The first published description of ribosome display was for peptide selection using prokaryotic ribosome display system. Until now, this system is still the most important system for ribosome display. As a leader in ribosome display, Creative Biolabs offers prokaryotic system for ribosome display, which is more time-saving and cost-effective.
Ribosome display is a cell-free system for the in vitro selection of proteins and peptides from large libraries. Ribosome display couples an individual nascent protein to its corresponding mRNA through the formation of stable protein-ribosome-mRNA (PRM) complexes. In this system, the ribosome itself serves as the connector and PRMs are exposed to immobilized target molecules. From those target-binding complexes, the mRNAs are isolated, reverse transcribed and amplified as DNA for further manipulation and protein expression. This permits the simultaneous isolation of a functional nascent protein, through affinity for a ligand, together with the encoding mRNA. Ribosome display can display very large libraries, suitable for generating toxic, proteolytically sensitive and unstable proteins, and also allows amino acids modification at defined position.
Creative Biolabs provides Escherichia coli S30 cell-free lysate for ribosome display, either with coupled or uncoupled transcription and translation.
Figure 1. E. coli ribosome display: (I) using coupled E. coli S30 extract and (II) using uncoupled E. coli S30 extract. (He and Taussig 2002)
Coupled E. coli S30 Extract
Coupled E. coli ribosome display system uses synthetic DNA library encoding random peptide sequences as the template to generate PRM. Chloramphenicol can be added to this system for stopping translation. Those PRM complexes displaying interacting peptide epitopes were captured with an immobilized ligand by panning on microtiter wells. The trapped PRM complexes are disrupted with EDTA to release the bound mRNA. The genotype mRNA was then converted and amplified into cDNA by RT-PCR.
Uncoupled E. coli S30 Extract
To avoid any possible disruptive effect of dithiothreitol (DTT) on the folding of proteins through reduction of disulfide bridges, prokaryotic ribosome display has been improved to carry out in an uncoupled format. “Uncoupled” means the transcription and translation stages are performed separately. In this system, mRNA is obtained from native sources or in vitro transcription, and it is introduced into an E. coli S30 translation system lacking DTT. In this system, a number of additional components need to be added. Take displaying folded single-chain antibody fragments (scFv) as an example, protein disulfide isomerase, vanadyl ribonucleoside complexes, and anti-ssrA anti-sense oligonucleotide are added to the translation mixture to promote folding of antibody fragments, stabilize mRNA and inhibit the action of ssrA RNA, respectively. The trapped PRM complexes are disrupted with EDTA to release the bound mRNA. The genotype mRNA was then converted and amplified into cDNA by RT-PCR.
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