Cloning, expression, purification and characterization of a sulfate transporter from Legionella pneumophila
Master thesis
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http://hdl.handle.net/11250/186526Utgivelsesdato
2013-09-09Metadata
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- Master's theses (KBM) [927]
Sammendrag
Legionella pneumophila is a gram-negative, non-encapsulated, rod-shaped facultative intracellular pathogen with a single, polar flagellum. The SO42- transporter protein (LPL0734) is a membrane protein of Legionella pneumophila. The LPL0734 protein has 12 trans-membrane helices and consists of 768 residues with the expected molecular weight of 84 kDa. LPL0734 belong to a sulfate transporter family. In this thesis, we attempt to study the characteristics of LPL0734; a suspected sulfate transporter in Legionella pneumophila by cloning, expressing, purifying and crystallizing this protein. Stability testing was also conducted.
In the first part of this research, we focused on the expression of a GFP-tagged SO42- transporter in Escherichia coli strains (Rosetta-II and C43 cells) and then purification of proteins (LPL0734) by metal-affinity chromatography on Ni-column followed by size-exclusion chromatography. Protein lipidation and detergent tests were also performed in order to check the stability of the proteins. In the second part of the research, we focused on cloning of the DNA encoding the SO42-transporter proteins of Legionella pneumophila into pETM11 expression vector.
The expressed protein showed aggregation during the size-exclusion chromatography step of purification. Therefore, crystallization was not possible. Result from stability test showed the LPL0734 protein was not stable at room temperature even after one day of incubation. This protein was more stable at 40C independent of the detergent conditions. Increased aggregation and degradation of the protein were observed when the lipid content was high. The result suggested that there is a need to optimize the temperature and buffer-detergent composition to improve the protein stability. It is worth noting that biochemical studies of LPL0734 have not come out with any clear results.