Question

Why do you repeat affinity selection (biopanning) 3-5 times in surface phage display?

Answer 1

Biopanning is a procedure of affinity selection that selects for peptides that can bind to a given target. All peptide sequences collected from biopanning are stored in peptide libraries.

Phage display is a process of presenting polypeptides on the surface of bacteriophage which is a virus-infected bacterial cell. A gene encoding the desired protein is injected into a phage coat protein gene, causing the phage to display the protein on the outside. There is also a gene for the protein inside the phage. This shows the connection between genotype and phenotype.

These displaying phages can be selected for other proteins, peptides or DNA sequences, to analyze the interaction between the displayed protein and those other molecules. So, it has become one of the most widely used laboratory techniques for the study of protein-protein, protein-peptide, and protein-DNA interactions.

With the help of rDNA technology, foreign cDNA is incorporated into phage DNA. Different sets of genes are injected into the genes of various phages. The gene for a phage coat protein is sliced so that the protein will be displayed on the outside of phage, and these separate phages will only display one protein, peptide, or antibody. Collections of these phages consist of libraries, such as a protein phage library, antibody phage library, or random phage library.

Then these phage libraries are displayed to selected targets and only some phages will interact and bind with targets. Unbound phages can be washed away, and only those which has an affinity for the receptors were left. Recovery of the target bound phage is done by elution (extraction).

Extracted phages showing specificity are selected to infect new host cells for the amplification process.

This phage display process is repeated 3-5 times for the selection of the most suitable binding sequence. After that, enrichment and purification of the phage collection are done by precipitation methods to increase the phage titer.