SGER: Novel Antimicrobial and Antiviral Properties of the Hok Killer Protein

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9615938 Wood The goal of this proposed research is to investigate the use of the Hok killer peptide as a completely new antibiotic and antiviral system. The potential efficacy of the Hok peptide as a broad, non-specific, antiparasitic drug will be determined by testing its activity against relevant bacteria, fungi, and phage. This research highly novel and exploratory since the extracellular antibiotic application of Hok peptide is largely untested. However, the antibiotic idea is sound since almost all bacteria tested to date are susceptible to killing by Hok when it is expressed intracellularly, and the PI has recently shown that phage T4 (a virus that destroys bacteria) can be excluded when the Hok protein is expressed in E. coli cells. In order to investigate the broad range effectiveness against human pathogens, the Hok peptide will be overexpressed using recombinant bacteria, purified using gel filtration and reverse-phase, high- performance-liquid chromatography, and identified through fast-atom-bombardment mass spectroscopy. Using the purified peptide, antimicrobial assays will be performed to determine if the peptide is capable of slowing or preventing bacterial and fungal growth. To thwart the propagation of virus in animal cells in a manner analogous to how the Hok protein excludes T4 phage by killing the host before the virus can propagate, a general suicide system for animal cells will be developed to combat virus-based diseases. This will require controllable expression of a killer protein nd activity in a wide variety of animal cells. Initially, the ability of Hok to kill insect cells (as representative animal cells) will be evaluated by using the baculovirus transfer vector pVL1392 for Hok expression in insect cells under the polyhedrin promoter. ***