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Protein-Binding Ligand Screening by Ribosome Display

Ribosome display can be used to select high affinity, specific ligand-binding peptides from combinatorial libraries. Although the identification of protein-binding ligands is commonly used by phage display, ribosome display can avoid library limitation and is capable of screening much larger libraries. Creative Biolabs has developed the in vitro display system for screening ligands from large libraries.

Ribosome display is a selection and evolution technology for proteins and peptides from large libraries, and it is performed entirely in vitro. In this technique, the genotype (RNA, DNA) and phenotype (protein) are linked during in vitro translation by stabilizing the complex consisting of the ribosome, the mRNA and the corresponding folded polypeptide (protein-ribosome-mRNA, PRM). The DNA library coding for a particular library of binding proteins is genetically fused to this system. Once the target protein is identified, the mRNAs are isolated from those target-binding complexes, reverse transcribed and amplified as DNA for further manipulation and protein expression. Ribosome display is especially suitable for generating toxic, proteolytically sensitive and unstable proteins, and also allows amino acids modification at defined position.

Creative Biolabs can use ribosome display technology to screen peptides binding to the antibody (antigen epitope), or other protein from very large combinatorial peptide libraries (As large as 1012-1014). We have extensive experience to generate high-quality antibody libraries, which can largely accelerate in vitro display process. After several rounds of affinity selection, sequences encoded peptides that bound specifically to the immobilized receptor and contained sequences similar to the epitope sequence can be identified. Both prokaryotic ribosome display system and eukaryotic ribosome display system can be used for ligand screening.

Ribosome display system for screening peptide libraries Figure 1. Ribosome display system for screening peptide libraries. (Coupled E. coli system, Mattheakis et al. 1994)

Typical Procedure

  1. Synthesizing a DNA library encoding a random NNK codon region.
  2. The DNA library is incubated in a prokaryotic or eukaryotic transcription/translation system.
  3. Stopping protein synthesis with chloramphenicol and isolating PRM complexes by centrifugation.
  4. PRM complexes are added to wells containing an immobilized receptor for affinity selection.
  5. Receptor-binding PRMs are disrupted with EDTA and mRNAs are recovered (for E. coli system) or using in situ RT-PCR procedure without dissociation of the ribosome complex (for rabbit reticulocyte lysate system).
  6. mRNAs inverse transcript to cDNA.
  7. cDNA is amplified by PCR.
  8. A portion of the enriched pool is cloned into a phagemid vector for ELISA and sequencing before repeating the cycle.

For more detailed information, please feel free to contact us or directly sent us an inquiry.

References

  1. He M and Taussig M J (2002) “Ribosome display: cell-free protein display technology.” Brief Funct Genomic Proteomic 1(2):204-212.
  2. Mattheakis L C, Bhatt R R and Dower W J (1994) “An in vitro polysome display system for identifying ligands from very large peptide libraries.” Proc. Natl. Acad. Sci. USA 91: 9022–9026.
  3. Gersuk G M, Corey M J, Corey E et al. (1997) “High-affinity peptide ligands to prostate-specific antigen identified by polysome selection.” Biochem. Biophys. Res. Commun. 232: 578–582.



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