title: Characterization of bacteriophage T7 DNA polymerase purified to homogeneity by antithioredoxin immunoadsorbent chromatography.
abstract: DNA polymerase of bacteriophage T7 is composed of two subunits, the gene 5 protein of the phage and the host-coded thioredoxin. We have purified T7 DNA polymerase to homogeneity from T7-infected Escherichia coli B cells with a novel technique based on immunoadsorbent affinity chromatography. The enzyme binds quantitatively to a column of anti-thioredoxin Sepharose 4B and remains as an active complex in the immobilized state. It is eluted in fully active and highly purified form by a pulse of buffer at pH 12. After a final phosphocellulose chromatography, T7 DNA polymerase of better than 99% purity, as estimated from sodium dodecyl sulfate polyacrylamide gel electrophoresis, is obtained. Determination of the molecular weight by sedimentation equilibrium centrifugation gives a value of 112,000. Denaturing gels showed that the enzyme is composed of gene 5 protein (Mr = 87,000 +/- 3,000) and thioredoxin (Mr = 12,000) in a 1:1 stoichiometry. The amino acid composition of the enzyme and its spectrum was determined. The DNA polymerase activity is dependent on sulfhydryl compounds, sensitive to salt, and shows a comparatively high Km value for the four deoxyribonucleotides. The enzyme preparation has an inherent 3' leads to 5' exonuclease activity, attacking both native and denatured T7 DNA; it is free from detectable endonuclease activity.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D002846:Chromatography, Affinity; D004259:DNA-Directed DNA Polymerase / Q000302:isolation & purification* / Q000378:metabolism; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology*; D005033:Ethylmaleimide / Q000494:pharmacology; D008970:Molecular Weight; D013056:Spectrophotometry, Ultraviolet; D013604:T-Phages / Q000201:enzymology*; D013879:Thioredoxins / Q000032:analysis
chemical_list: D000596:Amino Acids; D013879:Thioredoxins; D004259:DNA-Directed DNA Polymerase; D005033:Ethylmaleimide