Overproduction, purification, and characterization of EBNA1, the origin binding protein of Epstein-Barr virus.
The baculovirus expression system was used to overproduce the Epstein-Barr virus nuclear antigen, EBNA1, in insect cells. EBNA1 overproduced via baculovirus expression (baculoEBNA1) was followed during purification to homogeneity using its ability to specifically retain the family of repeats of the latent origin of replication, oriP, onto nitrocellulose filters. A two-column procedure was developed which yields more than 1 mg of homogeneous baculoEBNA1 from 9 x 10(8) insect cells (1.5 liters). Pure baculoEBNA1 had no detectable ATPase or helicase activity. BaculoEBNA1 was labeled with [32P]orthophosphate in vivo, and analysis showed detectable levels of phosphoserine; no phosphothreonine or phosphotyrosine could be detected. The baculoEBNA1 appeared dimeric in solution, and a stoichiometry of 56 baculoEBNA1 monomers per 24 EBNA1 binding sites in oriP suggests baculoEBNA1 binds its consensus site as a dimer. The binding of baculoEBNA1 to the dyad symmetry element of oriP (Kd approximately 2 nM) required more baculoEBNA1 and appeared less stable than the binding of baculoEBNA1 to the family of repeats in oriP (Kd approximately 0.2 nM).
Frappier
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Howard Hughes Medical Institute, Hearst Microbiology Department, Cornell University Medical College, New York, New York 10021.
O'Donnell
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D000596:Amino Acids / Q000032:analysis; D000956:Antigens, Viral / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019309:Epstein-Barr Virus Nuclear Antigens; D015967:Gene Expression Regulation, Viral; D005814:Genes, Viral; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000276:immunology* / Q000502:physiology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014779:Virus Replication
D000596:Amino Acids; D000956:Antigens, Viral; D019309:Epstein-Barr Virus Nuclear Antigens