Protocol | Published:

Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target

Nature Methods volume 4, pages 269279 (2007) | Download Citation

Subjects

Abstract

Ribosome display is an in vitro selection and evolution technology for proteins and peptides from large libraries1. As it is performed entirely in vitro, there are two main advantages over other selection technologies2,3. First, the diversity of the library is not limited by the transformation efficiency of bacterial cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, random mutations can be introduced easily after each selection round, as no library must be transformed after any diversification step. This allows facile directed evolution of binding proteins over several generations (Box 1). A prerequisite for the selection of proteins from libraries is the coupling of genotype (RNA, DNA) and phenotype (protein). In ribosome display, this link is accomplished during in vitro translation by stabilizing the complex consisting of the ribosome, the mRNA and the nascent, correctly folded polypeptide (Fig. 1). The DNA library coding for a particular library of binding proteins is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. The ribosomal complexes are allowed to bind to surface-immobilized target. Whereas non-bound complexes are washed away, mRNA of the complexes displaying a binding polypeptide can be recovered, and thus, the genetic information of the binding polypeptides is available for analysis. Here we describe a step-by-step procedure to perform ribosome display selection using an Escherichia coli S30 extract for in vitro translation, based on the work originally described and further refined in our laboratory1. A protocol that makes use of eukaryotic in vitro translation systems for ribosome display4,6,7 is also included in this issue8.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA 94, 4937–4942 (1997).

  2. 2.

    & Optimizing the affinity and specificity of proteins with molecular display. Mol. Biosystems 2, 49–57 (2006).

  3. 3.

    , & Selection of phage antibodies by binding affinity. Mimicking affinity maturation. J. Mol. Biol. 226, 889–896 (1992).

  4. 4.

    et al. High-affinity peptide ligands to prostate-specific antigen identified by polysome selection. Biochem. Biophys. Res. Commun. 232, 578–582 (1997).

  5. 5.

    , , & Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems. FEBS Lett. 450, 105–110 (1999).

  6. 6.

    et al. Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display. J. Immunol. Methods 231, 105–117 (1999).

  7. 7.

    & Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Res. 25, 5132–5134 (1997).

  8. 8.

    & Eukaryotic ribosome display with in situ DNA recovery. Nat. Methods 4, 281–288 (2006).

  9. 9.

    , , & Ribosome display: in vitro selection of protein-protein interactions. in Cell Biology, A Laboratory Handbook (ed. Celis, J.) 497–509 (Elsevier Academic Press, 2006).

  10. 10.

    & In vitro transcription: preparative RNA yields in analytical scale reactions. Anal. Biochem. 220, 420–423 (1994).

  11. 11.

    , & Selecting and evolving functional proteins in vitro by ribosome display. Methods Enzymol. 328, 404–430 (2000).

  12. 12.

    , & An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91, 9022–9026 (1994).

  13. 13.

    & Searching sequence space for high-affinity binding peptides using ribosome display. J. Mol. Biol. 329, 381–388 (2003).

  14. 14.

    et al. Functional selection of vaccine candidate peptides from Staphylococcus aureus whole-genome expression libraries in vitro. Infect. Immun. 71, 4633–4641 (2003).

  15. 15.

    , , & Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library. J. Immunol. Methods 281, 161–175 (2003).

  16. 16.

    , , , & Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc. Natl. Acad. Sci. USA 95, 14130–14135 (1998).

  17. 17.

    , , & Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat. Biotechnol. 18, 1287–1292 (2000).

  18. 18.

    et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296, 57–86 (2000).

  19. 19.

    et al. Selection of scFvs specific for HBV DNA polymerase using ribosome display. J. Immunol. Methods 284, 147–157 (2004).

  20. 20.

    et al. High-affinity binders selected from designed ankyrin repeat protein libraries. Nat. Biotechnol. 22, 575–582 (2004).

  21. 21.

    et al. In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei. Proc. Natl. Acad. Sci. USA 98, 8572–8577 (2001).

  22. 22.

    et al. Directed in vitro evolution and crystallographic analysis of a peptide-binding single chain antibody fragment (scFv) with low picomolar affinity. J. Biol. Chem. 279, 18870–18877 (2004).

  23. 23.

    , , , & Tailoring in vitro evolution for protein affinity or stability. Proc. Natl. Acad. Sci. USA 98, 75–80 (2001).

  24. 24.

    & Selection based on the folding properties of proteins with ribosome display. FEBS Lett. 539, 24–28 (2003).

  25. 25.

    et al. In vitro selection for catalytic activity with ribosome display. J. Am. Chem. Soc. 124, 9396–9403 (2002).

  26. 26.

    et al. Ribosome display for selection of active dihydrofolate reductase mutants using immobilized methotrexate on agarose beads. FEBS Lett. 514, 106–110 (2002).

  27. 27.

    & In vitro selection methods for screening of peptide and protein libraries. in Combinatorial Chemistry in Biology, Vol. 243 (eds. Famulok, M., Winnacker, E.-L. & Wong, C.-H.) 107–122 (Springer Verlag, Berlin Heidelberg, 1999).

  28. 28.

    , , , & In vitro selection and evolution of protein-ligand interactions by ribosome display. in Protein-Protein Interactions A Molecular Cloning Manual (eds. Golemis, E. & Adams, P.) 517–548 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2005).

  29. 29.

    & Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33 (1992).

  30. 30.

    Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 (1994).

  31. 31.

    , , & An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. J. Mol. Biol. 255, 589–603 (1996).

  32. 32.

    et al. Unbinding forces of single antibody-antigen complexes correlate with their thermal dissociation rates. Proc. Natl. Acad. Sci. USA 97, 9972–9977 (2000).

  33. 33.

    & Kinetics of protein-protein association explained by Brownian dynamics computer simulation. Proc. Natl. Acad. Sci. USA 89, 3338–3342 (1992).

  34. 34.

    & Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

  35. 35.

    et al. CDR walking mutagenesis for the affinity maturation of a potent human anti-HIV-1 antibody into the picomolar range. J. Mol. Biol. 254, 392–403 (1995).

Download references

Acknowledgements

We thank R. Skirgaila for originally suggesting and testing Phusion polymerase in ribosome display, as well as A. Batyuk, D. Ferrari, T. Huber, P. Martin Killias, P. Parizek, N. Sainz-Pastor and S.R. Wyss-Stoeckle for experimentally checking the protocol in detail and for many helpful suggestions and discussions, as well as former members of the Plückthun laboratory for establishing the protocol.

Author information

Affiliations

  1. Biochemisches Institut der Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.

    • Christian Zahnd
    • , Patrick Amstutz
    •  & Andreas Plückthun
  2. Molecular Partners AG, c/o Biochemisches Institut der Universität Zürich, Winterthurerstr. 190, CH-8057 Zürich, Switzerland.

    • Christian Zahnd
    •  & Patrick Amstutz

Authors

  1. Search for Christian Zahnd in:

  2. Search for Patrick Amstutz in:

  3. Search for Andreas Plückthun in:

Competing interests

A.P. is an inventor on a patent on ribosome display. Molecular Partners AG is using ribosome display commercially.

Corresponding author

Correspondence to Andreas Plückthun.

Supplementary information

About this article

Publication history

Published

DOI

https://doi.org/10.1038/nmeth1003

Further reading