Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag
Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag
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DOI：
10.1016/j.bbapap.2011.09.001
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发表时间：
2011-12-01
影响因子：
3.2
通讯作者：
Kuroda, Yutaka
中科院分区：
文献类型：
--
作者：
Rathnayaka, Tharangani;Tawa, Minako;Kuroda, Yutaka
高斯荧光素酶(GLuc)是已知的最小的生物发光蛋白，作为一种潜在的报告蛋白而备受关注。然而，其10个形成二硫键的半胱氨酸阻碍了重组GLuc的高效生产，从而限制了其在生物成像中的应用。在这里，我们证明了在标准表达温度下，在GLuc(GLuc-C9D)的C末端添加一个短的溶解性增强肽标签(SEP-Tag)显著增加了可溶性蛋白质的比例。表达时间大大缩短，GLuc-C9D的最终产量显著高于我们之前的pCoded表达系统。反相高效液相色谱分析表明，GLuc-C9D突变体在适当氧化后以单二硫键模式折叠。此外，GLuc-C9D的热变性是完全可逆的，CD光谱分析表明其二级结构含量在40℃之前保持不变。GLuc的H-1-核磁共振谱显示了天然折叠蛋白典型的、分散良好的尖峰。GLuc-C9D的生物发光活性很强，即使在高温下孵育也完全保持不变。这些结果表明，使用SEP标签的增溶作用对于生产大量含有多个二硫键的蛋白质是有用的。(C)2011年，由爱思唯尔出版。
Gaussia luciferase (GLuc) is the smallest known bioluminescent protein and is attracting much attention as a potential reporter protein. However, its 10 disulfide bond forming cysteines have hampered the efficient production of recombinant GLuc and thus limited its use in bio-imaging application. Here, we demonstrate that the addition of a short solubility enhancement peptide tag (SEP-Tag) to the C-terminus of GLuc (GLuc-C9D) significantly increased the fraction of soluble protein at a standard expression temperature. The expression time was much shorter, and the final yield of GLuc-C9D was significantly higher than with our previous pCold expression system. Reversed phase HPLC indicated that the GLuc-C9D variant folded with a single disulfide bond pattern after proper oxidization. Further, the thermal denaturation of GLuc-C9D was completely reversible, and its secondary structure content remained unchanged until 40 degrees C as assessed by CD spectroscopy. The H-1-NMR spectrum of GLuc indicated sharp well dispersed peaks typical for natively folded proteins. GLuc-C9D bioluminescence activity was strong and fully retained even after incubation at high temperatures. These results suggest that solubilization using SEP-Tags can be useful for producing large quantities of proteins containing multiple disulfide bonds. (C) 2011 Published by Elsevier B.V.