title: Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of a 12R-LOX-chaperone complex.
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 P2(1), 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 Å(3) 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.
authors.affiliation: X-ray Crystallography Facility, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institute of Health, Bethesda, MD 20892, USA. gou5skm2001@yahoo.com
authors.forename: Gouri
authors.initials: G
authors.lastname: Deb
authors.forename: Karen
authors.initials: K
authors.lastname: Boeshanes
authors.forename: William K
authors.initials: WK
authors.lastname: Idler
authors.forename: Bijan
authors.initials: B
authors.lastname: Ahvazi
mesh_terms: D001092:Arachidonate 12-Lipoxygenase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018835:Chaperonin 10 / Q000737:chemistry* / Q000378:metabolism; D018834:Chaperonin 60 / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D015870:Gene Expression; D006801:Humans; D011485:Protein Binding
chemical_list: D018835:Chaperonin 10; D018834:Chaperonin 60; D001092:Arachidonate 12-Lipoxygenase