One-pot chemo-enzymatic synthesis of reporter-modified proteins
One-pot chemo-enzymatic synthesis of reporter-modified proteins
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DOI：
10.1039/b512735a
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发表时间：
2006-01-01
影响因子：
3.2
通讯作者：
Burkart, MD
中科院分区：
文献类型：
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作者：
Worthington, AS;Burkart, MD
为了满足最近的进展，共价报告标记的蛋白质，我们提出了一个灵活的合成报告类似物。在这里，我们展示了一锅化学-酶促合成的标记蛋白，允许共价拴系的任何胺末端的荧光或亲和标记的载体蛋白或融合构建体。这两个反应序列由活化的泛酸盐偶联、生物合成转化为辅酶A（CoA）类似物和通过磷酸泛酰巯基乙胺基转移酶（PPT酶）进行的酶促载体蛋白修饰组成。我们还探测底物特异性辅酶AA，在途径中的第一个酶。用这种方法可以快速制备CoA类似物，从而允许来自各种分子种类的报告部分的区域特异性连接。
To meet recent advancements in the covalent reporter labeling of proteins, we propose a flexible synthesis for reporter analogs. Here we demonstrate a one-pot chemo-enzymatic synthesis of reporter-labeled proteins that allows the covalent tethering of any amine-terminal fluorescent or affinity label to a carrier protein or fusion construct. This two-reaction sequence consists of activated panthothenate coupling, biosynthetic conversion to the coenzyme A (CoA) analog, and enzymatic carrier protein modification via phosphopantetheinyltransferase (PPTase). We also probe substrate specificity for CoAA, the first enzyme in the pathway. With this approach CoA analogs may be rapidly prepared, thus permitting the regiospecific attachment of reporter moieties from a variety of molecular species.