Open AccessA New Procedure for the Simultaneous Large‐Scale Purification of Bacteriophage‐T4‐Induced Polynucleotide Kinase, DNA Ligase, RNA Ligase and DNA Polymerase
Author(s) -
DOLGANOV Gregory M.,
CHESTUKHIN Arsenii V.,
SHEMYAKIN Michael F.
Publication year - 1981
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1981.tb05143.x
Subject(s) - dna ligase , microbiology and biotechnology , biology , dna polymerase , biochemistry , dna polymerase i , rna ligase , dna , rna , reverse transcriptase , gene
A procedure for simultaneous large‐scale purification of the bacteriophage‐T4‐induced polynucleotide kinase, DNA ligase, RNA ligase and DNA polymerase has been developed. The method involves bacterial cell disruption by sonication, fractionation of cell extract with polymin P, salt elution from the polymin pellets, ammonium sulfate precipitation, and subsequent column chromatography purification of the enzymes. To enrich the enzyme content highly in the initial source non‐permissive Escherichia coli B‐23 cells infected with T4 amN82 phage were used. The procedure described is rapid, reproducible, high in yield, and able to handle preparations using from 1 g to 200 g cell paste. It can be easily scaled up. The method results in large amounts of the enzymes with very high specific activities, good stability essentially lacking exonuclease and endonuclease contamination. The final enzyme preparations were efficiently used in DNA sequencing and in multiple experiments on construction of various recombinant DNAs for cloning and expression in vivo .