Applications of Ribosome Display

In ribosome display, the physical link between genotype and phenotype is accomplished by mRNA-ribosome—protein complexes, which are directly used for selection. If a library of different mRNA molecules is translated, a protein library results in which each protein is produced from its own mRNA and remains connected to it. Since these complexes of the proteins and their encoding mRNAs are stable for several days under the appropriate conditions, very stringent selections can be performed. As all steps of ribosome display are carried out in vitro, reaction conditions of the individual steps can be tailored to the requirements of the protein species investigated, as well as the objectives of the selection or evolution experiment. Application of ribosome display has produced scFv fragments of antibodies with affinities in the picomolar range from libraries prepared from immunized mice (Hanes et al., 1998) and more recently from a naive, completely synthetic library (Hanes et al., 2000), and has been used to evolve improved off-rates and stability (Jermutus et al., 2000). 

The first successful application of ribosome display system was demonstrated for the display of a library of random peptide decamers... 

Yan, X. and Xu, Z. (2006) Ribosome-display technology applications for directed evolution of functional proteins. Drug Discov Today 11, 911-916. 

In vitro selection technology can be used in principle for three tasks. We will discuss these in the following sections for ribosome display, as currently the examples are mostly available from this approach. The first task is to identify a protein or a peptide from a pool of variants. Here, ribosome display is used exclusively as a method for selection, and changes are not actively introduced in the original pool. In this case, proofreading polymerases are used during the PCR amplification steps (see below). A second application is to evolve a given protein or peptide... 

The first success was demonstration in 1994, with the report of a large, diverse library of decapeptides displayed and selected while associated with E. coli S30 polysomes and RNA.262 The key to Dower s success was the application of natural product antibiotics that were known to interfere with protein synthesis by stabilizing the ribosome-mRNA-protein complex. Thus, rifampicin and chloramphenicol (for prokaryotic system) or cycloheximide (for eukaryotic system) were used.2 3 Because these antibiotics halt the translation at random locations, the ensuing libraries were composed of mostly truncated peptides and thus not really suitable for the generation of cDNA libraries. Later, removal of the stop codon from mRNA was used to stall the translation at the end of the mRNA.264,265 Several improvements have been made more recently to stabilize the... 

ARM display is the application of eukaiyotic ribosome display to selection and evolution of antibody combining sites. It potentially displays very large libraries... 

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