Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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


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.

A DNA library in the form of a PCR product ('library'; see Fig. 2 for construct details), coding for binding proteins, is ligated into the ribosome display vector pRDV, thereby genetically fusing it to a tolA spacer sequence in-frame, and providing a promoter and translation initiation region at the 5′ end. The final ribosome display construct is obtained by PCR amplification of both flanking regions and the library insert from the ligated vector. In vitro transcription of this PCR product yields mRNA that is used for in vitro translation. The ribosome stalls at the end of the mRNA and does not release the encoded and properly folded protein because of the absence of a stop codon. The mRNA-ribosome-protein ternary complexes are used for affinity selection on an immobilized target. mRNA of bound complexes is recovered after washing, reverse transcribed and amplified by PCR. Thereby the selected pools of binders can be used directly for the next cycle of ribosome display or analysis of single clones after cloning into expression vectors.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 2: The construct for ribosome display using E. coli ribosomes.


  1. 1

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

    CAS  Article  Google Scholar 

  2. 2

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

    CAS  Article  Google Scholar 

  3. 3

    Hawkins, R.E., Russell, S.J. & Winter, G. Selection of phage antibodies by binding affinity. Mimicking affinity maturation. J. Mol. Biol. 226, 889–896 (1992).

    CAS  Article  Google Scholar 

  4. 4

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

    CAS  Article  Google Scholar 

  5. 5

    Hanes, J., Jermutus, L., Schaffitzel, C. & Plückthun, A. Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems. FEBS Lett. 450, 105–110 (1999).

    CAS  Article  Google Scholar 

  6. 6

    He, M. 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).

    CAS  Article  Google Scholar 

  7. 7

    He, M. & Taussig, M.J. 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).

    CAS  Article  Google Scholar 

  8. 8

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

    Article  Google Scholar 

  9. 9

    Amstutz, P., Binz, H.K., Zahnd, C. & Plückthun, A. Ribosome display: in vitro selection of protein-protein interactions. in Cell Biology, A Laboratory Handbook (ed. Celis, J.) 497–509 (Elsevier Academic Press, 2006).

    Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Hanes, J., Jermutus, L. & Plückthun, A. Selecting and evolving functional proteins in vitro by ribosome display. Methods Enzymol. 328, 404–430 (2000).

    CAS  Article  Google Scholar 

  12. 12

    Mattheakis, L.C., Bhatt, R.R. & Dower, W.J. An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc. Natl. Acad. Sci. USA 91, 9022–9026 (1994).

    CAS  Article  Google Scholar 

  13. 13

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

    CAS  Article  Google Scholar 

  14. 14

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

    CAS  Article  Google Scholar 

  15. 15

    Yau, K.Y., Groves, M.A., Li, S. & Sheedy, C. Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library. J. Immunol. Methods 281, 161–175 (2003).

    CAS  Article  Google Scholar 

  16. 16

    Hanes, J., Jermutus, L., Weber-Bornhauser, S., Bosshard, H.R. & Plückthun, A. Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc. Natl. Acad. Sci. USA 95, 14130–14135 (1998).

    CAS  Article  Google Scholar 

  17. 17

    Hanes, J., Schaffitzel, C., Knappik, A. & Plückthun, A. Picomolar affinity antibodies from a fully synthetic naive library selected and evolved by ribosome display. Nat. Biotechnol. 18, 1287–1292 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Knappik, A. 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).

    CAS  Article  Google Scholar 

  19. 19

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

    CAS  Article  Google Scholar 

  20. 20

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

    CAS  Article  Google Scholar 

  21. 21

    Schaffitzel, C. 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).

    CAS  Article  Google Scholar 

  22. 22

    Zahnd, C. 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).

    CAS  Article  Google Scholar 

  23. 23

    Jermutus, L., Honegger, A., Schwesinger, F., Hanes, J. & Plückthun, A. Tailoring in vitro evolution for protein affinity or stability. Proc. Natl. Acad. Sci. USA 98, 75–80 (2001).

    CAS  Article  Google Scholar 

  24. 24

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

    CAS  Article  Google Scholar 

  25. 25

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

    CAS  Article  Google Scholar 

  26. 26

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

    CAS  Article  Google Scholar 

  27. 27

    Hanes, J. & Plückthun, A. 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).

    Google Scholar 

  28. 28

    Schaffitzel, C., Zahnd, C., Amstutz, P., Luginbühl, B. & Plückthun, A. 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).

    Google Scholar 

  29. 29

    Cadwell, R.C. & Joyce, G.F. Randomization of genes by PCR mutagenesis. PCR Methods Appl. 2, 28–33 (1992).

    CAS  Article  Google Scholar 

  30. 30

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

    CAS  Article  Google Scholar 

  31. 31

    Zaccolo, M., Williams, D.M., Brown, D.M. & Gherardi, E. An approach to random mutagenesis of DNA using mixtures of triphosphate derivatives of nucleoside analogues. J. Mol. Biol. 255, 589–603 (1996).

    CAS  Article  Google Scholar 

  32. 32

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

    CAS  Article  Google Scholar 

  33. 33

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

    CAS  Article  Google Scholar 

  34. 34

    Boder, E.T. & Wittrup, K.D. Yeast surface display for screening combinatorial polypeptide libraries. Nat. Biotechnol. 15, 553–557 (1997).

    CAS  Article  Google Scholar 

  35. 35

    Yang, W.P. 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).

    CAS  Article  Google Scholar 

Download references


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



Corresponding author

Correspondence to Andreas Plückthun.

Ethics declarations

Competing interests

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

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Zahnd, C., Amstutz, P. & Plückthun, A. Ribosome display: selecting and evolving proteins in vitro that specifically bind to a target. Nat Methods 4, 269–279 (2007).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing