Cell-free synthesis of cytochrome c oxidase, a multicomponent membrane protein
Cell-free synthesis of cytochrome c oxidase, a multicomponent membrane protein
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DOI：
10.1007/s10863-010-9285-8
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发表时间：
2010-06-01
影响因子：
3
通讯作者：
Shimada, Hideo
中科院分区：
文献类型：
--
作者：
Katayama, Yukie;Shimokata, Kunitoshi;Shimada, Hideo
无细胞蛋白质合成是一种有用的技术，可以特异性地将同位素标记的氨基酸结合到蛋白质中。这种结合对于用于阐明蛋白质功能的特定氨基酸残基的电子状态的红外分析是必不可少的。尽管已有17种膜蛋白通过无细胞系统在活性状态下合成，但迄今为止还没有用这种技术合成异亚基蛋白，这表明存在严重的技术局限性。在这里，我们报道了反硝化副球菌细胞色素c氧化酶的无细胞合成，细胞色素c氧化酶是一种由三个不同亚基组成的膜蛋白复合物，含有两个血红素a分子和两个氧化还原活性铜中心。合成的蛋白具有正常的可见光吸收光谱和铁细胞色素c氧化活性。
Cell-free protein synthesis is a useful technique that can site-specifically incorporate isotope-labeled amino acids into proteins. This incorporation is essential for infrared analyses of the electronic state of a specific amino acid residue used to elucidate protein function. Although 17 membrane proteins have been synthesized in their active state by cell-free systems, to date no hetero-subunit protein has been synthesized with this technique, suggesting that there are serious technical limitations. Here we report the cell-free synthesis of Paracoccus denitrificans cytochrome c oxidase, a membrane protein complex composed of three distinct subunits that contain two heme A molecules and two redox-active copper centers. The synthesized protein exhibited normal Soret/vis absorption spectra and ferrocytochrome c oxidation activity.