PA tag: a versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin.
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.
Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan.
Yuki
Y
Fujii
Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
Mika
M
Kaneko
Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama 230-0045, Japan.
Makiko
M
Neyazaki
Graduate School of Medical Life Science, Yokohama City University, Tsurumi, Yokohama 230-0045, Japan.
Terukazu
T
Nogi
Department of Regional Innovation, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. Electronic address: yukinarikato@med.tohoku.ac.jp.
Yukinari
Y
Kato
Laboratory of Protein Synthesis and Expression, Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan. Electronic address: takagi@protein.osaka-u.ac.jp.
Junichi
J
Takagi
D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification; D002846:Chromatography, Affinity / Q000295:instrumentation* / Q000379:methods; D006801:Humans; D007368:Interferometry; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D000098853:Podoplanin
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; C117468:PDPN protein, human; D000098853:Podoplanin
Immunoaffinity chromatography; Immunodetection; Mammalian cell expression; Monoclonal antibody; Peptide tag; Podoplanin; Purification method