Phage display screening without repetitious selection rounds
2012
’t Hoen, Peter A.C. | Jirka, Silvana M.G. | ten Broeke, Bradley R. | Schultes, Erik A. | Aguilera, Begoña | Pang, Kar Him | Heemskerk, Hans | Aartsma-Rus, Annemieke | van Ommen, Gertjan J. | den Dunnen, Johan T.
Phage display screenings are frequently employed to identify high-affinity peptides or antibodies. Although successful, phage display is a laborious technology and is notorious for identification of false positive hits. To accelerate and improve the selection process, we have employed Illumina next generation sequencing to deeply characterize the Ph.D.-7 M13 peptide phage display library before and after several rounds of biopanning on KS483 osteoblast cells. Sequencing of the naive library after one round of amplification in bacteria identifies propagation advantage as an important source of false positive hits. Most important, our data show that deep sequencing of the phage pool after a first round of biopanning is already sufficient to identify positive phages. Whereas traditional sequencing of a limited number of clones after one or two rounds of selection is uninformative, the required additional rounds of biopanning are associated with the risk of losing promising clones propagating slower than nonbinding phages. Confocal and live cell imaging confirms that our screen successfully selected a peptide with very high binding and uptake in osteoblasts. We conclude that next generation sequencing can significantly empower phage display screenings by accelerating the finding of specific binders and restraining the number of false positive hits.
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