Rapid purification of overexpressed T4 DNA polymerase.
A method for purifying T4 DNA polymerase from cells harboring overexpression plasmids is described. T4 DNA polymerase is precipitated from induced, lysed cells with polyethyleneimine, then extracted and fractionated further with (NH4)2SO4 before chromatography on a column of single-stranded DNA cellulose. This procedure can be completed in three days and consistently provides enzyme preparations which are at least 98% pure. When necessary, one further chromatography step provides T4 DNA polymerase suitable for recombinant DNA applications.
Rush
J
J
Department of Molecular Biophysics and Biochemistry, Yale University School of Medicine, New Haven, Connecticut 06510.
Konigsberg
W H
WH
D000645:Ammonium Sulfate; D011232:Chemical Precipitation; D002845:Chromatography; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004259:DNA-Directed DNA Polymerase / Q000096:biosynthesis / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D010957:Plasmids; D011094:Polyethyleneimine; D012995:Solubility; D013604:T-Phages / Q000201:enzymology*; D013696:Temperature
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D011094:Polyethyleneimine; D004259:DNA-Directed DNA Polymerase; D000645:Ammonium Sulfate