Before the website disappears, the latest description of ribosome display -
Ribosome Display is a cell-free display technology, which exploits in vitro the cellular components that are involved in protein synthesis, to create libraries containing billions (about 1013) of different human antibody fragments and from which in turn, antibodies to target molecules can be rapidly isolated.
Ribosome Display is based on the formation of stable antibody-ribosome-mRNA complexes and the technique has similarities with Phage Display in that the antibody protein is directly linked to its encoding DNA sequence.
A ribosome is a large, complex molecule that is involved in protein production, present in the cytoplasm of all cells. In a living cell, mRNA, carries the DNA code from the cell's nucleus and binds to a ribosome. The ribosome then moves along the mRNA reading the gene sequence and assembles an amino acid chain of expressed protein. In a living cell, the completed protein is usually released and the mRNA falls off the ribosome and is eventually degraded. In Ribosome Display this process is carried out in solution in vitro and the complex remains intact.
CAT has developed vast libraries of antibody genes extracted from human cells. The genes are copied by standard PCR; transcription occurs and creates a population of mRNA molecules, each encoding for a different antibody gene. The mRNA molecules are then incubated with lysate-based ribosomes (bacteria derived ribosomes and their protein making machinery), which translate the mRNA into protein complexes – a Ribosome Display library.
Such complexes, each displaying a different antibody, are mixed with the target antigen and those antibodies that are specific to the target bind to it and non-binders are washed away.
However, since Ribosome Display is not dependent on the use of phage or bacteria and the limitations of cellular processes, library generation and antibody selection are more efficient and multiple selection strategies can be parallel-tracked. Ribosome Display therefore offers the potential to create even larger antibody libraries than Phage Display currently allows with greater antibody diversity and efficiency in the development of antibody therapeutics. The technique also provides the opportunity to isolate antibodies to antigens that are toxic to bacteria. In addition, as Ribosome Display is a fully in vitro process, the protein folding conditions can be carefully controlled. These advantages are currently exploited in CAT's Antibody Optimisation processes.
Ribosome Display is currently used in addition to CAT's Phage Display capabilities, but it is emerging as a useful independent tool for providing antibodies with therapeutic potential. The technique is as effective as Phage Display and because it is amenable to automation and can produce even larger antibody libraries, it has the prospect of becoming an increasingly important tool in the advancement of antibody therapeutics... |
