Using Phage Display to Select Proteins with High Affinity to a Target Protein
Transcript
Begin phage display by adding a phage library to the wells of a multi-well plate, coated with target protein. The phage library contains genetically engineered phages, each displaying an individual protein variant fused to a coat protein on its surface. Incubate, allowing the phage and target proteins to interact.
Phages with proteins with a high target protein affinity bind to the target protein. Wash with a buffer, removing unbound phages. Add an acidic solution, disrupting the phage and target protein interactions, releasing phages into the solution. Neutralize the solution pH using a buffer, maintaining phage stability.
Transfer the phages to a fresh tube. Incubate with a blocking solution containing proteins, preventing non-specific interactions.
Transfer the phages to an actively-dividing E. coli suspension, infecting bacteria and initiating the phage replication cycle. Supplement with helper phages, which infect the bacteria, providing coat proteins necessary to package the progeny phages. Transfer the suspension into a selective medium, allowing phage-infected bacteria growth.
The progeny phages eventually get released into the medium. Centrifuge. Collect the supernatant containing phages. Add a precipitation solution, concentrating the phage particles. Centrifuge. Resuspend the phage pellet in a buffer, obtaining a homogenous suspension.
Perform subsequent phage selection rounds; repeat the phage-target protein binding, elution with an increased buffer wash stringency step, and amplification and centrifugation steps, resulting in the enrichment of phage pools displaying proteins with high affinity to the target protein.
