Development of bacterial display peptides for use in biosensing applications

Stratis‐Cullum, Dimitra N.; Kogot, Joshua M.; Sellers, Michael S.; Hurley, Margaret M.; Sarkes, Deborah A.; Pennington, Joseph M.; Val-Addo, Irene; Adams, Bryn L.; Warner, Candice R.; Carney, James; Brown, Raymond Albert Joseph; Pellegrino, Paul M.

doi:10.1117/12.919782

Cited by 11 publications

(12 citation statements)

References 5 publications

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“…Binding to SEB and SAPE for the R441 sample, as measured by median fluorescence, is comparable to the unlabeled controls for R338, R418, and R445. The result is similar to using percentage of cells outside of a gated, buffer‐only control, which is the method previously utilized in peptide bacterial display studies (Kogot et al ., ; Stratis‐Cullum et al, ). The R418 clone was bound at 11.6% to PA, 4.5% to SAPE, and approximately 11% to α HA, and 10.2% to NAPE.…”

Section: Resultsmentioning

confidence: 99%

Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting

Kogot

Pennington

Sarkes

et al. 2014

J of Molecular Recognition

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Bacterial peptide display libraries enable the rapid and efficient selection of peptides that have high affinity and selectivity toward their targets. Using a 15-mer random library on the outer surface of Escherichia coli (E.coli), high-affinity peptides were selected against a staphylococcal enterotoxin B (SEB) protein after four rounds of biopanning. On-cell screening analysis of affinity and specificity were measured by flow cytometry and directly compared to the synthetic peptide, off-cell, using peptide-ELISA. DNA sequencing of the positive clones after four rounds of microfluidic magnetic sorting (MMS) revealed a common consensus sequence of (S/T)CH(Y/F)W for the SEB-binding peptides R338, R418, and R445. The consensus sequence in these bacterial display peptides has similar amino acid characteristics with SEB peptide sequences isolated from phage display. The Kd measured by peptide-ELISA off-cell was 2.4 nM for R418 and 3.0 nM for R445. The bacterial peptide display methodology using the semiautomated MMS resulted in the discovery of selective peptides with affinity for a food safety and defense threat. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Journal of Molecular Recognition published by John Wiley & Sons, Ltd.

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Section: Resultsmentioning

confidence: 99%

Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting

Kogot

Pennington

Sarkes

et al. 2014

J of Molecular Recognition

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“…Using the MMS and a bacterial display library enables the rapid isolation and subsequent identification of a peptide reagent as an antibody alternative in less than one week [27]; each round of MMS sorting with bacterial display requires one-day for target binding, MMS sorting, and overnight growth of enriched, positively selected clones. Besides being semi-automated, the MMS sorting is performed in closed-system with a disposable cartridge to limit user exposure to dangerous biological targets [24].…”

Section: Resultsmentioning

confidence: 99%

Binding specificity and affinity analysis of an anti-protective antigen peptide reagent using capillary electrophoresis

Kogot¹,

Sarkes²,

Pennington³

et al. 2014

ABB

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Peptide biosensor reagents are emerging as an alternative to typical antibody-based detection methods. Peptides can be rapidly isolated using bacterial display methods for new and emerging biothreats and can be chemically synthesized for rapid, large-scale production. With the emergence of peptide biosensor reagents, there is a growing need to develop methods for characterizing binding interactions. Capillary electrophoresis (CE) is a free-solution separation method that is able to determine target and analyte binding association (K b ) and dissociation constants (K d ). In this study, the K b , K d , and peptide specificity of an isolated peptide binding reagent to protective antigen (PA) of Bacillus anthracis were evaluated using capillary electrophoresis at 10 and 20 kV. The relative binding specificity was rapidly assessed by measuring the peptide relative mobility shift at 20 kV at nonequilibrium using bovine serum albumin (BSA), horseradish peroxidase (HRP), and an anti-PA monoclonal antibody (mAb). The αPA peptide was shown to be highly specific for PA, with a K d = 177 nM measured at 20 kV and K d = 312 nM measured at 10 kV. These results show that peptides from bacterial display libraries can be rapidly tested for specificity and binding affinity, in solution, for use as a potential biosensor reagent against new and emerging biothreats.

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“…For example, antibodies (including engineered antibodies) usually require several weeks if not months to isolate from living hosts, whereas recent advances have demonstrated that synthetic routes can be employed using various synthetic peptide recognition element technologies (e.g., bacterial display and phage display) to produce bioreceptors in as little as a few days to a couple of weeks Stratis-Cullum et al, 2012. In this work, the extremely fast replication rate of bacteria and biological components (e.g., modified proteins) on an E. coli cell surface are harnessed to produce the peptide library of binders (Daugherty, 2007;Kogot et al, 2011;Stratis-Cullum et al, 2012). Although, several variations of combinatorial peptide technologies, including yeast, phage, and bacterial display and other chemically synthetic techniques, are currently used to isolate synthetic reagents, bacterial display with unconstrained bacterial peptide display technology shows great potential.…”

Section: Bacterial Displaymentioning

confidence: 99%