Vettone Antonella, Serpe Mario, Hidalgo Aurelio, Berenguer José, del Monaco Giovanni, Valenti Anna, Rossi Mosé, Ciaramella Maria, Perugino Giuseppe
Institute of Biosciences and BioResources, National Council of Research of Italy, Via P. Castellino 111, 80131 Naples, Italy
Extremophiles. 2016 Jan;20(1):1-13. doi: 10.1007/s00792-015-0791-9.
In the last decade, a powerful biotechnological tool for the in vivo and in vitro specific labeling of proteins (SNAP-tag™ technology) was proposed as a valid alternative to classical protein-tags (green fluorescent proteins, GFPs). This was made possible by the discovery of the irreversible reaction of the human alkylguanine-DNA-alkyl-transferase (hAGT) in the presence of benzyl-guanine derivatives. However, the mild reaction conditions and the general instability of the mesophilic SNAP-tag™ make this new approach not fully applicable to (hyper-)thermophilic and, in general, extremophilic organisms. Here, we introduce an engineered variant of the thermostable alkylguanine-DNA-alkyl-transferase from the Archaea Sulfolobus solfataricus (SsOGT-H5), which displays a catalytic efficiency comparable to the SNAP-tag™ protein, but showing high intrinsic stability typical of proteins from this organism. The successful heterologous expression obtained in a thermophilic model organism makes SsOGT-H5 a valid candidate as protein-tag for organisms living in extreme environments.
在过去十年中，一种用于体内和体外蛋白质特异性标记的强大生物技术工具（SNAP-tag™技术）被提出，作为经典蛋白质标签（绿色荧光蛋白，GFP）的有效替代方案。这是通过发现人烷基鸟嘌呤-DNA-烷基转移酶（hAGT）在苄基鸟嘌呤衍生物存在下的不可逆反应实现的。然而，嗜温性SNAP-tag™的温和反应条件和普遍不稳定性使得这种新方法不完全适用于（超）嗜热生物，一般来说也不适用于嗜极端生物。在这里，我们介绍了一种来自嗜热古菌嗜热栖热菌（SsOGT-H5）的热稳定烷基鸟嘌呤-DNA-烷基转移酶的工程变体，它表现出与SNAP-tag™蛋白相当的催化效率，但具有该生物体蛋白质典型的高内在稳定性。在嗜热模式生物中成功实现的异源表达使SsOGT-H5成为生活在极端环境中的生物体蛋白质标签的有效候选者。
