Effects of the Fc‐III tag on activity and stability of green fluorescent protein and human muscle creatine kinase
Effects of the Fc‐III tag on activity and stability of green fluorescent protein and human muscle creatine kinase
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DOI：
10.1002/pro.2282
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发表时间：
2013-07
期刊：
影响因子：
8
通讯作者：
S. Feng;Yiyi Gong;Gulishana Adilijiang;Haiteng Deng
中科院分区：
文献类型：
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作者：
S. Feng;Yiyi Gong;Gulishana Adilijiang;Haiteng Deng
Fc-III标签是一种新开发的融合标签，可应用于蛋白质纯化和检测。在目前的工作中，我们使用 Fc-III 标记的绿色荧光蛋白 (GFP) 和人肌肉肌酸激酶 (CK) 作为模型系统，研究 Fc-III 标记对表达的含多半胱氨酸蛋白的活性和稳定性的影响。我们的结果表明，与 His 标签蛋白相比，Fc-III 标签对 GFP 的荧光没有不利影响，并且减少了由于不正确的 Cys 氧化而导致 GFP 错误折叠的发生。 Fc-III 标记 CK 的活性和稳定性略低于无标记 CK，但高于 His 标记 CK（根据氧化 CK 与还原 CK 的比率确定）。 His 标记的 CK 的主要部分处于氧化形式，而 Fc-III 标记的 CK 的主要部分处于还原形式。提出了具有不同标签的 CK 折叠模型，这可能有助于了解 Fc-III 标签对二硫键蛋白构象的影响。
The Fc‐III tag is a newly developed fusion tag that can be applied to protein purification and detection. In the present work, we use the Fc‐III‐tagged green fluorescent protein (GFP) and human muscle creatine kinase (CK) as model systems to investigate effects of the Fc‐III tag on activities and stabilities of the expressed multicysteine‐containing proteins. Our results show the Fc‐III tag has no adverse effects on the fluorescence of GFP and reduces the occurrence of GFP misfolding due to incorrect Cys oxidation compared with the His‐tagged protein. The activity and stability of the Fc‐III‐tagged CK is slightly lower than that of the tag‐free CK, but is higher than that of the His‐tagged CK as determined by the ratio of the oxidized versus reduced CK. A major portion of His‐tagged CK is in its oxidized form, while that of the Fc‐III‐tagged CK is in its reduced form. A folding model of CK with different tags was proposed, which may provide insights into the effect of the Fc‐III tag on the conformations of disulfide‐bridged proteins.