Phage Display - General Protocol

General Protocol

Below is the sequence of events that are followed in phage display screening to identify polypeptides that bind with high affinity to desired target protein or DNA sequence:

1. Target proteins or DNA sequences are immobilised to the wells of a microtiter plate.
2. Many genetic sequences are expressed in a bacteriophage library in the form of fusions with the bacteriophage coat protein, so that they are displayed on the surface of the viral particle. The protein displayed corresponds to the genetic sequence within the phage.
3. This phage-display library is added to the dish and after allowing the phage time to bind, the dish is washed.
4. Phage-displaying proteins that interact with the target molecules remain attached to the dish, while all others are washed away.
5. Attached phage may be eluted and used to create more phage by infection of suitable bacterial hosts. The new phage constitutes an enriched mixture, containing considerably less irrelevant phage (i.e. non-binding) than were present in the initial mixture.
6. Steps 3 to 6 are optionally repeated one or more times, further enriching the phage library in binding proteins.
7. Following further bacterial-based amplification, the DNA within in the interacting phage is sequenced to identify the interacting proteins or protein fragments.