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Proceedings Paper

Analysis of protective antigen peptide binding motifs using bacterial display technology
Author(s): Deborah A. Sarkes; Brandi L. Dorsey; Dimitra N. Stratis-Cullum
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Paper Abstract

In today’s fast-paced world, a new biological threat could emerge at any time, necessitating a prompt, reliable, inexpensive detection reagent in each case. Combined with magnetic-activated cell sorting (MACS), bacterial display technology makes it possible to isolate selective, high affinity peptide reagents in days to weeks. Utilizing the eCPX display scaffold is also a rapid way to screen potential peptide reagents. Peptide affinity reagents for protective antigen (PA) of the biothreat Bacillus anthracis were previously discovered using bacterial display. Bioinformatics analysis resulted in the consensus sequence WXCFTC. Additionally, we have discovered PA binding peptides with a WW motif, one of which, YGLHPWWKNAPIGQR, can pull down PA from 1% human serum. The strength of these two motifs combined, to obtain a WWCFTC consensus, is assessed here using Fluorescence Activated Cell Sorting (FACS). While monitoring binding to PA, overall expression of the display scaffold was assessed using the YPet Mona expression control tag (YPet), and specificity was assessed by binding to Streptavidin R-Phycoerythrin (SAPE). The importance of high YPet binding is highlighted as many of the peptides in one of the three replicate experiments fell below our 80% binding threshold. We demonstrate that it is preferable to discard this experiment, due to questionable expression of the peptide itself, than to try to normalize for relative expression. The peptides containing the WWCFTC consensus were of higher affinity and greater specificity than the peptides containing the WW consensus alone, validating further investigation to optimize known PA binders.

Paper Details

Date Published: 22 May 2015
PDF: 8 pages
Proc. SPIE 9455, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XVI, 94550A (22 May 2015); doi: 10.1117/12.2179472
Show Author Affiliations
Deborah A. Sarkes, U.S. Army Research Lab. (United States)
Brandi L. Dorsey, Federal Staffing Resources (United States)
Dimitra N. Stratis-Cullum, U.S. Army Research Lab. (United States)

Published in SPIE Proceedings Vol. 9455:
Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XVI
Augustus Way Fountain III, Editor(s)

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