Purification of TaqI endonuclease from Thermus aquaticus

A purification procedure for the thermostable restriction enzyme TaqI was developed using high-performance ion-exchange liquid chromatography. The effects of various operating conditions on the separation behaviour of TaqI endonuclease from the cell extracts were investigated for optimisation and scaling up. The separation of the enzyme by HPLC was found to be strongly dependent on the sample volume, slope of linear gradient and order of the ion-exchange columns. The final yield of the enzyme is also dependent to a great extent upon the number of fractionation steps employed to purify the enzyme. In the present study, 4000 U TaqI endonuclease per mg protein was recovered from 2 g Thermus aquaticus cells with a two-step purification protocol in one day. The purification factor was 24. Compared to other classical methods of purification reported in literature with 4000 or 32,000 U enzyme from 200 g of Thermus aquaticus cells, HPLC yielded 190,000 U enzyme from 200 g cells using cation and anion HPLC columns sequentially and thus resulted in a higher efficiency.