福建农业科技2025，Vol.56Issue(5)：1-6,6.DOI:10.13651/j.cnki.fjnykj.2025.05.001
无大肠杆菌宿主DNA残留的Taq DNA聚合酶的表达与纯化
Expression and Purification of Taq DNA Polymerase Without Escherichia coli Host DNA Residues
摘要
Abstract
In order to completely remove the residual host DNA from Escherichia coli in Taq DNA polymerase,the aim of this study was to develop an efficient expression and purification strategy to meet the demand for high-purity Taq DNA polymerase in molecular biology research and clinical application.The pET-15b plasmid was used for the high-efficiency expression of Taq DNA polymerase in Escherichia coli BL21(DE3),with IPTG induction ensuring the high-level expression of the target protein.Then,the purification strategy combining Ni-NTA affinity chromatography and DEAE anion exchange chromatography was employed,in order to achieve the highly efficient purification of the expressed product.The results showed that the optimized process successfully obtained Taq DNA polymerase with a purity of more than 95％.The results of PCR detection of Escherichia coli 16S rRNA gene showed that the host DNA was not detected in the self-made Taq DNA polymerase,and the residual amount was equivalent to that of the commercially available Taq DNA polymerase.Compared to the traditional thermal inactivation combined with ammonium sulfate precipitation or single Ni-NTA affinity chromatography,the optimized purification process could more thoroughly remove the residual DNA of Escherichia coli host,which provided an efficient and feasible new strategy for the preparation of high-purity Taq DNA polymerase.关键词
Taq DNA聚合酶/表达与纯化/宿主DNA残留Key words
Taq DNA polymerase/Expression and purification/Host DNA residues分类
生物科学引用本文复制引用
吕新,刘兰英,李玥仁,罗土炎..无大肠杆菌宿主DNA残留的Taq DNA聚合酶的表达与纯化[J].福建农业科技,2025,56(5):1-6,6.基金项目
福建省科技计划省属公益类专项(2021R1022007) （2021R1022007）
福建省财政专项—福建省农业科学院科技创新团队建设项目(CXTD2021011-3) （CXTD2021011-3）
福建省人民政府、中国农业科学院"5511"协同创新工程(XTCXGC2021020). （XTCXGC2021020）
