Display ribosome

Hanes, J. and Pluckthun, A. (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proceedings of the National Academy of Sciences of the United States of America, 94, 4937-4942. 

He, M. and Khan, F. 2005. Ribosome display next generation display technologies for production of antibodies in vitro. Expert Review of Proteomics 2(3), 421-430. 

Hoffinullw, U. Schneider-MCTgenCT, J. (1998) In vitro evolutimi and selection of proteins ribosome display for larga libraries. Angew. Chem. Int. Ed. Engl., 37, 3141-3. 

Although its use in ligand selection for large scale of affinity chromatography is not wide, ribosome display and systematic evolution of ligands by exponential enrichment (SELEX) may... 

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

Hanes, J.,Jermutus, L., Schaffitzel, C., and Pluckthun, A. (1999). Comparison of Escherichia coli and rabbit reticulocyte ribosome display systems. FEBS Lett, 450(1-2),... 

Roberts, R. W. (1999). Totally In vitro protein selection using mRNA-protein fusions and ribosome display. Ourr. Opin. Chem. Biol. 3, 268-273. 

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). 

Ribosome display has thus two main advantages compared to in vivo selection systems on the one hand in vitro technologies allow one to screen very large libraries, since no transformation steps are necessary. On the other hand, subsequent diversification of the library is easy and convenient and every single clone present in the library can conceivably... 

The first experimental demonstration of the ribosome display technology was the selection of short peptides Irom a library using an E. coli S30 in vitro translation system (Mattheakis et al., 1994 Mattheakis et al., 1996). The concept pursued by Mattheakis and coworkers (1994) for peptides was then used for the development of ribosome display of functional proteins by use of the E. coli S30 in vitro translation system (Hanes and Pluckthun, 1997). However, for this purpose it was necessary to significantly modify and optimize the experimental conditions of ribosome display to make this technology efficient enough for the display of correctly folded, functional proteins. 

Fig. 2. Ribosome display. A library of proteins (e.g., scFv fragments of antibodies) is transcribed and translated in vitro. The resulting mRNA lacks a stop codon, giving rise to linked mRNA-ribosome-protein complexes. These are directly used for selection on the immobilized target. The mRNA incorporated in bound complexes is eluted and purified. RT-PCR can introduce mutations and yields a DNA pool enriched for binders that can be used for the next iteration.

The features of the ribosome display construct are summarized in Figure 3. On the DNA level, the construct requires a T7 promoter for efficient in vitro transcription to mRNA. On the mRNA level, the construct contains, as a regulatory sequence for translation, either a prokaryotic ribosome binding site (Shine and Dalgamo, 1975) if the E. coli... 

Another important prerequisite for efficient ribosome display in the E. coli system is the elimination of the lOSa-RNA (Ray and Apirion, 1979). lOSa-RNA is a stable bacterial RNA with a tRNA-like structure, but having an extended 11-loop (Komine et al., 1994). If a truncated... 

At both ends of the mRNA, the ribosome display construct should include stemloops, 5 - and 3 -stemloops are known to stabilize mRNA against RNases in vivo as well as in vitro. The presence of stemloops is important, especially in the E. coli ribosome display system, because the extract used for in vitro translation contains high RNase activities. To date, five of twenty E. coli RNases have been shown to contribute to mRNA degradation (Hajnsdorf et al., 1996), and they are probably all present in the S30 extract. The efficiency of ribosome display was in-... 

A protein tail, which is the same in all library members, is fused to the C-terminus of the ribosome display construct and serves as a spacer. This spacer has two main functions. First, it tethers the synthesized protein to the ribosome. Second, it keeps the structured part of the protein outside the ribosome and allows its folding and interaction with ligands, without clashing with the ribosomal tunnel. The ribosomal tunnel covers between 20 and 30 C-terminal amino acids of the nascent polypeptide chain during protein synthesis and can therefore prevent the folding of the protein (Malkin and Rich, 1967 Smith et al., 1978). 

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