Abstract
BackgroundBacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates. These libraries typically express a scaffold protein embedded within the outer membrane with a short, surface-exposed peptide that is either terminal or is incorporated into an outer loop, and can therefore interact with and bind to substrates of interest.ResultsIn this study, we employed a novel bacterial peptide display library which incorporates short 15-mer peptides on the surface of E. coli, co-expressed with the inducible red fluorescent protein DsRed in the cytosol, to investigate population diversity over two rounds of biopanning. The naive library was used in panning trials to select for binding affinity against 3D printing plastic coupons made from polylactic acid (PLA). Resulting libraries were then deep-sequenced using next generation sequencing (NGS) to investigate selection and diversity.ConclusionsWe demonstrated enrichment for PLA binding versus a sapphire control surface, analyzed population composition, and compared sorting rounds using a binding assay and fluorescence microscopy. The capability to produce and describe display libraries through NGS across rounds of selection allows a deeper understanding of population dynamics that can be better directed towards peptide discovery.
Highlights
Bacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates
Library members with inserts that resulted in a frame shift, contained stop codons, or that had an empty scaffold made up 27%, 21%, and 13% of the naive library population, respectively
While we demonstrate the enrichment of peptides with affinity for polylactic acid (PLA) in this work, and that the enriched peptide library is not purely "sticky" since it does not bind to sapphire with similar affinity (Fig. 8b), the primary goal of this work was to demonstrate how next generation sequencing (NGS) and coexpression of fluorescent proteins can be used to enhance data analysis of display libraries in general, rather than to provide an optimized peptide sequence for binding PLA
Summary
Bacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates. These libraries typically express a scaffold protein embedded within the outer membrane with a short, surface-exposed peptide that is either terminal or is incorporated into an outer loop, and can interact with and bind to substrates of interest. Selection of particular molecular interactions is key to natural and engineered antibody-antigen recognition for diagnostics, new Surface display libraries employ polypeptides (including peptides, scaffold proteins, and antibody fragments) typically presented at the surface of phage coats or bacterial, yeast, or mammalian cell membranes. After incubating a library with a substrate of interest, the unbound members are washed away while the remaining bound members are amplified and used for further
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