Van Reeth T, Drèze P L, Szpirer J, Szpirer C, Gabant P
Université Libre de Bruxelles, Rhode-Saint-Genèse, Belgium.
Biotechniques. 1998 Nov;25(5):898-904. doi: 10.2144/98255pf01.
Epitope tagging simplifies detection, characterization and purification of proteins. Gene fusion to combine the coding region of a well-characterized epitope with the coding region for a protein of interest generally requires several subcloning steps. Alternatively, a PCR strategy can be used to generate such a chimeric gene. In addition to its simplicity, this approach allows one to limit the size of the multiple cloning sites present in conventional expression vectors, thus reducing the introduction of artifactual amino-acid sequences into the fused protein. In this communication, we describe new vectors that allow PCR cloning and selection of chimeric genes coding for N- or C-terminal His-tagged proteins. These vectors are based on the control of cell death CcdB direct selection technology and are well adapted to the cloning of blunt-ended PCR products that were generated by using thermostable polymerases that provide proofreading activity.
表位标签技术简化了蛋白质的检测、特性鉴定及纯化过程。将一个已充分表征的表位的编码区与目标蛋白质的编码区进行基因融合，通常需要几个亚克隆步骤。或者，可以采用PCR策略来产生这样的嵌合基因。除了操作简单之外，这种方法还能让人们限制常规表达载体中多克隆位点的大小，从而减少人为氨基酸序列引入融合蛋白的情况。在本通讯中，我们描述了新的载体，这些载体允许PCR克隆和选择编码N端或C端带His标签蛋白质的嵌合基因。这些载体基于细胞死亡控制CcdB直接选择技术，非常适合克隆通过使用具有校对活性的热稳定聚合酶产生的平端PCR产物。
