Abstract
Ribosome display is a powerful polymerase chain reaction-based in vitro display technology that is well suited to the selection and evolution of proteins. This technology exploits cell-free translation to achieve coupling of phenotype and genotype by the production of stabilized ribosome complexes, whereby translated protein and their cognate mRNA remain attached to the ribosome. The Escherichia coli S30 extract for in vitro display of an mRNA library has proven to be very successful for the evolution of high-affinity antibodies and the optimization of defined protein characteristics. These methodologies will enable the end user to successfully perform ribosome display selections.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Hanes J, Plückthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci U S A 94: 4937–4942
Hanes J, Schaffitzel C, Knappik A, Plückthun A (2000) Picomolar affinity antibodies from a fully synthetic naïve library selected and evolved by ribosome display. Nat Biotechnol 18:1287–1292
Lamla T, Erdmann V (2001) In vitro selection of other proteins than antibodies by means of ribosome display. FEBS Lett 502:35–40
Jermutus L, Honeggar A, Schwesinger F, Hanes J, Plückthun A (2001) Tailoring in vitro evolution for protein affinity or stability. Proc Natl Acad Sci U S A 98:75–80
Amstutz P, Pelletier JN, Guggisberg A (2002) In vitro selection for catalytic activity with ribosome display. J Am Chem Soc 124: 9396–9403
Matsuura T, Plückthun A (2004) Strategies for selection from protein libraries composed of de novo designed secondary structure modules. Orig Life Evol Biosph 34:151–157
Thom G, Cockroft AC, Buchanan AG et al (2006) Probing a protein–protein interaction by in vitro evolution. Proc Natl Acad Sci U S A 103:7619–7624
Zahnd C, Wyler E, Schwenk JM et al (2007) A designed ankyrin repeat protein evolved to picomolar affinity to Her2. J Mol Biol 369: 1015–1028
Dower WJ, Cwirla SE (1992) Creating vast peptide expression libraries: electroporation as a tool to construct plasmid libraries of greater than 109 recombinants. In: Chang DC et al (eds) Guide to electroporation and electrofusion. Academic, San Diego, CA, pp 291–301
Vaughan TJ, Williams AJ, Pritchard K et al (1996) Human antibodies with sub-nanomolar affinities isolated from a large nonimmunized phage display library. Nat Biotechnol 14: 309–314
Zubay G (1973) In vitro synthesis of protein in microbial systems. Annu Rev Genet 7: 267–287
Jermutus L, Ryabova LA, Plückthun A (1998) Recent advances in producing and selecting functional proteins by using cell-free translation. Curr Opin Biotechnol 9:534–548
Hajnsdorf E, Braun F, Haugel-Nielsen J et al (1996) Multiple degradation pathways of the rpsO mRNA of Escherichia coli RNase E interacts with the 5′ and 3′ extremities of the primary transcript. Biochemie 78:416–424
Studier FW, Rosenberg AH, Dunn JJ, Dubendorff JW (1990) Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60–89
Reynolds R, Bermudez-Cruz RM, Chamberlin MJ (1992) Parameters affecting transcription termination by Escherichia coli RNA polymerase. I. Analysis of 13 rho independent terminators. J Mol Biol 224:31–51
McCafferty J, Fitzgerald KJ, Earnshaw J et al (1994) Selection and rapid purification of murine antibody fragments that bind a transition-state analog by phage display. Appl Biochem Biotechnol 47:157–171
Freedman RB, Hawkins HC, McLaughlin SH (1995) Protein disulfide-isomerase. Methods Enzymol 251:397–406
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Thom, G., Groves, M. (2012). Ribosome Display. In: Proetzel, G., Ebersbach, H. (eds) Antibody Methods and Protocols. Methods in Molecular Biology, vol 901. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-931-0_6
Download citation
DOI: https://doi.org/10.1007/978-1-61779-931-0_6
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-930-3
Online ISBN: 978-1-61779-931-0
eBook Packages: Springer Protocols