title: Purification, solution properties and crystallization of SIV integrase containing a continuous core and C-terminal domain.
abstract: The C-terminal two-thirds segment of integrase derived from the simian immunodeficiency virus has been cloned, expressed in Escherichia coli, and purified to greater than 95% homogeneity. The protein encompasses amino-acid residues 50-293 and contains a F185H substitution to enhance solubility. In dilute solutions at concentrations below 1 mg ml(-1), the enzyme is predominantly dimeric. At the higher concentrations (>10 mg ml(-1)) required to enable crystallization, the enzyme self-associates to form species with molecular weights greater than 200 kDa. Despite the apparent high aggregation in solution, the enzyme crystallizes from a 8%(v/v) polyethylene glycol (molecular weight 6000) solution in a form suitable for X-ray diffraction studies. The resulting single crystals belong to the space group P2(1)2(1)2(1), with unit-cell parameters a = 79.76, b = 99.98, c = 150.2 A, alpha = beta = gamma = 90 degrees and Z = 4. Under X-ray irradiation generated with a rotating-anode generator, the crystals diffract to 2.8 A resolution and allow collection of a native 3 A resolution diffraction data set.
authors.affiliation: Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Li
authors.forename: Y
authors.initials: Y
authors.lastname: Yan
authors.forename: J
authors.initials: J
authors.lastname: Zugay-Murphy
authors.forename: B
authors.initials: B
authors.lastname: Xu
authors.forename: J L
authors.initials: JL
authors.lastname: Cole
authors.forename: M
authors.initials: M
authors.lastname: Witmer
authors.forename: P
authors.initials: P
authors.lastname: Felock
authors.forename: A
authors.initials: A
authors.lastname: Wolfe
authors.forename: D
authors.initials: D
authors.lastname: Hazuda
authors.forename: M K
authors.initials: MK
authors.lastname: Sardana
authors.forename: Z
authors.initials: Z
authors.lastname: Chen
authors.forename: L C
authors.initials: LC
authors.lastname: Kuo
authors.forename: V V
authors.initials: VV
authors.lastname: Sardana
mesh_terms: D003001:Cloning, Molecular; D003460:Crystallization; D019281:Dimerization; D004926:Escherichia coli; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D009154:Mutation; D011092:Polyethylene Glycols; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification; D015302:Simian Immunodeficiency Virus / Q000201:enzymology*; D012984:Software; D012995:Solubility; D014461:Ultracentrifugation; D014961:X-Ray Diffraction
chemical_list: D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D019426:Integrases