Purification, solution properties and crystallization of SIV integrase containing a continuous core and C-terminal domain.
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.
Li
Y
Y
Department of Antiviral Research, Merck Research Laboratories, West Point, Pennsylvania 19486-0004, USA.
Yan
Y
Y
Zugay-Murphy
J
J
Xu
B
B
Cole
J L
JL
Witmer
M
M
Felock
P
P
Wolfe
A
A
Hazuda
D
D
Sardana
M K
MK
Chen
Z
Z
Kuo
L C
LC
Sardana
V V
VV
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
D011994:Recombinant Proteins; D011092:Polyethylene Glycols; D019426:Integrases