Overexpression, purification, and crystallization of the DNA binding and dimerization domains of the Epstein-Barr virus nuclear antigen 1.
The Epstein-Barr virus nuclear antigen (EBNA) 1 binds to and activates DNA replication from the latent origin of Epstein-Barr virus. Six different fragments of EBNA1 that retain DNA binding activity were expressed in bacteria, purified, and crystallized. Two fragments, EBNA470-619 and EBNA470-607, formed well ordered crystals that diffracted beyond 2.5-A resolution. Two different EBNA470-619 crystals were grown from sodium formate, pH 6-6.5. One crystal belonged to the trigonal space group P3 with unit cell dimensions a = b = 86.5 A and c = 31.8 A and with two molecules in the asymmetric unit. The other crystal, which appeared only twice and was likely related to the P3 crystal form, belonged to the trigonal space group P312 with cell dimensions a = b = 86.7 A and c = 31.8 A. Crystals of EBNA470-607 were grown by lowering the salt concentration to 0-100 mM NaCl at pH 6.0. These crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and had cell dimensions a = 59 A, b = 66.9 A, and c = 69.8 A with two molecules in the asymmetric unit.
Barwell
J A
JA
Institute for Molecular Biology and Biotechnology, McMaster University, Hamilton, Ontario, Canada.
Bochkarev
A
A
Pfuetzner
R A
RA
Tong
H
H
Yang
D S
DS
Frappier
L
L
Edwards
A M
AM
D000956:Antigens, Viral / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D001483:Base Sequence; D002848:Chromatography, DEAE-Cellulose; D003001:Cloning, Molecular; D003460:Crystallization; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D019309:Epstein-Barr Virus Nuclear Antigens; D004926:Escherichia coli; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D015183:Restriction Mapping; D013816:Thermodynamics
D000956:Antigens, Viral; D017931:DNA Primers; D004268:DNA-Binding Proteins; D019309:Epstein-Barr Virus Nuclear Antigens; D011994:Recombinant Proteins