Liu Bo, Archer Chase T, Burdine Lyle, Gillette Thomas G, Kodadek Thomas
Division of Translational Research, Departments of Internal Medicine and Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9185, USA.
J Am Chem Soc. 2007 Oct 17;129(41):12348-9. doi: 10.1021/ja072904r. Epub 2007 Sep 26.
A new label transfer method is presented that overcomes most of the limitations of current systems. A protein of interest is tagged with tetra-cysteine sequence (FlAsH Receptor Peptide (FRP)) that binds tightly and specifically to a chimeric molecule 3,4-dihydroxyphenylalanine – biotin – 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein (DOPA-biotin-FlAsH). Upon brief periodate oxidation, the DOPA moiety is cross-linked to nearby surface-exposed nucleophiles. Boiling the products in excess dithiol dissolves the FlAsH-FRP interaction, resulting in transfer of the biotin tag to the partner proteins, allowing them to be identified by standard methods.
提出了一种新的标记转移方法，该方法克服了当前系统的大多数局限性。将感兴趣的蛋白质用四半胱氨酸序列（荧光素砷结合肽（FRP））进行标记，该序列与嵌合分子3,4-二羟基苯丙氨酸-生物素-4′,5′-双（1,3,2-二硫代砷杂环戊烷-2-基）荧光素（DOPA-生物素-FlAsH）紧密且特异性结合。经过短暂的高碘酸盐氧化后，DOPA部分与附近暴露于表面的亲核试剂交联。在过量二硫醇中煮沸产物会破坏FlAsH-FRP相互作用，导致生物素标签转移至伴侣蛋白，从而能够通过标准方法对它们进行鉴定。
