Masters of Science / The three-dimensional structures of proteins are important in understanding their function and interaction with ligands and other proteins. In this work, the structures of two proteins, ImuA’ from mycobacterium tuberculosis and GH9 C1 cellulase from a metagenomic library, were analysed using structural biological and modelling techniques. The gene encoding ImuA’ was amplified by two-step PCR, cloned, and expressed in E. coli. The recombinant ImuA’ produced was found to be largely insoluble. The insoluble protein was successfully solubilized in 8M urea but refolding the protein to its native structure was unsuccessful. By homology modelling, a 3D model of ImuA’ was obtained from a partly homologous protein RecA. In comparison to RecA, ImuA’ appears to lack some loop amino acids critical for DNA binding. Hence ImuA’ is postulated to not bind DNA. The second protein, GH9 C1 cellulase, was produced in E. coli. The protein was purified by chromatographic techniques and crystallized in a precipitant to protein ratio of 1:2 by hanging and sitting drop crystallization methods. The reservoir solution was made up of 15-30% (w/v) PEG 3350, 200 mM salt and 100 mM Tris-HCL pH 7.5-8.5. The protein crystals only diffracted x-rays to 4 å resolution which could not be used to obtain a crystal structure of the protein. The diffraction data, however, showed the crystal to be monoclinic with space group P2. Homology modelling revealed GH9 C1 cellulase to be a two domain protein with a smaller N-terminal Ig-like domain and a larger catalytic domain.The catalytic domain retains two ca2+ binding sites, which potentially stabilize the active site conformation and increase thermostability of the protein. Overall GH9 C1 cellulase is structurally similar to other GH9 cellulases, suggesting that its catalytic mechanism may be conserved.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:uwc/oai:etd.uwc.ac.za:11394/4320 |
| Date | January 2014 |
| Creators | Anye, Valentine |
| Contributors | Schubert, Wolf-Dieter, Warner, Digby |
| Publisher | University of the Western Cape |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Rights | University of the Western Cape |
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