Rapid purification of truncated Taq DNA polymerase Stoffel fragment by boiling lysis of bacterial expression cultures.

Abstract:

A simplified and low-cost method, boiling lysis, was developed and used to purify the truncated DNA polymerase Stoffel fragment after overproduction of the enzyme in Escherichia coli. The method is based on the thermostable properties of the Stoffel fragment. Conditions such as the boiling time and number of cycles employed were identified as determinants with which to achieve a high yield and activity of this truncated Taq enzyme. Under established conditions, a period of 8 min consisting of two cycles of boiling and centrifuging was ideal for the purification of the Taq protein, followed by dibasic phosphate and ethanol extraction in a single type of storage buffer to remove contaminating nucleic acid. The whole purification process could be achieved within 1 or 2 h. A total yield of 80 mg of truncated Taq enzyme with an activity of 1.6x10(7) units was purified from l litre of bacterial culture. The purified Taq enzyme had a molecular mass of 61 kDa, a value consistent with the truncated DNA polymerase Stoffel fragment reported previously.

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