Honey S, Schneider B L, Schieltz D M, Yates J R, Futcher B
Cold Spring Harbor Laboratories, Cold Spring Harbor, NY 11724, USA.
Nucleic Acids Res. 2001 Feb 15;29(4):E24. doi: 10.1093/nar/29.4.e24.
A novel multiple affinity purification (MAFT) or tandem affinity purification (TAP) tag has been constructed. It consists of the calmodulin binding peptide, six histidine residues, and three copies of the hemagglutinin epitope. This 'CHH' MAFT tag allows two or three consecutive purification steps, giving high purity. Active Clb2-Cdc28 kinase complex was purified from yeast cells after inserting the CHH tag into Clb2. Associated proteins were identified using mass spectrometry. These included the known associated proteins Cdc28, Sic1 and Cks1. Several other proteins were found including the 70 kDa chaperone, Ssa1.
一种新型的多重亲和纯化（MAFT）或串联亲和纯化（TAP）标签已构建完成。它由钙调蛋白结合肽、六个组氨酸残基和三个血凝素表位拷贝组成。这种“CHH”MAFT标签允许进行两个或三个连续的纯化步骤，从而获得高纯度。将CHH标签插入Clb2后，从酵母细胞中纯化出了活性Clb2-Cdc28激酶复合物。使用质谱法鉴定了相关蛋白。这些蛋白包括已知的相关蛋白Cdc28、Sic1和Cks1。还发现了其他几种蛋白，包括70 kDa伴侣蛋白Ssa1。
