Phage display screening without repetitious selection rounds

Abstract

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.

Deep sequencing analysis of phage libraries using Illumina platform

“…The bias we observe is unlikely to be present in the naïve library, which should contain up to 10 9 clones according to the manufacturer (New England Biolabs). Indeed, sequencing of naïve (non-amplified) libraries demonstrated that there is little bias to specific sequences in the library [14].…”

“…Fields and co-worker used Illumina sequencing to characterize selection from libraries of WW-protein displayed on T7 phage [13]. Johan den Dunnen and co-worker used Illumina to characterize peptide libraries after one round of panning against cell surface receptors [14]. In this paper, we present a one-step PCR that converts a library of M13KE plasmids isolated from the phage library to a collection of short dsDNA sequences suitable for Illumina sequencing.…”

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

“…This methodology, although useful, is limited by the number of clones selected for individual Sanger sequencing after the last round of selection, and the selection of phages based on competitive growth advantages unrelated to enzyme specificity. The availability of high-throughput DNA sequencing technology (11) has facilitated detailed analysis of the changing complexity within a phage display library (12)(13)(14)(15)(16) without requiring multiple rounds of selection and amplification. By sequencing millions of phage particles, changes in the composition of the library can be precisely monitored.…”

Next generation phage display by use of pVII and pIX as display scaffolds

“…As there is a growing need for improved targeted therapeutic intervention [11,12], there is a strong incentive for continuous refinement of the technology, and initiatives such as the European Proteome Binders consortium [13] has been heavily depending on phage display as their primary discovery platform [14,15]. Also, other technological innovations are likely to improve performance and throughput beyond that seen today, such as automated selection [16] and next generation sequencing used in downstream screening [17][18][19][20].…”

“…As shown by several independent studies, clone dependent growth advantage is operational and an inherent part of phage display. This leads to unwanted target independent repertoire bias during selection [19,67,68]. The result is that clones with potentially desirable phenotypes are lost, either during the initial library packaging or later, during selection.…”