Ribosome Display - Ribosome Display Process

Ribosome Display Process

Ribosome display begins with a naive library of DNA sequences coding for polypeptides (Mattheakis, Bhatt and Dower (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci USA 91, 9022-9026). Each sequence is transcribed, and then translated in vitro into polypeptide. However, the DNA library coding for a particular library of binding proteins is genetically fused to a spacer sequence lacking a stop codon. This spacer sequence, when translated, is still attached to the peptidyl tRNA and occupies the ribosomal tunnel, and thus allows the protein of interest to protrude out of the ribosome and fold. What results is a complex of mRNA, ribosome, and protein which can bind to surface-bound ligand. This complex is stabilized with the lowering of temperature and the addition of cations such as Mg2+.

During the subsequent binding, or panning, stages, the complex is introduced to surface-bound ligand. This can be accomplished several ways, for example using an affinity chromatography column with a resin bed containing ligand, a 96-well plate with immobilized surface-bound ligand, or magnetic beads that have been coated with ligand. The complexes that bind well are immobilized. Subsequent elution of the binders via high salt concentrations, chelating agents, or mobile ligands which complex with the binding motif of the protein allow dissociation of the mRNA. The mRNA can then be reverse transcribed back into cDNA, undergo mutagenesis, and iteratively fed into the process with greater selective pressure to isolate even better binders.