|M.Sc Thesis||Department of Chemistry|
|Supervisor:||Prof. Adir Noam|
|Full Thesis text|
Cyanobacteria are oxygenic photosynthetic organisms, which require manganese for photosynthetic water splitting. In the cyanobacterium Synechocystis sp. PCC6803, high affinity manganese import is carried out by an ABC transporter - MntABC. The structure of the substrate binding protein - MntC, has been determined using X-ray crystallography. The structures of the ATP-binding protein - MntA and the transmembrane protein - MntB, are yet to be solved. In fact, no high resolution structure of a metal ion ABC transport system has been solved to date.
The research goals of this study were to clone, express, isolate and crystallize the MntB membrane protein from Synechocystis in order to solve its 3D molecular structure through X-ray crystallography. This project takes part in an overall research goal of our lab, to solve and analyze the structure and function of the entire MntABC transport system, in hope to promote drug design development of novel antibacterial agents.
A variety of different over- expression and purification trials were tested. The mntB gene was cloned into several vectors, all of which have a T7 promoter and all of which allow an attachment of a 6xHis tag to the protein, allowing purification of the protein by chelation - chromatography. Three different E. coli hosts for the over- expression of the protein were tested. Further more, co-expression trials of the MntB protein together with highly soluble easily expressed proteins (one of them being the MntA protein) was attempted in hope to improve MntB protein amounts during expression.
The results showed that the best amounts of isolated purified MntB protein were achieved when the mntB gene was cloned into a pET28b expression vector and when over-expression took place in an E. coli host type C43. The protein purification process that gave considerably better results then all other trials was with the use of the detergent sodium dodecyl sulfate for the initial steps of cell disruption and protein isolation. The detergent was then changed to dodecyl-β-d-maltoside in order to get a properly folded protein. Native polyacrylamide gel electrophoresis tests have assured that the protein is homogeneous and a highly purified protein solution was set for crystallization. The protein crystallized in the sitting drop technique and many protein crystals were obtained. Dissolved crystals were then analyzed by SDS PAGE and the bands obtained were identified as MntB by mass spectrometry. The crystals were subjected to X-ray diffraction in the ESRF and diffracted to ~12Å.