Chen B P, Hai T
Ohio State Biochemistry Program, Ohio State University, Columbus 43210.
Gene. 1994 Feb 11;139(1):73-5. doi: 10.1016/0378-1119(94)90525-8.
We have constructed two convenient vectors to produce foreign proteins in Escherichia coli. The first vector was developed to produce histidine (His)-tagged fusion proteins. In addition to encoding six contiguous His residues, it contains three unique restriction sites that allow cloning of a blunt-ended DNA fragment in three different reading frames. Therefore, one can clone any gene of interest in this vector to make a fusion protein tagged with six His at the N terminus. The His-tag allows purification of the fusion protein to almost homogeneity by a nickel-chelating column in a single step. The second vector is a derivative of the first vector; it encodes two tandem phosphorylation sites for heart muscle kinase (HMK) immediately downstream from the His residues. Therefore, the resulting fusion protein can be radiolabeled using [gamma-32P]ATP and HMK in vitro. The labeled protein can then be used as a probe to detect protein-protein interaction.
我们构建了两种便捷的载体，用于在大肠杆菌中生产外源蛋白。第一种载体用于生产带有组氨酸（His）标签的融合蛋白。除了编码六个连续的His残基外，它还包含三个独特的限制性酶切位点，可在三个不同的阅读框中克隆平端DNA片段。因此，人们可以将任何感兴趣的基因克隆到该载体中，以制备在N端带有六个His标签的融合蛋白。His标签能够通过镍螯合柱在一步操作中将融合蛋白纯化至几乎均一的状态。第二种载体是第一种载体的衍生物；它在His残基的紧邻下游编码两个用于心肌激酶（HMK）的串联磷酸化位点。因此，所得到的融合蛋白可以在体外使用[γ-32P]ATP和HMK进行放射性标记。然后，标记后的蛋白可作为探针用于检测蛋白质-蛋白质相互作用。
