一种采用阴离子交换柱快速纯化Taq DNA聚合酶的方法

Rapid Purification of Taq DNA Polymerase Using Anion-exchange Column

通过37℃恒温振荡培养含有Taq DNA聚合酶基因的E.coli菌株,并用IPTG诱导该基因表达获得TaqDNA聚合酶蛋白,利用40%硫酸铵沉淀该蛋白后溶解于storage buffer中。此法获得的Taq DNA聚合酶带负电荷,因此采用阴离子交换柱纯化蛋白。试验结果表明,此法与传统的透析方法相比能快速地去除生物小分子杂质,同时去除透析方法无法除去的杂蛋白;既能保证Taq DNA聚合酶的生物学活性,同时能缩短纯化时间、提高Taq DNA聚合酶的纯度。以土壤微生物DNA、水稻cDNA为模板进行PCR扩增并对扩增产物进行测序,结果显示纯化后的Taq DNA聚合酶具有较高的扩增效率和保真性。

In this study, the Escherichia coli strain contains the gene encoding Taq DNA Polymerase was shaking- cultured in constant temperature incubator shaker at 37℃ for synthesizing of the Polymerase, using IPTG as an inducer. The enzyme was then precipitated by 40% ammonium sulfate. Taq DNA Polymerase obtained in this way was negatively charged, and anion-exchange column was used to purify the protein. It was found that this performance can remove the unneeded small biological molecules more quickly than the traditional method of dialysis; meanwhile, it not only keeps biological activities of Taq DNA Polymerase, but also shortens time of purification and improves pure degree of Taq DNA Polymerase. The rice cDNA, rice genomic DNA and soil microorganism DNA were used as template to PCR amplification and the PCR products were sequenced. The results showed that the polymerase had great activity and fidelity.