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 CIS display does not require cloning into cells but uses the extracted biochemical machinery from within the cells and a simple linear double stranded DNA template.
CIS display is adapted from a natural system in which a DNA binding protein (RepA) binds to the same template DNA from which it was derived. RepA binds to a site known as ori but an element known as CIS is necessary for this activity. It is thought that the CIS element stalls the RNA polymerase and enables loading of the translated protein onto ori.
By fusing peptide or protein libraries to RepA, the expressed peptide is attached to its coding DNA through attachment of RepA to ori. Therefore the sequence of the peptide can be determined by the sequence of its encoding DNA.
For large libraries of sequences, cycles of ‘panning’ are necessary in order to enrich the binding population and reduce the number of clones from trillions to hundreds which can be screened.
The process involves
1. Building of the library cassette
2. Libraries expressed in vitro –
3. Protein-DNA complexes subjected to affinity selection
4. Eluted complexes regenerated by PCR
Optimal ligands after 3-5 successive rounds
The selection can be monitored polyclonally by direct
measurement of DNA concentration. Cloning out and
screening isolated the best clones.
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