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Description
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.
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Creative Biolabs
protein production;hybridoma;immunoassay;tissue array;antibody engineering
Address: 45-16 Ramsey Road Shirley,
New York, New York
United States, 11967
Tel: 1-631-871-5806
Fax: 1-631-207-8356
