PA tag: A versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin

Yuki Fujii, Mika Kaneko, Makiko Neyazaki, Terukazu Nogi, Yukinari Kato, Junichi Takagi

Research output: Contribution to journalArticlepeer-review

93 Citations (Scopus)

Abstract

Peptide-based epitope tagging technology is universally used in nearly all kind of research projects that involve biochemical characterization of a target protein, but not many systems are fully compatible with purification purpose. By utilizing an anti-human podoplanin antibody NZ-1, we constructed a novel epitope tag system. NZ-1 possesses exceptionally high affinity toward a dodecapeptide dubbed "PA tag", with a characteristic slow dissociation kinetics. Because of its high affinity, PA-tagged proteins in a dilute sample can be captured by immobilized NZ-1 resin in a near complete fashion and eluted by a solution of free PA peptide. This enabled efficient one-step purification of various proteins including soluble (an ectodomain fragment of neuropilin-1) and membrane (epidermal growth factor receptor) proteins expressed in mammalian cells. Mild regeneration condition of the peptide-bound antibody ensures repeated use of the antibody resin, indicating a cost-efficient nature of the system. Together with its outstanding performance in the immunodetection experiments (i.e., Western blotting and flow cytometry), PA tag/NZ-1 system will offer a great chance to facilitate protein production in many biomedical research projects.

Original languageEnglish
Pages (from-to)240-247
Number of pages8
JournalProtein Expression and Purification
Volume95
DOIs
Publication statusPublished - 2014 Mar

Keywords

  • Immunoaffinity chromatography
  • Immunodetection
  • Mammalian cell expression
  • Monoclonal antibody
  • Peptide tag
  • Podoplanin
  • Purification method

ASJC Scopus subject areas

  • Biotechnology

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