Date of Award

2013

Document Type

Master Thesis

Degree Name

Masters of Science (Research)

Department

Biological Sciences

First Advisor

Dr. Aidan Coffey

Abstract

This project investigated an anti-staphylococcal bacteriophage-derived peptidoglycan hydrolase enzyme, namely a cysteine-histidine amydohydrolase/peptidase (CHAPk). The study focused firstly on optimizing the production of recombinant CHAPk, which was previously cloned in an E. coli expression system, and sets out the resulting optimal Standard Operating Procedures (SOPs) for production, purification and determination of concentration and activity of the resulting protein stock. A typical yield resulting from the investigation was 300pg of CHAPk from one litre of E.coli culture. The activity of the purified enzyme was typically in the region of 190U nmol ’. Secondly, the project investigated the application of CHAPk to eliminate S. aureus from blood. It was found that concentrations of 200 and lOOμg mP' of the enzyme eliminated S. aureus DPC5246 (2.5 x l05 CFU ml -1) within 30 minutes and one hour, respectively. In the third and final part of the project, the gene encoding CHAPk was cloned into the Lactococcus lactis-derived vector pNZ44 with the aim of introducing it into a probiotic Lactobacillus strain as a model for secretion of endolysins in lactic acid bacteria. This vector uses the pediocin operon to drive the secretion of recombinant protein. A variety of cloning strategies were investigated.

Access Level

info:eu-repo/semantics/openAccess

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