大肠杆菌中Taq DNA聚合酶表达的优化
Optimization of Taq DNA polymerase enzyme expression in Escherichia coli.
作者信息
Moazen Fateme, Rastegari Ali, Hoseini Sayed Mehdi, Panjehpour Mojtaba, Miroliaei Mehran, Sadeghi Hamid Mir Mohammad
机构信息
Department of Pharmaceutical Biotechnology, Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Sciences, Isfahan University of Medical Science, Isfahan, Iran.
出版信息
Adv Biomed Res. 2012;1:82. doi: 10.4103/2277-9175.103004. Epub 2012 Oct 31.
BACKGROUND
In the present study, we optimized the experimental conditions using pET15b expression vector to obtain large amounts of Taq DNA polymerase.
MATERIALS AND METHODS
Correct framing of the gene in the expression vector pET15b and its orientation were analyzed by digestion and sequencing. Production of Taq DNA polymerase in Escherichia coli BL21 (DE3) cells was induced by incubation with different concentrations of IPTG. Optimum production occurred with the addition of 1mM IPTG for 2h. The activity of the obtained enzyme was measured by comparing the intensities of the produced DNA bands in PCR reactions.
RESULTS
Recombinant plasmid containing taq polymerase gene was confirmed by restriction digestion and DNA sequencing. Purified protein was identified by Western blotting. Optimum condition for the production of the enzyme was induction with 1mM IPTG for 23h. Addition of NP40 increased enzyme stability.
CONCLUSION
We expressed the recombinant Taq DNA polymerase in E. coli using a T7based promoter system and obtained an active and stable enzyme.
背景
在本研究中，我们使用pET15b表达载体优化实验条件以获得大量的Taq DNA聚合酶。
材料与方法
通过酶切和测序分析表达载体pET15b中基因的正确读框及其方向。用不同浓度的异丙基-β-D-硫代半乳糖苷（IPTG）孵育诱导大肠杆菌BL21（DE3）细胞产生Taq DNA聚合酶。添加1mM IPTG孵育2小时可实现最佳产量。通过比较PCR反应中产生的DNA条带强度来测定所得酶的活性。
结果
通过限制性酶切和DNA测序确认了含有Taq聚合酶基因的重组质粒。通过蛋白质免疫印迹法鉴定纯化的蛋白质。产生该酶的最佳条件是用1mM IPTG诱导23小时。添加NP40可提高酶的稳定性。
结论
我们使用基于T7的启动子系统在大肠杆菌中表达了重组Taq DNA聚合酶，并获得了一种活性且稳定的酶。
Zotero 插件