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Rapid purification of high-activity Taq DNA polymerase

1993; Oxford University Press; Volume: 21; Issue: 20 Linguagem: Inglês

10.1093/nar/21.20.4850

1362-4962

Fred G. Pluthero,

Animal Genetics and Reproduction

The method described here is derived from that of Engelke et al. (1), and uses the same cloned form of Thermus aquaticus {Taq) DNA polymerase to produce this enzyme in E.coli. The modified purification method described here is quite simple, however it is important to note that factors such as the bacterial strain used, induction time and protein concentration during isolation have been optimized. Deviation from the established parameters can have large effects on enzyme yield and activity. E.coli strain INVlalphaF' (Invitrogen Corp., San Diego, California) was transformed with the pTaq plasmid, which contains the Taq gene expressed under control of the tac promoter (1). Large-scale cultures of E.coli containing the pTaq plasmid were initiated by adding 500 n of an overnight culture to one litre of LB broth with ampicillin (80 mg/1). These cultures were grown at 37°C for 11 hours to an ODeoo of approx. 0.8, and then IPTG was added to a concentration of 125 mg/1. After 12 hours of induction the cells were harvested by centrifugation and washed in 100 ml per litre of original culture volume of buffer A (50 mM Tris-HCl pH 7.9, 50 mM dextrose, 1 mM EDTA; all reagents used in the purification were molecular biology grade, and care was taken to avoid contamination with biological material or metal ions). Cells were recovered by centrifugation and resuspended in 50 ml per litre of original culture volume of pre-lysis buffer (buffer A plus 4 mg/ml lysozyme). After 15 minutes at room temperature an equal volume of lysis buffer was added (10 mM Tris-HCl pH 7.9, 50 mM KC1, 1 mM EDTA, 1 mM PMSF, 0.5 % Tween 20, 0.5 % Nonidet P40) and the lysis mixture was incubated in 200 ml aliquots in pyrex flasks at 75 °C for 1 hour. The lysis mixture was then transferred to plastic bottles for centrifugation at 15,000 rpm for 10 minutes at 4°C, and the clarified lysate was transferred to a clean pyrex flask.