A novel approach of protein immobilization for protein chips using an oligo-cysteine tag
| Autor: | Saemi Ohshiro, Daisuke Sato, Yasuhiro Kuramitsu, Teruhisa Ichihara, Shuichi Kamei, Junko Akada, Masanori Fujimoto, Kazuyuki Nakamura |
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| Rok vydání: | 2006 |
| Předmět: |
Proteomics
Chemistry Recombinant Fusion Proteins Green Fluorescent Proteins Protein Array Analysis Substrate (chemistry) General Chemistry Protein tag medicine.disease_cause Biochemistry Fusion protein Green fluorescent protein FLAG-tag medicine Stathmin Cysteine Escherichia coli Oligopeptides Myc-tag DNA Primers |
| Zdroj: | Journal of proteome research. 5(9) |
| ISSN: | 1535-3893 |
| Popis: | 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, diamond-like, 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. |
| Databáze: | OpenAIRE |
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