Custom Ribosome Display Services

Ribosome display is a powerful in vitro method for directed evolution, which is widely used in selection of peptides, antibodies or proteins of unique binding properties from large libraries.

A prerequisite for library screening is the coupling of genotype (RNA or DNA) and phenotype (protein). In ribosome display, this link is the complex formed during in vitro translation, which consists of a ribosome, an mRNA and a nascent, correctly folded polypeptide. In a ribosome display library, protein-encoding DNA 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 to protrude out of the ribosome and fold. What results is a complex of mRNA, ribosome and protein. During the subsequent binding, or panning, stages, the complexes are screened against a surface-bound ligand. The complexes whose proteins bind well are immobilized; whereas non-bound complexes are washed away. Subsequent elution of the binders via high salt concentrations, chelating agents, or mobile ligands allows dissociation of the mRNA. The mRNA can then be reverse transcribed back into cDNA, PCR amplified or undergone mutagenesis, and iteratively fed into the screening process with greater selective pressure to isolate even better binders. After screening, the mRNA can be recovered by RT-PCR for expression and identification of the proteins.

Ribosome display library screening enables the isolation of peptide, antibodies or proteins against virtually any targets, including self-molecules, certain drugs, potent toxins and haptens, which would normally be impossible to raise with in vivo based systems because of toxicity and/or lack of immunogenicity.

Since it is performed entirely in vitro, ribosome display has two main advantages over other selection technologies. First, the diversity of the library (10¹³-10¹⁵) is not limited by the transformation efficiency of in vivo systems, e.g. bacterial host cells, but only by the number of ribosomes and different mRNA molecules present in the test tube. Second, additional sequence diversification can be introduced easily by PCR amplification of the mRNA after each round of screening. For the same reason, directed evolution of the binding proteins can be performed over several generations by using an error-prone polymerase or by shuffling the library DNA between rounds of screening.

Fig 1. Illustration of ribosome display cycle.

Applications
           •   Peptide screening
           •   Antibody screening
           •   Antibody affinity maturation and
           •   Directed evolution of proteins

Featured Services

Ribosome Display Library Construction

• Ribosome display peptide library
Ribosome display can be used to select high affinity, specific ligand-binding peptides from combinatorial libraries. Peptides binding to a monoclonal antibody with affinities ranging from 7.2 to 140 nM were selected from a random decapeptide library of 10¹² members using a coupled E. coli S30 system. Similarly, peptides binding to specific antigen with affinities of 0.8–20 nM were obtained from a random 20-mer peptide library (10¹² members) using an uncoupled wheat germ system.
• Ribosome display antibody library
Ribosome display is employed in immunized, na"ive, semi-synthetic and synthetic antibody libraries. Using an E. coli display system, picomolar affinity antibody fragments against peptides and proteins were selected from an immunized mouse library and a human synthetic antibody library. Antibody fragments, which recognize different DNA structures/ conformations formed by identical sequences, were obtained. Eukaryotic ribosome display was used to select fully human antibody fragments from an immunized transgenic mouse library.

Ribosome Display Library Screening

• Prokaryotic ribosome display system
The prokaryotic systems use the Escherichia coli S30 extractor the PURE (protein synthesis using recombinant elements) system, composed of purified E. coli proteins and factors that are essential for translation.
• Eukaryotic ribosome display system
For eukaryotic ribosome display, either the rabbit reticulocyte lysate system or wheat germ extracts can be used.