Optimization Of Preparation Technology Of Taq DNA Polymerase

Along with the extensive application of PCR technique in medical diagnosis, molecular biology, agriculture, environment protection and so on, the usage of Taq DNA polymerase is increasing every year. At the beginning, Taq DNA polymerase was isolated from one of hydric thermophilic bacillus (Thermits aquaticus). The output of Taq DNA polymerase preperation was not high enough, because the original bacillus strain was difficult to cultivate, the procedure for the enzyme purification was complicated, and selectivity deposition and ion exchange chromatogram isolation were needed. At present, the commercial product of Taq DNA polymerase is transgenic preperation. The preparation of Taq DNA polymerase by transgenic bacillus strain is beneficial to increase of the output and simplification of the purification procedure. The commercial production of Taq DNA polymerase is beneficial to cost decrease and extensive application of PCR technique.Although the commercial production of Taq DNA polymerase has come true for many years and a lot of technique improvement has been made, the activity ratio of the enzyme preparation is not high, some methods of procedure too complicated, or the production cost is high. A lot of researches have been conducting, in order to increase the output and activity of enzyme and simplify the purification procedure.The Taq DNA polymerase used in this experiment consists of 832 amino acids with a molecular weight of 94kDa, a good stability to heat and a polymerization speed of about 150 nt/sec at 75-80℃. This Taq DNA polymerase is widely used in polymerase chain reaction (PCR).In this study, E.coli strains were transformed with plasmid pTrc99A containing the gene of Taq DNA polymerase and Taq DNA polymerase was prepared after IPTG inducement expression. Through screening engineering bacterium strains, transformation methods, inducement expression time, IPTG concentration and amount of ammonium sulphate to optimize the preparation techniques of Taq DNA polymerase and increase enzymic ratio activity. The results of SDS-PAGE electrophoresis and PCR amplification indicated that it is the optimized technique combination consists of E.coli strain JM109, electric transformation, and inducement expression of 1.0mmol/L IPTG for 12 hours. Prepared with this combination, the enzymic protein expression, enzyme activity and activity ration are significantly higher than other treatments of concentration and time. The purification, enzyme activity, sensitivity and specifivity of the Taq DNA polymerase preparation meet the demand of molecular biological experiment. This preparation has some advantages comparing with other products, with a ration activity of 2651U/mg.