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Introduction

After SELEX, ribosome display (RD) is probably the earliest in vitro selection techniques with its first variant reported in 1994. It was developed largely as a quick and simple alternative to the phage display. To this day, it is probably the most common in vitro selection method which is typically used in finding protein- (e.g. single-chain antibodies) and peptide-binders.

Principle of the Ribosome Display

RD is based on the cell-free protein synthesis reaction except that the translated product is trapped on the translating ribosome along with its corresponding mRNA. This way both newly translated protein and the mRNA encoding it are kept physically connected throughout the selection procedure. Sequences of the proteins that bind tightest to the bait which is normally attached to a surface (chromatography column) get extracted from mRNA by firstly reverse transcribing it and then sequencing obtained DNA. There are both prokaryotic and eukaryotic versions of this protocol as well as one that is based on a fully reconstituted translation system PURE.

The most popular RD protocol is based on the extract from Escherichia coli as it is relatively easy to make and many versions are commercially available. The use of crude E. coli extract provides both experimentally and financially viable route to this technology making this technique available to many laboratories.

Challenges and limitations

All-in vitro techniques, while being useful, come with an extensive optimisation burden. In contrast to the selection in living cells, in vitro systems do not inherently possess self-regulatory mechanisms thus conditions for a given in vitro selection system must be meticulously reproduced.

Secondly, success in setting up useful RD depends on two factors: high activity cell-free system (gladly, you simply can buy it) and stability of nascent chainRibosomemRNA complex. While the use of the crude extracts (E. coli or rabbit reticulocyte) are perfectly suitable for RD, the stability of proteinribosomemRNA complexes is heavily compromised. Ribosome complexes are dissociated due to the activity of E. coli endogenous factors that help cells recover from ribosomes stalling during cell growth. The mRNA is particularly prone for RNAse cleavage, while the nascent chain can be proteolytically degraded.

A eukaryotic version of RD is particularly useful for selection of hard to fold proteins or whenever an environment of eukaryotic cytosol is required.

Examples

Ribosome display is a potent tool for the selection of binders that has been developed further:

  • RD was used in high-throughput protein-protein interactions studies.
  • The use of the fully reconstituted translation mix (PURE) which is largely void of protease and nuclease activities has been finding application since the early 2000s. The notion of using PURE mix is particularly promising, as the production of this translation mixture was recently democratised.
  • Recently, RD coupled with ‘in vitro integrated synthesis, assembly, and translation’ (iSAT) was used in the engineering of the ribosome. Generally, RD is perfectly suitable for directed evolution of components of the translation apparatus.

Links to the protocols and references

The first report on the use of Ribosome display

Mattheakis, L. C., Bhatt, R. R., & Dower, W. J. (1994). An in vitro polysome display system for identifying ligands from very large peptide libraries. Proceedings of the National Academy of Sciences of the United States of America, 91, 9022–9026 https://doi.org/10.1073/pnas.91.19.9022

Following protocols are the most useful and detailed.

The most comprehensive protocols which are based on crude E. coli extract

Use of PURE translation mix is demonstrated in this methodological paper

A good example of the use eukaryotic RD is here