Expression, purification and in silico characterization of BPSL2774, a hypothetical protein from Burkholderia pseudomallei K96243 /
Melioidosis is a disease that infects human and animals and can be detrimental in humans. Mortality rate from melioidosis septic shock due to infection from gram-negative Burkholderia pseudomallei in endemic regions of Malaysia and Thailand remain high despite available antimicrobial therapy. Differ...
Saved in:
| Main Author: | |
|---|---|
| Format: | Thesis |
| Language: | English |
| Published: |
Kuantan, Pahang :
Kulliyyah of Science, International Islamic University Malaysia,
2018
|
| Subjects: | |
| Online Access: | Click here to view 1st 24 pages of the thesis. Members can view fulltext at the specified PCs in the library. |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Summary: | Melioidosis is a disease that infects human and animals and can be detrimental in humans. Mortality rate from melioidosis septic shock due to infection from gram-negative Burkholderia pseudomallei in endemic regions of Malaysia and Thailand remain high despite available antimicrobial therapy. Different strategies are being utilized to identify essential genes and drug targets in this bacterium for improvement in current antimicrobial therapies. This particular concern is due to the resistance of B. pseudomallei to many available and commercial antibiotics as well as the lack exposure about the pathogenicity of this bacterium. In this study, five target genes predicted to be essential for B. pseudomallei by transposon-directed insertion site sequencing (TraDIS) technique were selected and amplified using nested polymerase chain reaction (PCR) for subsequent GatewayTM cloning protocols. Currently, positive clones have been verified for one target gene, BPSL2774 using colony PCR, BsrG1 restriction digest and deoxyribonucleic acid (DNA) sequencing. The essential gene BPSL2774, obtained from B. pseudomallei K96243 were expressed in Escherichia coli BL21(DE3) for protein production. Large scale protein preparations in high density cultures were made according to the auto-induction method to express the soluble target protein. The target protein was successfully separated and expressed in soluble form by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Mass spectrometry analysis shown the soluble BPSL2774 protein was successfully expressed with the correct mass of 35102 kDa. After confirmation of the purified protein identity, in silico structural and functional prediction on BPSL2774 protein was performed. Secondary and tertiary structure of BPSL2774 protein was predicted. BLASTp to protein databank (PDB) database showed that BPSL2774 protein have conserved domains of glycosyltransferase GT-B type superfamily. This correlates with the secondary and tertiary structure model of BPSL2774 that displayed two β/α/β Rossmann fold domains with six parallel beta strands found in each domain, indicative of the same fold. By using consensus approach (COACH) meta-server, the top prediction for ligand binding was α-D-glucose, Uridine diphosphate (UDP) and N-Acetylglucosamine (NAG). The refined structural model of BPSL2774 protein validated by Ramachandran plot was used for docking simulations with UDP, GDP and NAG. The docking results from both AutoDock4.2 and AutoDock Vina did not show significant binding affinity of the three tested ligands to BPSL2774 protein, with binding affinity values ranging from -4.4 kcal/mol to -6.9 kcal/mol. This was consistent with the challenges of characterizing glycosyltransferases due to the various sugar donor and acceptor specificity. Taking all the results into account, the functional annotation of BPSL2774 protein as a glycosyltransferase is recommended, though future validation from biochemical experiments or a more exhaustive docking simulation experiments were needed to support this. In the future, BPSL2774 protein can be further purified through additional purification steps following initial GST-tagged Affinity Chromatography for subsequent functional assay and biophysical experiments. |
|---|---|
| Physical Description: | xvi, 136 leaves : colour illustrations ; 30cm. |
| Bibliography: | Includes bibliographical references (leaves 104-114). |