The production and purification of the 1,4-dihydroxy-2-naphthoate octaprenyltransferase enzyme from Escherichia coli
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Typ
Examensarbete för masterexamen
Master Thesis
Master Thesis
Program
Publicerad
2012
Författare
Szabo, Emelie
Modellbyggare
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Sammanfattning
The aim of the project was to produce, purify and crystallize the 1,4-dihydroxy-2- naphthoate octaprenyltransferase enzyme from Escherichia coli. This is a key enzyme in the biosynthesis of bacterial menaquinone and is encoded by the gene menA. Menaquinone is an electron carrier in the bacterial electron transport chain and constitute, together with phylloquinone, the available natural forms of Vitamin K. Since humans lack the genes for producing menaquinone, menA has strong potential as a new drug target for multi-drug resistant bacteria. Obtaining a crystal structure would serve as a valuable tool for further optimization of inhibitor molecules against the enzyme. The menA was cloned in a vector under control of the T7 promoter and transformed into the BL21*(DE3) strain. Protein production was done with shake-flask cultivation and induction with IPTG. Purifications included IMAC and size exclusion chromatography where different detergents were tested. The menA enzyme was successfully overproduced and it could be shown that it was also functional in the membrane. Initial purification revealed degradation of menA, both as precipitation when concentrating and as an obvious pattern on SDS-PAGEs. Specific protease inhibitors hindered the degradation to take place, yielding pure menA from the size exclusion chromatography. However, the protein concentration was too low to set up crystal plates. The growth conditions need to be optimized in order to obtain a larger amount of protein for structural studies.
Beskrivning
Ämne/nyckelord
Livsvetenskaper , Industriell bioteknik , Life Science , Industrial Biotechnology