In vitro compartmentalization and deep sequencing enable discovery of functional cell-binding ligands by phage display

• Author / Creator

  Matochko, Wadim L

• Phage display is a technique that accelerated the discovery of peptide and protein-based ligands to numerous targets in academia and industry. Many FDA-approved antibodies and peptides on the market have originated from phage display experiments. However, one of the main drawbacks to this technique is that there are two independent steps in the selection process, an enrichment step for ligands with binding affinity towards a target and an amplification step to amplify the enriched phage clones. The amplification step introduces a bias towards enriching phage clones with a different phenotype based on growth rate. The implication of this bias is that target-binding ligands are enriched from a small subset, with low diversity, of the phage library that contains fast growing phage clones. Furthermore, selection from the fast growing phage population gives rise to a large number of target unrelated ligands. This undesired amplification bias is especially detrimental when selecting for ligands against multi-site binding target, such as cell or tissues. In this thesis, we examine whether the collapse of diversity can be prevented in phage libraries by amplifying these libraries in emulsions. We show that preventing the diversity collapse, we can identify more ligands than the standard selection method and speed up the discovery of ligands to targets with multiple binding sites. This thesis first describes the development of the emulsion amplification technique. We describe the manufacturing of the microfluidic devices and synthesis of the perfluoro-surfactant needed to maintain stable emulsions throughout the amplification process. To analyze the amplification process, we develop a method for deep-sequencing of phage display libraries using Illumina and Ion Torrent platforms, as well as MATLAB scripts, to analyze deep-sequencing data. We applied deep sequencing to examine how diversity of peptides in phage display libraries changes as a result of amplification of libraries in bacteria. Using a Ph.D.-12 library as our model library, we observed that amplification enriches ~150 clones, which dominate ~20% of the library. Deep sequencing, for the first time, characterized the collapse of diversity in phage libraries. We extend the use of next-generation sequencing to characterize the Ph.D. 7 library. Using Illumina and Ion Torrent sequencing and multiple biological replicates of amplification of the Ph.D.-7 library, we identified a focused population of 770 sequences that grow quickly, we term these sequences ‘parasites’. In all, 197 sequences from this population have been identified in literature reports that used Ph.D.-7 library. Many of these enriched sequences have confirmed function (e.g., target binding capacity). The bias in the literature, thus, can be viewed as a selection with two different selection pressures: i) target-binding selection, and ii) amplification-induced selection. Enrichment of parasitic sequences could be minimized if amplification bias is removed. Here, we demonstrate that emulsion amplification in libraries of ~106 diverse clones prevents the selection of parasitic clones. We examine if emulsion-amplification can prevent enrichment of parasitic clones in selection against a multi-site target, here we use MDA-MB-231 breast cancer cells. We perform selection using the standard method to amplify phage libraries in one common bulk solution (bulk amplification). We reproducibly identified peptide ligands for breast cancer cells from a ~0.0001% sub-population of the library, which harbors fast-growing, “parasitic” phage. Replacing bulk with emulsion-amplification dramatically altered the selection landscape and yielded ligands from the regions of the library not accessible to bulk-amplification selection by preventing diversity collapse during amplification. We propose incorporating emulsion-amplification into selection against multi-site targets (cells, antibody mixtures, etc.), can lead to the discovery of ligands missed by conventional selection strategies.

• Subjects / Keywords

  • Phage display
  • Deep-sequencing
  • In vitro compartmentalization

• Graduation date

  Fall 2015

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R30C4SS9B

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.