Browsing by Author "Boonzaier, Jeremy"

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    Structural Analysis of Induced Mutagenesis Protein B from Mycobacterium tuberculosis Jeremy
    (University of the Western Cape, 2016) Boonzaier, Jeremy; Schubert, Wolf-Dieter
    Knowing the three-dimensional structure of a protein may be useful in understanding its function. In this study, induced mutagenesis protein B (ImuB) from Mycobacterium tuberculosis was analyzed using molecular biology and molecular modelling techniques. The Rv3394c gene expressing ImuB was obtained from the group of Prof. Digby Warner at the Institute of Infectious Diseases and Molecular Medicine, University of Cape Town. Rv33974c was amplified from an expression plasmid using polymerase chain reaction (PCR) and inserted into multiple expression vectors. The pMal-c2X-Rv3394c construct was most successful in producing ImuB as a fusion protein with N-terminal maltose binding protein in an E. coli expression systems. Attempts were undertaken to refold insoluble ImuB. Soluble MBP-ImuB was purified by affinity chromatography and size-exclusion chromatography. Purified MBP-ImuB was concentrated and used for hanging drop crystallization experiments. Crystallization of ImuB remained elusive as protein crystals did not form. A homology model of ImuB was generated based on structurally related Y-family DNA polymerases. ImuB, however, lacks the catalytic residues required for DNA replication. Sequence analysis an identified a potentially disordered C-terminal domain. Together, this would suggest that ImuB is not directly responsible for induced mutagenesis but is required as an accessory protein for induced mutagenesis to occur.