Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:A novel protein tag from herpes simplex virus type 1 DNA polymerase. 1039 99

The baculovirus replication factors LEF-1 and LEF-2 of the Autographa californica multinucleocapsid nucleopolyhedrovirus were overexpressed as fusions containing a hemagglutinin (HA) epitope and a HIS(6) tag using recombinant baculoviruses. LEF-1 was purified to near homogeneity and found to have primase activity in an indirect assay employing Escherichia coli DNA polymerase I (Klenow enzyme) and poly(dT) template. The LEF-1 primase products were also directly characterized by electrophoresis in 20% polyacrylamide-8 M urea gels and agarose gels. Primer synthesis was time dependent, and products of several hundred nucleotides or more were observed from the M13 single-stranded DNA (ssDNA) template. The LEF-1 primase was absolutely dependent on divalent cations (Mg(2+)), and optimal activity was supported by 10 mM MgCl(2). An alkaline pH (8.8 to 9.4) was optimal, whereas monovalent salt (KCl) was inhibitory. Mutation of an invariant aspartic acid in a putative primase domain caused LEF-1 activity to be abolished. Upon ultracentrifugation in glycerol gradients, LEF-1 was found to have a sedimentation coefficient of 3S that is consistent with its being present as a monomer. Elution profiles of LEF-1 and LEF-2 from ssDNA-cellulose and DEAE resin suggested that LEF-2 may bind to both DNA and LEF-1.
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PMID:Baculovirus replication factor LEF-1 is a DNA primase. 1183 7