Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction studies of
Nazia Nasir1, Rajan Vyas, Chetna Chugh
1Protein Crystallography Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi 110 067, India.
Abstract:
HisC2 from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel-nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion technique from a condition consisting of 7 mg ml(-1) HisC2 (in 20 mM Tris pH 8.8, 50 mM NaCl and 5% glycerol), 1 M succinic acid pH 7.0, 0.1 M HEPES pH 7.0 and 1%(w/v) polyethylene glycol monomethyl ether 2000. The crystals belonged to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 255.98, b=77.09, c = 117.97 Å. X-ray diffraction data were recorded to 2.45 Å resolution from a single crystal using the in-house X-ray facility.
