| Summary: | Magister Scientiae - MSc (Biotechnology) === 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.
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