For the generation of Affitins, we use ribosome display, a poweful technique for the selection and evolution of protein/ligand interactions. Originally, it has been developped for the selection of peptides by Mattheakis et al and its potential has been extended to proteins by Hanes et al. In the ribosome display system, the link between the phenotype and the genotype is ensured by the ribosome. Its main advantage is to fully take place  in vitro. Thus,  by skiping transformation step to make DNA library enter into cells, large libraries of more than 1012 variants can be easlily created in few days. Moreover, additional diversity can be introduced during selections via an error-prone PCR for instance), making ribosome display an evolution method. Selections are usually performed against pure recombinant proteins. Recently, we showed that ribosome display selections can be performed against whole-bacteria to isolate binders against surface-exposed epitopes.

A selection round consists in the following steps:

  • transcription of the DNA library
  • translation of the mRNA library
  • binding of mRNA-ribosome-protein ternary complexes to the target of interest
  • washings to discard ternary complexes without affinity for the target
  • elution of selected mRNA
  • reverse transcription of mRNA into cDNA
  • amplification of selected sequences by PCR

Usually three to five rounds are necessary to get a sufficient enrichment for the screening of clones. This screening step can be done by ELISA or FACS, for instance.

A round of ribosome display selection