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

被引：108

作者：

Fujii, Yuki ^{[1
]}

Kaneko, Mika ^{[2
]}

Neyazaki, Makiko ^{[3
]}

Nogi, Terukazu ^{[3
]}

Kato, Yukinari ^{[2
]}

Takagi, Junichi ^{[1
]}

机构：

[1] Osaka Univ, Inst Prot Res, Lab Prot Synth & Express, Suita, Osaka 5650871, Japan

[2] Tohoku Univ, Grad Sch Med, Dept Reg Innovat, Aoba Ku, Sendai, Miyagi 9808575, Japan

[3] Yokohama City Univ, Grad Sch Med Life Sci, Yokohama, Kanagawa 2300045, Japan

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2014年 / 95卷

关键词：

Purification method; Immunoaffinity chromatography; Monoclonal antibody; Podoplanin; Peptide tag; Immunodetection; Mammalian cell expression; MONOCLONAL-ANTIBODY; RECOMBINANT PROTEINS; PURIFICATION; IDENTIFICATION; GLYCOSYLATION; PEPTIDE;

D O I：

10.1016/j.pep.2014.01.009

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

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-I, 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. (C) 2014 Elsevier Inc. All rights reserved.

引用

页码：240 / 247

页数：8

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