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Förslaget inkom 2005-06-01

The role of glutaredoxin 2 in the life of Escherichia coli

Intracellular sulfhydryl homeostasis is maintained in Escherichia. coli (E. coli) through the glutaredoxin (Grx) and thioredoxin systems. In the Grx system, reducing equivalents are transferred from NADPH to glutathione reductase (GR), glutathione (GSH) and finally to the three different Grxs (Grx1, 2, 3). Grx1 can reduce ribonucleotide reductase 1a (RR1a), and PAPS reductase. Grx3 has 5 % of the catalytic activity of Grx1 in reducing RR and no catalytic activity to PAPS reductase. Grx2 does not reduce PAPS reductase or RR1a but is the most potent antioxidant redoxin. In E. coli crude extracts, Grx2 comprises more than 80 % of total GSH-disulfide oxidoreductase activity and prevents oxidative damage (carbonylation) of E. coli proteins by hydrogen peroxide. The antioxidant function of Grx2 has been demonstrated also in rat neurons, where the protein affects signal transduction pathways leading to the activation of NF-kappaB via Ref-1, preventing thus dopamine-induced apoptosis. In relation to its role as an antioxidant, Grx2 is upregulated at the stress conditions of stationary phase to up to 1 % of total protein by ppGpp and RpoS. However, the physiological function of Grx2 is unknown. We intend to elucidate its function by identifying the physiological substrates of Grx2.

We will use two approaches.

1. Biochemical approach.
Using a monothiol Grx2 fused to a His tag, we will make a stable complex of monothiol Grx2 with its substrates. The complex will be purified using an affinity matrix, then reduced to release the Grx2 partner which will finally be identified with electrospray mass spectrometry.

2. Molecular biology approach.
We will use the currently available (Stratagene) two-hybrid system for bacteria to identify potential proteins interacting with Grx2.

Candidates from combined results from the two approaches will be overexpressed and purified and their interaction with Grx2 confirmed with in vitro assays measuring consumption of NADPH in the presence of GSH, and GR. Future work will include the construction of appropriate null mutants.

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