A novel approach of protein immobilization for protein chips using an oligo-cysteine tag
A novel approach of protein immobilization for protein chips using an oligo-cysteine tag
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DOI：
10.1021/pr0504889
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发表时间：
2006-09-01
影响因子：
4.4
通讯作者：
Nakamura, Kazuyuki
中科院分区：
文献类型：
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作者：
Ichihara, Teruhisa;Akada, Junko K.;Nakamura, Kazuyuki
蛋白质芯片技术是实现高通量功能蛋白质组学的必要技术。我们开发了一种新的蛋白质标签，由蛋白质末端的五个串联半胱氨酸重复序列（Cys-tag）组成。Cys-tag被设计成允许蛋白质共价附着在马来酰亚胺修饰的、类金刚石的、碳涂层的硅衬底表面。作为模型蛋白，我们创建了一个增强型绿色荧光蛋白（EGFP）和一个EGFP-stathmin融合蛋白，两者都含有一个Cys-tag。我们还加入了一个低聚组氨酸标签，以便使用Ni珠纯化，并在大肠杆菌中表达该蛋白。纯化的cys标记的EGFP可以有效地捕获在马来酰亚胺包被的底物上，因此融合蛋白的荧光检测为50 pg，而结合增强化学发光的免疫检测仅为5 pg。这种高度敏感的免疫检测可能是由于cys -标签与底物的强共价结合以及蛋白质在周围溶液中的有效暴露。因此，Cys-tag应该有助于开发一种新的蛋白质芯片的蛋白质打印方法，这种方法需要非常少的蛋白质，并且可以用于蛋白质-配体相互作用的高性能分析。
Protein chip technology is essential for high-throughput functional proteomics. We developed a novel protein tag consisting of five tandem cysteine repeats (Cys-tag) at termini of proteins. The Cys-tag was designed to allow covalent attachment of proteins to the surface of a maleimide-modified, diamondlike, carbon-coated silicon substrate. As model proteins, we created an enhanced green fluorescent protein (EGFP) and an EGFP-stathmin fusion protein, both of which contained a Cys-tag. We also included an oligo-histidine tag to allow its purification by the use of Ni beads, and we expressed the protein in Escherichia coli. The purified Cys-tagged EGFP could be captured on the maleimide-coated substrate efficiently so that 50 pg of the fusion protein was detected by fluorescence, and as little as 5 pg was immunodetected by combination with enhanced chemiluminescence. This highly sensitive immunodetection may be due to the strong covalent binding of the Cys-tag to the substrate combined with efficient exposure of the protein to the surrounding solution. Thus, the Cys-tag should be useful for developing a novel protein printing method for protein chips that requires very low amounts of protein and can be used for high-performance analysis of protein-ligand interactions.