One-step column purification of herpes simplex virus 1 helicase-primase subcomplex using C-terminally his-tagged UL5 subunit.
A protocol for purification of the two-subunit complex of herpes simplex virus type 1 (HSV-1) helicase-primase by metal affinity chromatography is presented. In order to bind the enzyme complex consisting of UL5 and UL52 gene functions to the affinity column, the C-terminus of the UL5 gene of HSV-1 strain ANG was fused in-frame with a sequence encoding six histidines, resulting in a His6-tagged DNA helicase (UL5his) when expressed via recombinant baculovirus. In addition, hybridoma cell lines producing anti-UL5 IgG were generated for screening of DNA helicase expression. Initial purification trials revealed that the presence of low concentrations of imidazole in the wash buffers interfered with the binding of the helicase-primase subunit complex to the metal affinity resin. Alternative means, such as high salt, altered pH, and substitution of imidazole by histidine tetrapeptide (His4), were tested. From those, the addition of His4 in combination with an acidic pH turned out to be very efficient for the removal of protein contaminants from a Ni2+-NTA (nitrilotriacidic acid) affinity resin. By applying only one column step, the present protocol yields a helicase-primase preparation, which is suitable for inhibitor screening and further functional studies. The final preparation is free of interfering enzyme activities, and exerts each of the enzymatic functions described for a two subunit complex, i.e., DNA-dependent ATPase, DNA primase, and DNA helicase activities.
Schreiner
Uwe
U
Forschungsschwerpunkt Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, INF 242, Heidelberg 69120, Germany.
Theune
Myriam
M
Althof
Frank
F
Kehm
Elke
E
Knopf
Charles W
CW
D000255:Adenosine Triphosphate / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002846:Chromatography, Affinity / Q000379:methods*; D004265:DNA Helicases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D019915:DNA Primase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005822:Genetic Vectors; D018259:Herpesvirus 1, Human / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D019915:DNA Primase; C062021:helicase-primase, Human herpesvirus 1; D004265:DNA Helicases