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 [1]. 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 [2]. 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 [3] and Escherichia
coli [4] 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).
References
[1] M.S. Paget, V. Molle, G. Cohen, Y.
Aharonowitz, M.J. Buttner, Mol. Microbiol. 42 (2001)
1007-20.
[2] J.L. Martin, Structure 3 (1995)
245-250.
[3] A. Weichsel, J.R. Gasdaska, G. Powis, W.R.
Montfort, Structure 4 (1996) 735-51.
[4]
S.K. Katti, D.M. LeMaster, H. Ecklund, J. Mol.Biol. 212 (1990)
167-184.