Burgos-Muñiz, Ricardo

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    Isolation of interacting peptides to Bacillus anthracis lethal toxin (LF) by T7 phage display
    (2010) Burgos-Muñiz, Ricardo; Ríos-Velázquez, Carlos; College of Arts and Sciences - Sciences; Martínez-Cruzado, Juan C.; Santos Flores, Carlos J.; Department of Biology; Suárez, O. Marcelo
    During the last decade, Bacillus anthracis has reemerged as a biological weapon due to the occurrence of the first terrorist attack in U.S. soil with anthrax spores. Bacillus anthracis is the etiological agent of anthrax, a zoonotic deadly disease. The pathogenicity and virulence reside in the presence of a capsule and a three component protein exotoxin. The three components of anthrax exotoxin are the protective antigen (PA), the lethal factor (LF) and the edema factor (EF). The anthrax lethal factor is a protease that cleavage several isoforms of mitogen-activated protein kinase kinase (MAPKK). It is not yet known if the cleave sites of LF are exclusive to MAPKK, granting opportunities to map new interacting partners. In order to search for such interactions, functional proteomics and combinatorial chemistry approaches such as phage display have been developed using biomolecules of interest as main targets. T7 phage display system is an in vitro technique where a peptide is genetically fused to a coat protein of a bacteriophage to identify polypeptides by affinity selection, and has been used to identify novel interacting proteins of several pathogens. The main purpose of this research was to identify interacting partners to LF by T7 phage display. Four different screenings of human cDNA libraries from brain, heart, liver and lungs were performed using two variants of LF, a wild type and a mutant, as targets. Three rounds of biopanning were done to isolate phage particles displaying peptides with affinity to LF. The DNA of 380 phages was isolated and the cloned fragments amplified by PCR. The size of the clones varied between 400 and 1000 base pairs. In silico analysis of 161 sequenced fragments was done to search for the displayed peptides homologies and identities using available databases. Some of the predicted peptides sequences encode for kinases, phosphorylation proteins, tafazzin and ferritin. Also, specificity tests were done using different concentrations of LF, and other targets such as bovine serum albumin, ribonuclease and lysozyme. The results suggested that the peptides recognized and bound specifically only to LF. Interacting peptides isolated in this study will serve as baseline to understand new possible paths and interacting partners of pathogenicity, allowing the development of biomarkers, as well as blocking agents to the anthrax toxin action, pursuing new vaccines prototypes.