An anti-peptide monoclonal antibody recognizing the tobacco etch virus protease-cleavage sequence and its application to a tandem tagging system

Sanae Tabata, Yu Kitago, Yuki Fujii, Emiko Mihara, Keiko Tamura-Kawakami, Naoko Norioka, Katsu Takahashi, Mika K. Kaneko, Yukinari Kato, Junichi Takagi

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)94-99
Number of pages6
JournalProtein Expression and Purification
Volume147
DOIs
Publication statusPublished - 2018 Jul

Keywords

  • Affinity tag
  • Monoclonal antibody
  • Sandwich ELISA
  • Tag removal
  • Tandem tag
  • Tobacco etch virus protease

ASJC Scopus subject areas

  • Biotechnology

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