Purification and Functional Characterization of PlyN74 Endolysin for Antibacterial Applications

Abstract

Antibiotic resistance is a growing problem in the medical field, which requires the development of alternative medicines to treat patients with bacterial infections. Bacteriophage endolysin proteins such as N74 have been shown to target Bacillus cereus as well as Bacillus anthracis (anthrax). This study aims to characterize the effectiveness of N74 at facilitating lysis of Bacillus cereus and how lysis can be augmented through use of endolysins plyG and P56, in conjunction with N74. A recombinant version of the N74 protein with a 6x-HIS tag was overexpressed and purified from E. coli using sonication and Ni-NTA columns. We used a BCA assay to measure protein concentration. tested the activity of the protein by conducting spectrophotometric lysis assays and log-fold killing assays were used to characterize the lytic activity of N74 against B.cereus. Our results show that PlyN74 is indeed an effective protein to lyse B. cereus. The BCA assay was successful in showing that N74 was purified and the SDS-PAGE showed the proper band size in the gel. The log-fold killing assays showed a decrease in bacterial growth. Our results demonstrated the effectiveness of PlyN74 at lysing Bacillus cereus and characterized the effects of endolysins PlyG and P56 on lysis when used alongside PlyN74. While previous studies have shown that endolysins that lyse Bacillus cereus have been able to lyse Bacillus anthracis, further testing is required to determine the effectiveness of PlyN74 on Bacillus anthracis. The results will ultimately aid in the discovery of new alternatives to antibiotics.