A low-cost affinity purification system using β-1,3-glucan recognition protein and curdlan beads

Horiuchi, Masataka; Takahasi, Kiyohiro; Kobashigawa, Yoshihiro; Ochiai, Masanori; Inagaki, Fuyuhiko

A low-cost affinity purification system using β-1,3-glucan recognition protein and curdlan beads

Horiuchi, Masataka; Takahasi, Kiyohiro; Kobashigawa, Yoshihiro et al.

Protein Engineering, Design and Selection (PEDS), 2012, 25(8), 405-413

Permalink : https://hdl.handle.net/2115/49735

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アクセス権	open access
URI	https://hdl.handle.net/2115/49735
タイトル	A low-cost affinity purification system using β-1,3-glucan recognition protein and curdlan beads
著者	Horiuchi, Masataka ; Takahasi, Kiyohiro ; Kobashigawa, Yoshihiro ; Ochiai, Masanori ; Inagaki, Fuyuhiko
e-Rad_Researcher 90322825 著者名 Horiuchi, Masataka
著者名 Takahasi, Kiyohiro
著者名 Kobashigawa, Yoshihiro
e-Rad_Researcher 10241382 著者名 Ochiai, Masanori
e-Rad_Researcher 70011757 著者名 Inagaki, Fuyuhiko
言語	英語
キーワード	β-1,3-glucan recognition protein
	affinity tag
	curdlan
	glutathione S-transferase
	recombinant protein
発行日	2012-08
出版者	Oxford University Press
収録物名	Protein Engineering, Design and Selection (PEDS)
巻(号)	25(8)
ページ	405-413
抄録	Silkworm β-1,3-glucan recognition protein (βGRP) tightly and specifically associates with β-1,3-glucan. We report here an affinity purification system named the 'GRP system', which uses the association between the β-1,3-glucan recognition domain of βGRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble β-1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (4-6). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.
資源タイプ	学術雑誌論文
出版タイプ	Version of Record
PISSN	1741-0126
関連情報 (isIdenticalTo)	出版社版DOI https://doi.org/10.1093/protein/gzs028