Integrated tandem affinity protein purification using the polyhistidine plus extra 4 amino acids (HiP4) tag system.

Autor: Ino Y; Advanced Medical Research Center, Yokohama City University, Yokohama, Kanagawa, Japan.; Department of Health Science, Gunma Paz University Graduate School of Health Sciences, Takasaki, Gunma, Japan., Yamaoka Y; Life Science Laboratory, Technology and Development Division, Kanto Chemical Co. Inc., Isehara, Kanagawa, Japan.; Department of Microbiology, School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan., Tanaka K; Department of Microbiology, School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan., Miyakawa K; Department of Microbiology, School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan.; Center for Influenza and Respiratory Virus Research, National Institute of Infectious Diseases, Tokyo, Japan., Nishi M; Department of Microbiology, School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan., Hatayama Y; Advanced Medical Research Center, Yokohama City University, Yokohama, Kanagawa, Japan.; Department of Microbiology, School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan., Kimura H; Department of Health Science, Gunma Paz University Graduate School of Health Sciences, Takasaki, Gunma, Japan., Kimura Y; Advanced Medical Research Center, Yokohama City University, Yokohama, Kanagawa, Japan., Ryo A; Advanced Medical Research Center, Yokohama City University, Yokohama, Kanagawa, Japan.; Department of Microbiology, School of Medicine, Yokohama City University, Yokohama, Kanagawa, Japan.; Department of Virology III, National Institute of Infectious Diseases, Tokyo, Japan.
Jazyk: angličtina
Zdroj: Proteomics [Proteomics] 2023 Jun; Vol. 23 (11), pp. e2200334. Date of Electronic Publication: 2023 Mar 03.
DOI: 10.1002/pmic.202200334
Abstrakt: Peptide tag systems are a robust biophysical and biochemical method that is widely used for protein detection and purification. Here, we developed a novel tag system termed "HiP4" (histidine plus four amino acids) whose epitope sequence comprises only seven amino acids (HHHDYDI) that partially overlap with the conventional 6x histidine tag (6xHis-tag). We produced a monoclonal antibody against the HiP4 tag that can be used in multiple immunoassays with high specificity and affinity. Using this system, we developed a tandem affinity purification (TAP) and mass spectrometry (TAP-MS) system for comprehensive protein interactome analysis. The integrated use of nickel bead purification followed by HiP4 tag immunoprecipitation made it possible to reduce nonspecific binding and improve selectivity, leading to the recovery of previously unrecognized proteins that interact with hepatitis B virus X (HBx) protein or TAR DNA-binding protein 43 (TARDBP or TDP-43). Our results indicate that this system may be viable as a simple and powerful tool for TAP-MS that can achieve low background and high selectivity in comprehensive protein-protein interaction analyses.
(© 2023 Wiley-VCH GmbH.)
Databáze: MEDLINE
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