An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system
| Autor: | Junichi Takagi, Mika K. Kaneko, Emiko Mihara, Katsu Takahashi, Yuki Fujii, Yu Kitago, Yukinari Kato, Keiko Tamura-Kawakami, Naoko Norioka, Sanae Tabata |
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| Rok vydání: | 2018 |
| Předmět: |
Models
Molecular 0301 basic medicine medicine.drug_class Recombinant Fusion Proteins medicine.medical_treatment Peptide Cleavage (embryo) Monoclonal antibody law.invention Rats Sprague-Dawley Epitopes Mice 03 medical and health sciences 0302 clinical medicine Protein Domains Recognition sequence law Endopeptidases Escherichia coli medicine Animals Humans Amino Acid Sequence Adaptor Proteins Signal Transducing chemistry.chemical_classification Protease biology Tobacco etch virus Antibodies Monoclonal biology.organism_classification HEK293 Cells 030104 developmental biology Biochemistry chemistry Bone Morphogenetic Proteins Recombinant DNA Female Peptides Linker 030217 neurology & neurosurgery Protein Binding Biotechnology |
| Zdroj: | Protein Expression and Purification. 147:94-99 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2018.03.004 |
| Popis: | Peptide-based affinity tags are commonly used in recombinant production/purification of proteins, and are often preceded or followed by a protease recognition sequence to allow tag removal. We describe a rat monoclonal antibody 2H5 recognizing an undecapeptide tag called "eTev", which contains a recognition sequence for Tobacco Etch Virus (TEV) protease. In the crystal structure of 2H5-eTev complex, the long eTev peptide assumes compact α-helical conformation in the binding groove, exposing both ends to the solution. This architecture allowed us to connect eTev with another peptide tag called PA tag via linker sequence, ensuring the simultaneous access of two anti-tag antibodies. When this tandem double tag was attached at one end of various proteins, it enabled highly sensitive and protein-independent detection by sandwich ELISA. Utilizing this system during a rapid cell line screening, we succeeded in isolating stable cell clones expressing high level of mouse Wise protein. |
| Databáze: | OpenAIRE |
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