Analysis of protective antigen peptide binding motifs using bacterial display technology

Deborah A. Sarkes; Brandi L. Dorsey; Dimitra N. Stratis-Cullum

doi:10.1117/12.2179472

22 May 2015 Analysis of protective antigen peptide binding motifs using bacterial display technology

Deborah A. Sarkes, Brandi L. Dorsey, Dimitra N. Stratis-Cullum

Author Affiliations +

Proceedings Volume 9455, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XVI; 94550A (2015) https://doi.org/10.1117/12.2179472
Event: SPIE Defense + Security, 2015, Baltimore, MD, United States

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.

Citation Download Citation

Deborah A. Sarkes, Brandi L. Dorsey, and Dimitra N. Stratis-Cullum "Analysis of protective antigen peptide binding motifs using bacterial display technology", Proc. SPIE 9455, Chemical, Biological, Radiological, Nuclear, and Explosives (CBRNE) Sensing XVI, 94550A (22 May 2015); https://doi.org/10.1117/12.2179472

ACCESS THE FULL ARTICLE

INSTITUTIONAL
Select your institution to access the SPIE Digital Library.

SELECT YOUR INSTITUTION

PERSONAL
Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.

PERSONAL SIGN IN

No SPIE Account? Create one

;

PURCHASE THIS CONTENT

SUBSCRIBE TO DIGITAL LIBRARY

50 downloads per 1-year subscription

Members: $195

Non-members: $335 ADD TO CART

25 downloads per 1 - year subscription

Members: $145

Non-members: $250 ADD TO CART

PURCHASE SINGLE ARTICLE

Includes PDF, HTML & Video, when available

Members: $17.00

Non-members: $21.00 ADD TO CART

PROCEEDINGS
8 PAGES

DOWNLOAD PAPER SAVE TO MY LIBRARY

GET CITATION

RIGHTS & PERMISSIONS

Get copyright permission Get copyright permission on Copyright Marketplace

KEYWORDS

Luminescence

Display technology

Proteins

Analytical research

Bioinformatics

Biological detection systems

Biological research

Show All Keywords

Keywords/Phrases

Search In:

Publication Years