Purification, solution properties and crystallization of SIV integrase containing a continuous core and C-terminal domain

Acta Crystallogr D Biol Crystallogr. 1999 Nov;55(Pt 11):1906-10. doi: 10.1107/s0907444999009610.

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.

MeSH terms

  • Cloning, Molecular
  • Crystallization
  • Dimerization
  • Escherichia coli
  • Integrases / chemistry*
  • Integrases / genetics
  • Integrases / isolation & purification
  • Mutation
  • Polyethylene Glycols
  • Protein Conformation
  • Recombinant Proteins / isolation & purification
  • Simian Immunodeficiency Virus / enzymology*
  • Software
  • Solubility
  • Ultracentrifugation
  • X-Ray Diffraction

Substances

  • Recombinant Proteins
  • Polyethylene Glycols
  • Integrases