A simple and efficient method for extraction of Taq DNA polymerase

Background Thermostable DNA polymerase (Taq Pol ?) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. The method used ammonium sulfate to precipitate the enzyme, and it saved effort and money but not time. Moreover, we found that 30-40% activity of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small amount of DNA. Results We provided a novel, simplified and low-cost method to purify the Taq Pol ? after overproduction of the enzyme in Escherichia coli, which used ethanol instead of ammonium sulfate to precipitate the enzyme. The precipitate can be directly dissolved in the storage buffer without dialysis. In addition, DNA and RNA contamination was removed with DNase I and RNase A before precipitation, and the extraction procedure was optimized. Our improvements increase recovery rate and specific activity of the enzyme, and save labor, time, and cost. Conclusions Our method uses ethanol, DNase I, and RNase A to purify the Taq Pol ?, and simplifies the operation, and increases the enzyme recovery rate and quality.

Production and evaluation of Taq DNA polymerase.

Taq DNA polymerase is an enzyme essential in performing Polymerase Chain Reaction (PCR) which has recently become a basic technology in research and diagnostic laboratories. In order to reduce the cost of research work in Thailand, recombinant Taq DNA polymerase was locally produced from pTaq cloned in E. coli. The enzyme was characterized and evaluated in comparison with the commercial Taq DNA polymerase produced by Perkin Elmer Cetus, U.S.A. The yield of enzyme was 6.72 mg/ml and the activity of 9,524 units/mg protein with the total of 448,000 units/litre of the bacterial culture. The preparation was free of DNase based upon its ability to degrade Lambda DNA evaluated by gel electrophoresis. Although the enzyme produced gave a high DNA polymerase activity, the preparation was not as pure as the enzyme produced by Perkin Elmer Cetus. Immunoblot analysis indicated that the enzyme preparation contained the products of enzyme degradation obtained during preparation and bacterial protein contaminations. In spite of the existence of bacterial proteins in the preparation, the Taq enzyme produced was proved to be applicable in performing PCR such as the PCR-SSP (Sequence Specific Primers) typing for HLA-DR. The cost of enzyme preparation was about 256 times less than that of the commercial enzyme. Economically, the locally produced Taq DNA polymerase can be used efficiently in the research laboratories performing PCR based typing of the HLA genes.