TY - JOUR
T1 - PA tag
T2 - A versatile protein tagging system using a super high affinity antibody against a dodecapeptide derived from human podoplanin
AU - Fujii, Yuki
AU - Kaneko, Mika
AU - Neyazaki, Makiko
AU - Nogi, Terukazu
AU - Kato, Yukinari
AU - Takagi, Junichi
N1 - Funding Information:
We would like to thank Keiko Tamura-Kawakami, Emiko Mihara, and Rika Oi for their excellent technical support, and Mie Sakai for preparation of the manuscript. This work was supported by the Platform for Drug Discovery, Informatics, and Structural Life Science grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT).
PY - 2014/3
Y1 - 2014/3
N2 - 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.
AB - 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.
KW - Immunoaffinity chromatography
KW - Immunodetection
KW - Mammalian cell expression
KW - Monoclonal antibody
KW - Peptide tag
KW - Podoplanin
KW - Purification method
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U2 - 10.1016/j.pep.2014.01.009
DO - 10.1016/j.pep.2014.01.009
M3 - Article
C2 - 24480187
AN - SCOPUS:84893717643
SN - 1046-5928
VL - 95
SP - 240
EP - 247
JO - Protein Expression and Purification
JF - Protein Expression and Purification
ER -