An improved purification method and a physical characterization of phage T7 DNA polymerase

Eur J Biochem. 1982 Nov 15;128(2-3):445-9. doi: 10.1111/j.1432-1033.1982.tb06984.x.

Abstract

The immunoadsorbent used to purify T7 DNA polymerase contains antibodies directed towards thioredoxin. Elution of the enzyme is made by a pulse of buffer at pH 12.0. This decreases the binding capacity of the column. Binding experiments with [3H]thioredoxin showed that the effect was caused by reduction of the antibodies by thiols in alkaline buffers. T7 DNA polymerase aggregated and irreversibly lost activity in buffers of low ionic strength. Experiments with gel chromatography and glycerol density gradient centrifugation showed that 0.2 M sodium chloride was required to keep the enzyme in its monomeric form. The sedimentation coefficient and the Stokes' radius are 5.3 S and 4.6 nm respectively, evaluated by gel chromatography and glycerol density gradient centrifugation techniques. The frictional ratio of 1.49 indicates that the T7 DNA polymerase is an asymmetrical protein.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA-Directed DNA Polymerase / isolation & purification*
  • DNA-Directed DNA Polymerase / metabolism
  • Escherichia coli / enzymology*
  • Macromolecular Substances
  • Molecular Weight
  • Protein Conformation
  • T-Phages / enzymology*

Substances

  • Macromolecular Substances
  • DNA-Directed DNA Polymerase