Development of INSOL-tag for proteome-wide protein handling and its application in protein array analysis.

Autor: Fukuda E; Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Koto-ku, Tokyo, Japan.; Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki, Japan., Mori M; Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Koto-ku, Tokyo, Japan., Shiku H; Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Tsu, Mie, Japan., Miyahara Y; Department of Immuno-Gene Therapy, Mie University Graduate School of Medicine, Tsu, Mie, Japan., Kawamura Y; Japan Biological Informatics Consortium, Koto-ku, Tokyo, Japan., Ogawa K; Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Koto-ku, Tokyo, Japan., Ogura T; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki, Japan., Goshima N; Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Koto-ku, Tokyo, Japan.
Jazyk: angličtina
Zdroj: Genes to cells : devoted to molecular & cellular mechanisms [Genes Cells] 2020 Jan; Vol. 25 (1), pp. 41-53. Date of Electronic Publication: 2019 Dec 03.
DOI: 10.1111/gtc.12735
Abstrakt: Proteomic analysis requires protein tags that enable high-throughput handling; however, versatile tags that can be used in in vitro expression systems are currently lacking. In this study, we developed an insoluble protein tag, INSOL-tag, derived from human transcription factor MafG. The INSOL-tagged target protein is expressed in a eukaryotic in vitro expression system and recovered as a pellet following centrifugation at 19,000 × g for 20 min. Comparisons of the target protein recovery rates of GST-tag and INSOL-tag using 111 cytoplasmic proteins revealed a fourfold increase in the yield of INSOL-tagged proteins. Using 267 cancer antigens purified with INSOL-tag, we subsequently developed an INSOL-CTA array method, for profiling autoantibodies in sera of cancer patients. The detection limit of the array was approximately 11.1 pg IgG, and the correlation with ELISA was high (R 2  = .993, .955). Moreover, when autoantibody profiling of digestive cancer patient sera was performed, antigen spreading was observed. These data suggest that INSOL-tag is a versatile tag that can insolubilize a wide range of target proteins. It is therefore expected to become a powerful tool in comprehensive protein preparation for protein arrays, antibody production, and mass spectrometry.
(© 2019 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)
Databáze: MEDLINE
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