Directed ribosome display

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

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). 

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.

Protein disulfide isomerase (PDI) was found to be important in catalyzing disulfide bond formation of antibody fragments, and it improved the efficiency of E. coli ribosome display of antibodies threefold when used during the in vitro translation reaction (Hanes and Pluckthun, 1997) (see also Section II, B). A fourfold improvement of ribosome display was observed when 1 OSa-RNA, which is involved in degradation of truncated proteins (see Section III, B, 2), was inhibited by using an antisense DNA oligonucleotide directed against the lOSa-RNA (Hanes and Pluckthun, 1997). 

In contrast to the other in vitro selection technologies discussed below, where to date mostly model enrichments have been reported, more examples on directed evolution are available for ribosome display, and these are reported in a separate section (see Section IV). Briefly, experiments on peptide and protein libraries are summarized, and the directed evolution of distinct biophysical properties is discussed. 

In a direct comparison of the rabbit reticulocyte system to the E. coli ribosome display system in a model study, the E. coli system turned out to be more efficient for the display of the model scFv constructs tested (Hanes et al., 1999). 

Directed evolution consists of cycles of diversification and selection. Because ribosome display takes place entirely in vitro, it can ideally be combined with in vitro methods of generating sequence diversity. Since true evolution requires diversification in each cycle, this facile alternation between in vitro mutagenesis and in vitro selection is one of the attractions of the ribosome display method. 

If certain amino acids (such as disulfide bonds or crucial amino acids in the hydrophobic core) are indispensable for protein stability, these positions can be changed by site-directed mutagenesis (Proba et al., 1998). To avoid back-mutations during the evolution process or the selection of a residual wild-type contamination, the pool is amplified after each round of ribosome display with a primer that reintroduces the destabilizing mutation. If the mutation is not close to one of the termini, the coding sequence has to be amplified in two parts, which are then reassembled by PCR. Thus, to evolve improved stabilities this strategy first removes known crucial stabilizing factors to select for compensatory mutations at different positions. 

Ribosome display uses the unmodified DNA library, which is transcribed and translated in vitro. The link occurs between the evolved protein and mRNA, and is achieved by stalling the translating ribosome at the end of the mRNA, which lacks a stop codon. Without a stop codon, the protein is not released by the ribosome, and the complex formed by the mRNA, protein (usually correctly folded) and ribosome is used directly for selection against an immobilized target. This method is shown schematically in Figure 8.7(a). 

Ribosome display (Figure 2) has been used to select protein-binding partners but, to our knowledge, never been used to find the unknown protein-binding partner for a small molecule.268 272-275 Fiowever, a recent paper by Pluckthun clearly indicates that this is possible. In this report, the authors show, for the first time, that it is possible to use ribosome display to select for catalytic activity based on catalytic turnover (directed evolution). In their experiment, they displayed variants of RTEM-/3-lactamase and used biotinylated ampicillin sulfone (80) to select for catalytically active variants (Figure 3)276... 

Figure 2 In ribosome display, mRNA (A) extracted from a cell is converted into a cDNA library (B) is transcribed back into mRNA with no stop codons. Prokaryotic or eukaryotic proteosomes are added and the ribosome then travels down the mRNA (C) translating until it reaches the end of the mRNA molecule (D), where the ribosome halts. With no stop codon, the release factor proteins cannot bind and so the protein, ribosome, and mRNA are physically associated and can be stabilized by high Mg2+ and low temperatures. This complex could then be bound directly to an immobilized natural product (E), the nonbinding library members washed away and the bound members eluted with EDTA (F), which destabilizes the ribosomal complexes by removing Mg2+. The purified sublibrary is converted into cDNA by reverse transcription (RT-PCR) and amplified by regular PCR (B). The/n vitro transcription and translation can be repeated for another round of selection or the cDNA can be analyzed by agarose electrophoresis and/or sequencing.

With the advent of recombinant DNA technology, antibody genes can be amplified and selected through phage display, cell-surface display, or cell-free display systems (i.e., ribosome display). A major advantage shared by these systems is the direct... 

Perhaps the most interesting conclusion from the peptidyltransferase assays is the evidence that ribosomes from extremely thermophilic archaea display significant peptidyltransferase activity (both uncoupled and coupled) at temperatures (37°C) well below that required for poly(U)-directed polypeptide synthesis. Therefore, in the thermophile systems high temperature appears to activate reactions other than peptide bond formation. 

A single mRNA may be processed simultaneously by numerous ribosomes to increase the rate of protein synthesis. The structure so formed is called a polyribosome or polysome. Since mRNA molecules are translated in the 5 ->3 direction, the ribosome bound nearest to the 5 -end displays the shortest polypeptide chain. 

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