RAPID PURIFICATION OF HIGH ACTIVITY Taq POLYMERASE

The method described here is very simple, however it is important to note that all of the parameters of the purification have been optimized, and deviation from them can have marked effects on yield and activity of enzyme.

Method - first transform a suitable strain of E. co/i with the pTaq plasmid, which contains the Taq gene expressed under control of the tac promoter (Engelke et al. Anal. Biochem. 191:396-400, 1990). Strains DM1 and DM5 give good yields, as does 1NV1F' (Invitrogen Corp., San Diego, California). The pTaq plasmid is maintained by growing transformed cells in medium containing 80 ug/mL ampicillin. All reagents used in the purification should be molecular biology grade, and care must be taken to avoid contamination with biological material or metal ions.

1.Cultures are initiated by adding 500 ml of an overnight culture per litre of LB broth and grown at 37 0C for 11 hours (OD600 approx. 0.8) before IPTG is added to a concentration of 125 mg/l.

2. After 12 hours cells are harvested by centrifugation, washed in 100 ml/l (original culture volume) of buffer A (50 mM tris-MCl pM 7.9, 50 mM dextrose, 1 mM EDTA).

3.Re-centrifuge and resuspended in 50 ml/l of pre-lysis buffer (buffer A plus 4 mg/ml lysozyme).

4.After 15 minutes at room temperature an equal volume of lysis buffer is added (10 mM tris-HCl pH 7.9, 50 mM KCl, 1mM EDTA, 1mM PMSF, 0.5% Tween 20, 0.5% Nonidet P40) and cells are incubated in 200 ml aliquots in pyrex flasks at 75 0C for 1 hour.

5.The lysis mixture is then transferred to plastic bottles for centrifugation at 15,000 rpm for 10 minutes at 4 0C, after which the clarified lysate is transferred to a clean pyrex flask.

6.Taq polymerase is recovered from the clarified lysate by adding 30g of powdered (NH4)2S04 per 100mls while stirring rapidly at room temperature.

7.Centrifuge at 15000 rpm for 10 minutes, harvest protein precipitate (both in pellets and as surface precipitate) and resuspend in 20 mls of A buffer per 100 mls of lysate.

8.The resuspended protein is then dialyzed against 2 changes of at least 12 hours each of storage buffer (50 mM tris-HCl pH 7.9, 50 mM KCI, 0.1 mM EDTA, 1mM DTT, 0.5 mM PMSF, 50% glycerol) at 4 0C. After dialysis the resulting protein can be diluted 1:1 with sterilized storage buffer and stored at -70 0C.

Results - typical activity of undiluted purified polymerase is 100 units/ul, and yields of 106 units/litre of culture are average. It is important to titrate the final product against commercial enzyme to find the correct operating dilution, since excess enzyme can cause a decrease in activity. The cloned Taq polymerase shows a 90kD band on SDS-PAGE, and activity of pure enzyme is on the order of 1 unit/l00ng of protein.

Note that this is a 1992 protocol and a more rapid way of purifying it has been published in Biotechniques 19:780-784 (Nov 1995)

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