  An in vivo library-versus-library selection of optimized protein–protein interactions , 1999, Nature Biotechnology.

[77]  M. Ehrenberg,et al.  tmRNA.SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes. , 2010, Journal of structural biology.

[78]  R. Barrett,et al.  Peptides on phage: a vast library of peptides for identifying ligands. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[79]  R. Sauer,et al.  The tmRNA system for translational surveillance and ribosome rescue. , 2007, Annual review of biochemistry.

[80]  Y. Chen,et al.  Antibody mimics based on human fibronectin type three domain engineered for thermostability and high-affinity binding to vascular endothelial growth factor receptor two. , 2005, Protein engineering, design & selection : PEDS.

[81]  Philippe Mondon,et al.  Human antibody libraries: a race to engineer and explore a larger diversity. , 2008, Frontiers in bioscience : a journal and virtual library.

[82]  J. Kaufman,et al.  cDNA clones for the heavy chain of HLA-DR antigens obtained after immunopurification of polysomes by monoclonal antibody. , 1982, Proceedings of the National Academy of Sciences of the United States of America.

[83]  M. Ehrenberg,et al.  Termination of translation: interplay of mRNA, rRNAs and release factors? , 2003, The EMBO journal.

[84]  A. Lim,et al.  Directed evolution of high-affinity antibody mimics using mRNA display. , 2002, Chemistry & biology.

[85]  Burckhard Seelig,et al.  Selection and evolution of enzymes from a partially randomized non-catalytic scaffold , 2007, Nature.

[86]  M J Corey,et al.  High-affinity peptide ligands to prostate-specific antigen identified by polysome selection. , 1997, Biochemical and biophysical research communications.

[87]  Andreas Plückthun,et al.  Identification of a functional epitope of the Nogo receptor by a combinatorial approach using ribosome display. , 2005, Journal of molecular biology.

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

[89]  Hiroshi Yanagawa,et al.  In vitro evolution of single-chain antibodies using mRNA display , 2006, Nucleic acids research.

[90]  A. Plückthun,et al.  Antigen recognition by conformational selection , 1999, FEBS letters.

[91]  A. Fersht,et al.  Rapid, electrostatically assisted association of proteins , 1996, Nature Structural Biology.

[92]  Charles Duyckaerts,et al.  Llama VHH antibody fragments against GFAP: better diffusion in fixed tissues than classical monoclonal antibodies , 2009, Acta Neuropathologica.

[93]  A. Plückthun,et al.  Trinucleotide phosphoramidites: ideal reagents for the synthesis of mixed oligonucleotides for random mutagenesis. , 1994, Nucleic acids research.

[94]  Nikolaos E. Labrou,et al.  Random mutagenesis methods for in vitro directed enzyme evolution. , 2009 .

[95]  W. Dower,et al.  Cell-free synthesis of peptide libraries displayed on polysomes. , 1996, Methods in enzymology.

[96]  M. Groves,et al.  Affinity maturation of phage display antibody populations using ribosome display. , 2006, Journal of immunological methods.

[97]  Y. Cho,et al.  A decade of yeast surface display technology: where are we now? , 2008, Combinatorial chemistry & high throughput screening.

[98]  Andreas Plückthun,et al.  Folding and unfolding mechanism of highly stable full-consensus ankyrin repeat proteins. , 2008, Journal of molecular biology.

[99]  Andreas Plückthun,et al.  Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display , 2000, Nature Biotechnology.

[100]  Frances H. Arnold,et al.  Molecular evolution by staggered extension process (StEP) in vitro recombination , 1998, Nature Biotechnology.

[101]  A. Plückthun,et al.  Rapid selection of high-affinity binders using ribosome display. , 2012, Methods in molecular biology.

[102]  A. Plückthun,et al.  Tailoring in vitro evolution for protein affinity or stability. , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[103]  M. Taussig,et al.  Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. , 1997, Nucleic acids research.

[104]  H. Blau,et al.  Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[105]  A. Spirin,et al.  Functional antibody production using cell-free translation: Effects of protein disulfide isomerase and chaperones , 1997, Nature Biotechnology.

[106]  G. Georgiou,et al.  Production and fluorescence-activated cell sorting of Escherichia coli expressing a functional antibody fragment on the external surface. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[107]  B. Luisi,et al.  Endonucleolytic initiation of mRNA decay in Escherichia coli. , 2009, Progress in molecular biology and translational science.

[108]  A. Plückthun,et al.  Her2-specific multivalent adapters confer designed tropism to adenovirus for gene targeting. , 2011, Journal of molecular biology.

[109]  T. Ueda,et al.  Ribosome display with the PURE technology. , 2010, Methods in molecular biology.

[110]  Koreaki Ito,et al.  The Ribosomal Exit Tunnel Functions as a Discriminating Gate , 2002, Cell.