Polysome display

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

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... 

Figure 5 Starting from natural mRNA, a cDNA library (A blue) is produced and like ribosomal display, the cDNA is transcribed into mRNA (B) with no stop codons. The 3 -end of each mRNA molecule is ligated to a short synthetic DNA linker (C) and sometimes a polyethyleneglycol spacer, which terminates with a puramycin molecule (small red sphere). The ligation is stabilized by the addition of psoralen (green clamp), which is photoactivated to covalently join both strands. Addition of crude polysomes or purified ribosomes (D) results in translation of the mRNA into protein, but the ribosome stalls at the mRNA-DNA junction. Since there are no stop codons, release factors cannot function and instead the puromycin enters the A-site of the ribosome (A). Because puramycin is an analog of tyrosyl-tRNA, the peptidyl transferase subunit catalyzes amide bond formation between the puromycin amine and the peptide carboxyl terminus, but is unable to hydrolyze the amide link (which should be an ester in tyrosyl-tRNA) to release the dimethyladenosine. The ribosome is dissociated to release the mRNA-protein fusion (E), which is protected with complementary cDNA using RT-PCR (F). The mRNA library can then be selected against an immobilized natural product probe (G), nonbinding library members washed away and the bound mRNA (H) released with SDS. PCR amplification of the cDNA provides a sublibrary (A) for another round of selection or for analysis/ sequencing.

The recently described method of ribosome display has been proposed as a method to affinity-mature antibodies. This approach involves the translation of proteins in vitro and their selection while attached to ribosomes [88,89]. This can occur if the mRNA lacks a stop codon. In this case, the ribosome is not able to detach, and mRNA and encoding protein remain attached together and can be selected on a relevant ligand. After selection, the mRNA from selected polysomes is converted into cDNA, amplified by PCR, and used for the next transcription, translation, and selection round. Ribosome display has been used for the display [89,90] and evolution of a scFv antibody in vitro [89], but no improvement in affinity was reported. However, the combination of this display technique (which potentially allows access to unlimited genetic diversity, unlike phage display) with methods such as DNA shuffling will see greater affinity improvements... 

Initially, display of a peptide libraiy on prokaiyotic polysomes in the E. cdi S30 system was described (6, 7). To produce a population of stalled polysomes, agents such as rifampicin or chloramphenicol, which block prokaiyotic translation, were used. (The term stalled implies that the process of translation has been interrupted such that the ribosome, mRNA, and nascent protein remain associated.) The main example was of screening a large peptide libraiy with 10 members and selection of epitopes by a specific antibody. Peptide libraries have also been expressed and selected in the wheat germ system (8). 

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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