Purification of the T4 endonuclease V

K. M. Higgins, R. S. Lloyd

Research output: Contribution to journalArticle

11 Scopus citations

Abstract

A new purification protocol has been developed for the rapid isolation to physical homogeneity of T4 endonuclease V. The enzyme was purified from an Escherichia coli strain which harbors a plasmid containing the T4 denV structural gene downstream of the λ rightward promoter. The purification of the enzyme was monitored by pyrimidine dimer-specific nicking activity, Western blot analysis and silver or Coomassie Blue staining of SDS-polyacrylamide gels. Milligram quantities of the enzyme have been purified by the following procedure. After sonication of cells and removal of major cell debris, total protein and nucleic acids were passed over a single-stranded DNA agarose column. Endonuclease V was eluted at 650 mM KCl with a linear salt gradient yielding enzyme of approximately 20% purity and following dialysis, was applied to a chromatofocusing column. The enzyme elutes at pH 9.4 is > 90% homogeneous at this step. The final purification step is CM-Sephadex chromatography which attains > 98% homogeneity.

Original languageEnglish (US)
Pages (from-to)117-121
Number of pages5
JournalMutation Research DNA Repair Reports
Volume183
Issue number2
DOIs
StatePublished - Mar 1987

Keywords

  • (Escherichia coli)
  • Endonuclease V
  • Pyrimidine dimer-specific nicking activity
  • SDS-polyacrylamide gels
  • T4 endonuclease V
  • T4 purification
  • Western blot analysis
  • purification

ASJC Scopus subject areas

  • Medicine(all)

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