PURIFICATION OF THERMUS-AQUATICUS DNA-POLYMERASE EXPRESSED IN ESCHERICHIA-COLI
PURIFICATION OF THERMUS-AQUATICUS DNA-POLYMERASE EXPRESSED IN ESCHERICHIA-COLI
复制标题
DOI：
10.1016/0003-2697(90)90238-5
复制
发表时间：
1990-12-01
影响因子：
2.9
通讯作者：
GINSBURG, D
中科院分区：
文献类型：
--
作者：
ENGELKE, DR;KRIKOS, A;GINSBURG, D
水热菌DNA聚合酶因其在DNA扩增和DNA测序方案中的应用而成为分子生物学中常用的试剂。本文描述了一种在大肠杆菌中过量生产后分离重组Taq酶的简化方法。纯化需要8至10小时，需要热处理和清除大肠杆菌裂解物，然后用聚乙烯亚胺沉淀酶，并在单盐步骤中从Bio Rex 70离子交换树脂中洗脱。所得到的酶制备含有一个单一的、几乎均匀的蛋白质，与先前建立的Taq DNA聚合酶的大小一致，每升细胞培养物的蛋白质产量为40-50毫克。
DNA polymerase from Thermus aquaticus has become a common reagent in molecular biology because of its utility in DNA amplification and DNA sequencing protocols. A simplified method is described here for isolating the recombinant Taq enzyme after overproduction in Escherichia coli. Purification requires 8 to 10 h and entails heat treating and clearing the E. coli lysate, followed by precipitation of the enzyme with polyethyleneimine and elution from Bio Rex 70 ion exchange resin in a single salt step. The resulting enzyme preparation contains a single, nearly homogeneous protein consistent with the previously established size of the Taq DNA polymerase in a yield of 40-50 mg of protein per liter of cell culture.