Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of a 12R-LOX–chaperone complex
| Autor: | William K. Idler, Bijan Ahvazi, Gouri Deb, Karen Boeshanes |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Biophysics
Gene Expression Crystal structure macromolecular substances Arachidonate 12-Lipoxygenase Crystallography X-Ray Biochemistry Affinity chromatography Structural Biology Genetics Chaperonin 10 Humans Cloning Molecular biology Chemistry GroES Chaperonin 60 Condensed Matter Physics GroEL Crystallography enzymes and coenzymes (carbohydrates) Crystallization Communications Chaperone (protein) X-ray crystallography biological sciences biology.protein health occupations Chaperone complex bacteria Monoclinic crystal system Protein Binding |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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