Show simple item record

dc.contributor.advisorBeukes, Mervyn.
dc.contributor.advisorWatson, Gregory.
dc.creatorMia, Rizwana.
dc.date.accessioned2014-06-05T13:33:38Z
dc.date.available2014-06-05T13:33:38Z
dc.date.created2010
dc.date.issued2010
dc.identifier.urihttp://hdl.handle.net/10413/10840
dc.descriptionThesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2010.en
dc.description.abstractThe disease listeriosis is caused by Listeria monocytogenes. This common food-borne disease has been responsible for about 0.1 to 10 cases per million inhabitants per year. However, this disease is serious with its high fatality rates of 20% - 30%, and 40% of all cases reported have been in pregnant women suffered from a foetal abortion. Recently the organism has acquired resistance to antibiotic treatment and the development of an alternative treatment is necessary. Class IIa bacteriocins such as leucocin A have been shown to be active against L. monocytogenes. However, the leucocin A receptor molecule responsible for growth inhibition within L. monocytogenes remains unclear. Various studies have implicated the mannose PTS permease (EIIt Man) of L. monocytogenes as the putative receptor for class IIa bacteriocins. The results from studies reviewed indicate that the EIIt Man of L. monocytogenes could be the chiral receptor needed for bacteriocin interaction at the surface of targeted cells. Specifically, the membrane associated IIDMan and IICMan subunits were implicated in direct interaction with class IIa bacteriocins. Our study focused on cloning, expression and purification of the subunits of the mannose PTS permease of L. monocytogenes EGD. Primers were designed to amplify the subunit genes of the mptACD operon. The mptC, mptD and mptAB genes which were then successfully cloned into pET28a expression vector and transformed into E. coli JM109(DE3) host strain. Recombinant plasmids were screened using colony PCR. Subsequently recombinant pET28-C, pET28-D and pET28-AB was once again transformed and expressed in the E. coli BL21(DE3) pLysS expression host strain. After an induction at 30°C for 5 hours, IICMan and IIDMan were found to be expressed in the cell membrane, whilst IIABMan was expressed in the cytosol of the host expression strain. Membrane proteins His-IICMan, His- IIDMan, and cytosol associated His-IIABMan were purified using Ni2+-NTA affinity chromatography. Results for His-IICMan yielded a 28 kDa protein and a 55 kDa co-purified protein. Results for His-IIDMan yielded a 31 kDa protein and a 60 kDa co-purified protein. Results for His-IIABMan yielded a 35 kDa protein and a 68 kDa co-purified protein. A western blot analysis revealed that all proteins purified carried an attached His-tag as detected by an anti-mouse peroxidase conjugate anti-His-tag antibody.en
dc.language.isoen_ZAen
dc.subjectListeria monocytogenes.en
dc.subjectBacteriocins.en
dc.subjectListeria monocytogenes--Geneticsen
dc.subjectMannose.en
dc.subjectProteins.en
dc.subjectListeriosis.en
dc.subjectTheses--Genetics.en
dc.titleCloning, expression and purification of the subunits of the Mannose PTS Permease of Listeria monocytogenes EGD.en
dc.typeThesisen


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record