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Acta Crystallogr Sect F Struct Biol Cryst Commun. Aug 1, 2011; 67(Pt 8): 903–906.
Published online Jul 19, 2011. doi:  10.1107/S1744309111021361
PMCID: PMC3151124
Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of a 12R-LOX–chaperone complex
Gouri Deb,a* Karen Boeshanes,a William K. Idler,a and Bijan Ahvazia
aX-ray Crystallography Facility, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institute of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA
Correspondence e-mail: gou5skm2001/at/yahoo.com
Received March 24, 2011; Accepted June 2, 2011.
Abstract
Lipoxygenases are a family of nonheme iron-containing dioxygenases. An Escherichia coli expression system producing the bacterial chaperones GroES and GroEL was engineered and successfully used to produce large quantities of recombinant human 12R-LOX (LOXR; MW 80.34 kDa; 701 amino-acid residues). The co-overproduction of the two chaperones with 12R-­LOX resulted in increased solubility of 12R-­LOX and allowed the purification of milligram amounts of active enzyme for structural studies by X-ray diffraction. The lipoxygenase protein was purified on an affinity column and a gel-filtration column with chaperone protein (MW 57.16 kDa). The LOXR–chaperone complex was crystallized with ligand by the hanging-drop vapor-diffusion method using 1.5 M ammonium hydrogen phosphate as precipitant. The crystals belonged to the monoclinic system, space group P21, with unit-cell parameters a = 138.97, b = 266.11, c = 152.26 Å, β = 101.07°. Based on the calculated Matthews coefficient (3.1 Å3 Da−1), it is estimated that one molecule of LOXR complexed with two molecules of chaperone is present in the asymmetric unit of the crystal lattice. X-ray diffraction data were collected to 4 Å resolution using synchrotron radiation.
Keywords: 12R-LOX, lipoxygenases, chaperones
Articles from Acta Crystallographica Section F: Structural Biology and Crystallization Communications are provided here courtesy of
International Union of Crystallography