A novel protein tag from herpes simplex virus type 1 DNA polymerase.
An epitope (HPOL) derived from the so-called thumb region of the herpes simplex virus type 1 DNA polymerase in combination with a monoclonal antibody (MAb 1051c) was tested for protein tagging. Using a conventional expression vector, a DNA cassette encoding the HPOL epitope was fused to the C-terminus of the dihydrofolate reductase (DHFR) gene such that the recombinant DHFR contained both a N-terminal HIS-tag and a C-terminal HPOL tag. Expression of recombinant DHFR in Escherichia coli cells was compared by Western blot analysis using either mouse RGS.HIS antibody or MAb 1051c. Immunostaining revealed that both antibodies reacted specifically with DHFR, but the detection sensitivity achieved with MAb 1051c was about 15-fold greater using a standard staining protocol. An HPOL antibody column was successfully applied for affinity purification of DHFR, demonstrating the usefulness of the HPOL epitope/MAb 1051c system for protein tagging, expression monitoring and purification of HPOL-tagged recombinant proteins.
Deutsches Krebsforschungszentrum, Genomforschung und Bioinformatik, Heidelberg, Deutschland.
U
U
Schreiner
J
J
Hansen
F J
FJ
Kühn
C W
CW
Knopf
D000911:Antibodies, Monoclonal / Q000378:metabolism*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D000939:Epitopes / Q000737:chemistry / Q000378:metabolism*; D005090:Exodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D018259:Herpesvirus 1, Human / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011994:Recombinant Proteins; D014764:Viral Proteins; D013762:Tetrahydrofolate Dehydrogenase; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; C043417:DNA polymerase, Simplexvirus