Cloning, Expression, Purification And Structure Modelling Of Β-Ketoacyl-Acp Synthase I Of Salmonella Typhi
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Date
2015-07
Authors
Fadzil, Nurulhani Shakila
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Abstract
Salmonella enterica serovar Typhi (S. typhi) is a human-specific pathogen that causes enteric typhoid fever. As years passed by, this bacteria has developed multidrug-resistance towards current antibiotics. Therefore, a new drug target is needed to overcome the emergence of new drug resistant S. typhi strains. One of the promising targets for the discovery of new antibiotics is β-ketoacyl-ACP synthase I (FabB) of S. typhi as it is one of the essential enzymes in fatty acid biosynthesis. This study attempts to clone, express and purify FabB protein from S. typhi and also to analyze its model structure. The 1.2 kb FabB gene from S. typhi genome was successfully amplified and cloned into an expression vector (pCold I DNA) for protein expression. The FabB gene was induced with 1 mM of β-D-thiogalactopyranoside (IPTG) at 15°C for 24 hours in E. coli (BL21 DE) host cells producing a recombinant protein with a molecular mass of 42 kDa. TALON metal affinity chromatography and size exclusion chromatography were then performed and it was found that the native molecular mass of FabB was approximately 72 kDa, indicating that the enzyme was in homodimeric form. Hampton Research Crystal Screen and Crystal Screen 2 were used in crystal screening of FabB, however, no crystal was found as the conditions and the concentration of the protein was not suitable for crystal formation. The homology modelling between FabB of S. typhi structure using E. coli FabB as the model template gave a RMSD value of 0.095
showing a high similarity between these two protein structures. This can be a good prediction of the real protein structure as it is important to know the protein structure of the target protein in order to design ligands that would interact with the active site. This project has successfully expressed the β-ketoacyl-ACP synthase I (FabB) of S. typhi, developed purification method and modelled the structure, paving the way for virtual high throughput screening of ligands.
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Keywords
Cloning , Salmonella Typhi