The expression and purification of GlgE and GlgB, two enzymes involved in the synthesis of α-glucan in mycobacteria AND Large subunit ribosomal protein 41 is preferentially found in Drosophilia melanogaster larval midgut stem cells

Taylor, Sarah Maria (2015). The expression and purification of GlgE and GlgB, two enzymes involved in the synthesis of α-glucan in mycobacteria AND Large subunit ribosomal protein 41 is preferentially found in Drosophilia melanogaster larval midgut stem cells. University of Birmingham. M.Res.

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Abstract

Project 1:
The four step α-glucan synthesis GlgE pathway is involved in capsular layer production in \(Mycobacterium\) \(tuberculosis\) and two of the enzymes in the pathway, GlgE and GlgB, which are essential to cell viability have been determined as potential targets for new drugs to treat TB. This project involved expressing and purifying \(Mycobacterium\) \(tuberculosis\) and \(Mycobacterium\) \(smegmatis\) GlgE and GlgB using Ni-NTA chromatography, ion exchange chromatography and size exclusion chromatography in order to be able to characterise the enzymes. An activity assay indicated both proteins are active and small integrated crystals of \(M.\) \(tuberculosis\) GlgE were grown by vapour diffusion.

Project 2:
Ribosomal proteins have an essential role in ribosome biogenesis and function but research is beginning to show they can also have extra ribosomal functions while not part of the ribosome. This project characterised a number of transgenic \(Drosophila\) \(melanogaster\) strains expressing a number of ribosomal proteins tagged with YFP at their endogenous loci in salivary gland, fat body and midgut cells. Within the midgut RpL41-YFP showed strong expression in adult midgut precursor (AMP) stem cells. An immunostain using an antibody against the endogenous RpL41 confirmed higher expression in AMP stem cells and also in a small area within the nucleus of differentiated cells, but the specificity of the antibody used has yet to be confirmed. Visualisation of these proteins was also carried out during larval development and expression within the enterocyte nuclei was detected only during early second larval instar.

Type of Work:

Thesis (Masters by Research > M.Res.)

Award Type:

Masters by Research > M.Res.

Supervisor(s):