Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins.
Biotination of proteins in vivo. A post-translational modification to label, purify, and study proteins.
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DOI：
10.1016/s0021-9258(18)86949-6
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发表时间：
1990-06
期刊：
影响因子：
--
通讯作者：
J. Cronan
中科院分区：
文献类型：
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作者：
J. Cronan
用生物素对蛋白质进行翻译后修饰提供了在体内特异性标记蛋白质和从粗细胞裂解产物中纯化蛋白质的方法。经生物素连接酶修饰的羧基末端蛋白片段具有很强的保守性。我们已经证明，通过一些异源蛋白与这些羧基末端序列的翻译基因融合而编码的蛋白质在体内被生物化。允许融合蛋白生物定位所需的蛋白质片段的最小大小为75个氨基酸。这种生物定位序列虽然起源于细菌，但在酿酒酵母和大肠杆菌中都起作用。融合蛋白在体内很容易被生物素标记，而且由于生物标记蛋白种类稀少(不到5种)，标记具有高度的特异性。通过与单体亲和素结合，然后用含有生物素的缓冲液洗脱，可以很容易地以天然形式纯化生物标记的融合蛋白。或者，蛋白质可以在1%十二烷基硫酸钠或8M尿素存在下以变性形式纯化。因此，这项技术允许通过亲和层析来纯化任何可以连接生物定位序列的蛋白质。体内特定标记蛋白质的能力在细胞内蛋白质运输和细胞骨架动力学等研究中应该是有用的。
Post-translational modification of proteins with biotin provides the means to specifically label proteins in vivo and to purify proteins from crude cell lysates. The carboxyl-terminal protein segments modified by reaction with biotin ligase are strongly conserved in nature. We have demonstrated that the proteins encoded by translational gene fusions of a number of heterologous proteins to these carboxyl-terminal sequences become biotinated in vivo. The minimum size of the protein segment needed to allow biotination of fusion proteins is 75 amino acids. This biotination sequence, although of bacterial origin, functions in Saccharomyces cerevisiae as well as in Escherichia coli. Fusion proteins are readily labeled with [3H]biotin in vivo and the labeling is highly specific due to the scarcity (less than 5) of biotinated protein species. Biotinated fusion proteins can be readily purified in native form by binding to columns of monomeric avidin followed by elution with buffers containing biotin. Alternatively, proteins can be purified in a denatured form in presence of 1% sodium dodecyl sulfate or 8 M urea. Thus, this technology allows purification by affinity chromatography of any protein to which a biotination sequence can be attached. The ability to specifically label a protein in vivo should have utility in studies such as intracellular protein trafficking and cytoskeletonal dynamics.