在多链内插入的新型His-tag变体
Anastasiia G Tarabarova1, Anton Lopukhov2, Alexey N Fedorov3
1A N Bach Institute of Biochemistry of the Russian Academy of Sciences, Leninskii prosp 33/2, Moscow 119071, Russian Federation.
新设计的内部His标签,具有histidine和glycine残留物,最大限度地减少蛋白质结构的影响. 这些可插入的标签提供了一种新的方法来净化各种蛋白质,而无需N或C端融合.
科学领域:
- 生物化学 生物化学
- 分子生物学分子生物学
- 蛋白质工程是指蛋白质工程.
背景情况:
- 六西丁 (His-tag) 亲和度净化是一种标准的技术.
- N-或C-终端的His-tag融合可以对蛋白质结构和功能产生负面影响.
- 探索了内部标签插入,以减轻N / C终端融合问题.
研究的目的:
- 设计和描述新的内部His标签.
- 为了最大限度地减少固体障碍和对蛋白质结构/功能的影响.
- 扩大像eGFP和GrAD207.7这样的融合蛋白的实用性.
主要方法:
- 设计了内部的His标签,其中有两个由甘氨酸残留物分离的histidine三胞胎.
- 通过将它们内部融合到增强绿色光蛋白 (eGFP) 中,测试了标签的适用性.
- 经过验证的标签功能与修改的GroEL陪伴域 (GrAD207).
主要成果:
- 成功地将内部His标签纳入eGFP和GrAD207.7中.
- 证明与终端标签相比,内部标签可以最大限度地减少硬体障碍.
- 显示了在不同蛋白质类别中广泛适用的潜力.
结论:
- 内部His标签为N/C终端融合提供了一个可行的替代方案.
- 设计的标签可以调整以有效净化各种蛋白质.
- 这种方法扩大了聚变蛋白的功能,但不损害术语.
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