Isolation and crystallisation of the key protein important in the redox control of Streptomyces coelicolor
P. ©tefanková1, J. Maderová1,2, I. Barák3, M. Kollárová1, D.R. Tomchick2, Z. Otwinowski2
1Department of Biochemistry, Faculty of Natural Sciences, Comenius University, Mlynska dolina CH-1, 842 15 Bratislava, Slovak Republic
2Department of Biochemistry, Southwestern Medical Center at Dallas, University of Texas, 5323 Harry Hines Blvd., 75 390 Dallas, Texas
3Institute of Molecular Biology, Slovak Academy of Sciences, Dubravska cesta, 842 51 Bratislava, Slovak Republic
Thioredoxins are ubiquitous proteins that serve as reducing agents and general protein disulfide reductases in living cells. Thioredoxin together with thioredoxin reductase and coenzyme NADPH formed the cellular redox enviroment in the Streptomyces . Streptomyces are Gram-positive spore-forming soil bacteria with specific life cycle. Streptomyces coelicolor is geneticaly best known representative of the genus. It is suitable model for study of the proteins important in thiol-disulfide status in the cell because glutathione in not made by actinomycetes.
Thioredoxins belong to the structural family that includes glutaredoxin, glutathion peroxidase, bacterial protein disulfide isomerase and N-terminal domain of glutathione transferase . The primary structure of many thioredoxins are known, and show 27-69 % sequence identity. 3D structure of thioredoxins from a number of species, including man  and Escherichia coli  are known, too. In all these 3D structure thioredoxin is a compact globular protein with a five-stranded β sheet surrounded by four α helices.
In our work we over-expressed thioredoxin 1 of S. coelicolor His.Taq fusion protein in Escherichia coli. The protein was purified using single step metal chelate affinity chromatography. Homogenous protein was used for study of the protein stability, its role in the redox control in the cell and the control of protein – protein interaction. Purified protein was used for crystallization trials, too. The crystallization conditions for thioredoxin 1 of S. coelicolor was determinated and we have obtained the crystals suitable for x-ray diffraction analyses. The premilitary x-ray study has showed that protein crystallized in P222 space group with cell parameters 33.2, 43.5, 143.9, 90.0, 90.0, 90.0.
This work was supported by the Slovak Scientific Grant Agency VEGA (grants No.1/0035/03 and 2/4053/04).
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