RESUMEN
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 80.34â 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 57.16â kDa). The LOXR-chaperone complex was crystallized with ligand by the hanging-drop vapor-diffusion method using 1.5â M ammonium hydrogen phosphate as precipitant. The crystals belonged to the monoclinic system, space group P2(1), with unit-cell parameters a = 138.97, b = 266.11, c = 152.26â Å, ß = 101.07°. Based on the calculated Matthews coefficient (3.1â Å(3)â 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 4â Å resolution using synchrotron radiation.