Purification and properties of Epstein-Barr virus DNase expressed in Escherichia coli.
A cDNA corresponding to the BGLF5 open reading frame of the Epstein-Barr virus (EBV) genome and coding for an early DNase was inserted into the procaryotic expression vector pKK223-3. One bacterial clone producing the expected 52-kilodalton DNase was used as a source of EBV DNase. The 52-kilodalton Dnase was purified in the active form to near homogeneity by ammonium sulfate precipitation and successive chromatographies on phosphocellulose, DNA-cellulose, and gel filtration columns. The purified enzyme exhibited both exonuclease and endonuclease activities, an absolute requirement for divalent cations, an alkaline pH preference, and a typical residual activity in presence of 300 mM KCl. Moreover, the enzyme was specifically inhibited by human sera with high antibody titers to EBV early antigens. These properties are similar to those observed for EBV-induced DNase from lymphoblastoid cell extracts. In addition, the enzyme was recognized by both immunoglobulin G and A serum fractions from patients with nasopharyngeal carcinoma (NPC). From these results and previous studies which demonstrated the value of antibody titers to this viral DNase as an NPC marker, it appears that EBV-encoded DNase produced in a heterologous expression system could be used in the development of a specific and early NPC diagnosis test.
Stolzenberg
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MC
Laboratoire de Virologie Moléculaire, UM30, CNRS, Faculté de Médecine Alexis Carrel, Lyon, France.
Ooka
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D001483:Base Sequence; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003851:Deoxyribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005814:Genes, Viral; D004854:Herpesvirus 4, Human / Q000201:enzymology / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013379:Substrate Specificity; D015678:Viral Structural Proteins / Q000235:genetics*
D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D003851:Deoxyribonucleases