Polyarginine as a multifunctional fusion tag
Polyarginine as a multifunctional fusion tag
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DOI：
10.1110/ps.051393805
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发表时间：
2005-06-01
期刊：
影响因子：
8
通讯作者：
Raines, RT
中科院分区：
文献类型：
--
作者：
Fuchs, SM;Raines, RT
与阳离子肽如九精氨酸（R-9）的融合提供了将分子货物递送到哺乳动物细胞中的手段。在这里，我们提供了一个模型蛋白质：牛胰腺核糖核酸酶（RNase A）的属性上的R9标签的效果进行了彻底的分析。R9标签降低了RNA酶A的构象稳定性（在磷酸盐缓冲盐水中Δ T-m = -8 ℃）。这种影响几乎可以通过添加盐来减轻。该标签不损害RNase A的酶活性。R-9标签有助于通过阳离子交换色谱法纯化RNA酶A，并使RNA酶A能够吸附在载玻片和硅胶树脂上，同时保留酶活性。用羧肽酶B处理可以精确和完全地去除标签。最后，R-9标签增加RNA酶A的细胞摄取和G88 R RNA酶A的细胞毒性，G88 R RNA酶A是一种逃避胞质核糖核酸酶抑制蛋白的变体。因此，我们得出结论，聚精氨酸是一种通用的蛋白质融合标签。
Fusion to cationic peptides, such as nonaarginine (R-9), provides a means to deliver molecular cargo into mammalian cells. Here, we provide a thorough analysis of the effect of an R9 tag on the attributes of a model protein: bovine pancreatic ribonuclease (RNase A). The R9 tag diminishes the conformational stability of RNase A (Delta T-m = -8 degrees C in phosphate-buffered saline). This effect is nearly mitigated by the addition of salt. The tag does not compromise the enzymatic activity of RNase A. An R-9 tag facilitates the purification of RNase A by cation-exchange chromatography and enables the adsorption of RNase A on glass slides and silica resin with the retention of enzymatic activity. The tag can be removed precisely and completely by treatment with carboxypeptidase B. Finally, the R-9 tag increases both the cellular uptake of RNase A and the cytotoxicity of G88R RNase A, a variant that evades the cytosolic, ribonuclease inhibitor protein. Thus, we conclude that polyarginine is a versatile protein fusion tag.