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Crystallization and preliminary crystallographic study of a trypsin‐resistant catalytic domain of human calcineurin

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Calcineurin, a Ca2+/calmodulin‐dependent serine/threonine protein phosphatase, plays a key role in a number of cellular pathways, including T‐cell activation, and is an important molecular target of the immunosuppressive drugs cyclosporin A and FK506. To understand the structural basis underlying the activation of calcineurin by calmodulin, X‐ray crystallography was employed to solve the three‐dimensional structure of the free calcineurin catalytic domain (residues 20–347 of the A subunit). To accomplish this, a bacterially expressed glutathione S‐transferase (GST) fusion protein of the human calcineurin catalytic domain was first purified by GST‐affinity chromatography. After limited digestion by trypsin, the catalytic domain (Cncat) was purified using anion‐exchange and size‐exclusion chromatography. Crystallization of Cncat was achieved by the hanging‐drop vapour‐diffusion method at pH 6.5 using PEG 6000 as precipitant. The diffraction results showed that the Cncat crystal belonged to the orthorhombic space group P21212, with unit‐cell parameters a = 161.6, b = 87.4, c = 112.0 Å. There are four Cncat molecules in the asymmetric unit, with 49.5% solvent content. An X‐ray diffraction data set was collected to 2.87 Å resolution and a clear molecular‐replacement solution was obtained. The active site of Cncat is open to the solvent channels in the crystal packing.

Document Type: Research Article


Publication date: 2012-05-01

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