以冻融法快速纯化高活力基因工程Taq DNA聚合酶

Rapid Purification of High-activity Recombinant Taq DNA Polymerase Using Freezing-thawing Method

用含有TaqDNA聚合酶基因的 pTaq表达质粒转化E .coliDH5α菌株 ,IPTG诱导表达TaqDNA聚合酶。利用该酶的热稳定性 ,反复 2次 - 70℃、75℃交替冻融以裂解细胞释放胞浆 ;以高速离心除去冻融变性的细胞碎片及核酸蛋白的复合物以达到快速纯化TaqDNA聚合酶的目的。PCR扩增反应和SDS -PAGE分析表明所制备的TaqDNA聚合酶的纯度、活力、敏感性、特异性均达到试验要求。该方法具有快速简便的优点。

The strain of E.coli DH5α was transformed with p Taq expressing plasmid within which the Taq DNA polymerase gene was inserted. The expression of the gene could be induced with IPTG. Exploiting the thermal stability property of this enzyme, the authors developed a rapid method to isolated this enzyme by two cycles of -70 ℃ freezing and 75 ℃ thawing, which ruptured the cell membrane and released the cell content. After a high speed centrifugation, the denatured macromolecules precipitated and the Taq DNA polymerase could be collected in the supernatant. SDS-PAGE and PCR amplification analysis demonstrated that the purified enzyme was very high in purity, activity, specificity and sensitivity. The purification method was more convenient and rapider than those reported previously.