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.

Polymerases:

T7

Topics:

Historical Protein Properties (MW, pI, ...), Other Enzymatic Activities, Accessory Proteins/Complexes, RNase H Activity, Nucleotide Incorporation, Exonuclease Activity, Terminal Transferase, Source / Purification

Status:

new topics/pols set partial results complete validated

Results:

Polymerase Reference Property Result Context
T7 Nordström B1981 Molecular Weight 112kD
T7 Nordström B1981 3-5' Exonuclease (proofreading) Yes
T7 Nordström B1981 Cloned or native Cloned in E. coli
T7 Nordström B1981 3-5' Exo Specific Activity 2.4E+04 units/mg
T7 Nordström B1981 Tagged No
T7 Nordström B1981 Full length or truncated Full length
T7 Nordström B1981 Specific Activity 5150 units/mg
T7 Nordström B1981 Other accessory protein(s) E. coli thioredoxin

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