串联亲和纯化标签分类与优化

质谱技术高速发展,检测灵敏度不断提高,但区分特异性与非特异性相互作用仍然是研究相互作用蛋白的瓶颈,获得高纯度的蛋白复合体是鉴定相互作用蛋白的限制性因素。近年来串联亲和纯化（TAP）技术的产生和发展有效解决了相互作用蛋白鉴定中的特异性问题。TAP技术是将N端或C端TAP标签与目的蛋白融合并导入靶细胞进行表达,裂解细胞释放融合蛋白,在接近生理状态下利用标签两步特异性亲和洗脱得到蛋白复合体。其中,TAP标签蛋白的选择和优化是该技术成功的关键。

Studies of Protein Tags Used in Tandem Affinity Purification

The problem of distinguishing specific from nonspecific interacting proteins remains, although improvements to mass spectrometry instrumentation generally increase peptide detection sensitivities. Thus ultimately, the limiting factor in the identification of specific interacting proteins lies with the purification itself. An effort to resolve this specificity issue has been made with the introduction of the tandem affinity purification（TAP）. The TAP method involves fusion of the TAP tag to N- or C-terminal of the target protein and introduction of the construct into the host cell. The fusion protein is recovered from cell extracts by two sequential purification steps under close-to-physiological base on the TAP tag, so the key is the tag. In a word, optimization of tags is one of the effective means to improve the effectiveness of TAP.