The next generation of biopanning: next gen sequencing improves analysis of bacterial display libraries

Abstract

Background: 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 therefore interact with and bind to substrates of interest. Results: In 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. Conclusions: We 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.

Discovery of Targeted Material Binding Microorganisms Using a Centrifugal Microfluidic Platform

“…Next‐generation sequencing (NGS) was performed to examine trends in the amino acid content of the peptide sequences associated with gold and ITO binding. [ 44 ] Using NGS data, the frequency of amino acid occurrence in the eCPX 3.0 library and the samples from round 4 of library sorting against gold and ITO was examined using an analysis similar to that employed by Adams et al. [ 21 ] and by Thai et al.…”

Generation of peptides using phage display technology for cancer diagnosis and molecular imaging

“…The last step is to repeat the aforementioned steps several times, normally three to four times in order to ensure the enrichment of a population of best binders. Sequencing of the individual phage clone genome that encodes the displayed peptide can be carried out to determine the ligands that bind specifically to the target receptors [22].…”

High-Throughput Monoclonal Antibody Discovery from Phage Libraries: Challenging the Current Preclinical Pipeline to Keep the Pace with the Increasing mAb Demand

“…During the amplification step, however, these clones can incorporate protein pIII supplemented by the helper phage, thus acquiring an enhanced growth rate. Moreso, clones bearing truncated forms of fusion proteins (e.g., clones bearing large in-frame deletions of scFv) can acquire a growth rate advantage due to lower interference between pIII and F pilus of bacteria needed for amplification [ 71 , 72 , 73 ]. This well-known issue can be at least in part circumvented by the exploitation of helper phages lacking effective infectivity domains of protein pIII to make phages infective only in the presence of full-length scFv-pIII fusion protein [ 74 ].…”