title: Enhancement of solubility and yield of a β-glucan receptor Dectin-1 C-type lectin-like domain in Escherichia coli with a solubility-enhancement tag.
abstract: Dectin-1 is a C-type lectin-like pattern recognition receptor for β(1-3)-glucans. It plays a crucial role in protecting against fungal invasion through binding to β-glucans which are commonly present on the fungal cell wall. To probe its ligand binding mechanism by NMR, we expressed the recombinant murine Dectin-1 C-type lectin-like domain (CTLD) in E. coli using pCold vector and purified it. However, the high concentration of Dectin-1 CTLD required for NMR analysis could not be attained due to its inherent low solubility and low bacterial expression. In this study, we tried to increase expression and solubility of Dectin-1 CTLD by codon optimization and fusion of a GB1 tag (B1 domain of streptococcal Protein G). GB1 was inserted on either the N-terminal (NT) or C-terminal end as well as both terminal ends of human and mouse Dectin-1 CTLDs. A pure monomeric sample was only obtained with NT-GB1 fused mouse Dectin-1. Expression of mouse Dectin-1 CTLD yielded 0.9 ± 0.2 mg/L culture, codon optimized mouse Dectin-1 CTLD produced 1.4 ± 0.2 mg/L, and the tag-fused domain 7.1 ± 0.3 mg/L. The tag also increased solubility from 0.1 mM to 1.4 mM. The recombinant protein was correctly folded, in a monomeric state, and specifically bound β-glucan laminarin. These results indicate that fusing GB1 to the N-terminus of mouse Dectin-1 domain advantageously increases yield and solubility, allows retention of native structure, and that the site of fusion is critical.
authors.affiliation: Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-0034, Japan.
authors.forename: Hari Prasad
authors.initials: HP
authors.lastname: Dulal
authors.affiliation: Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Masamichi
authors.initials: M
authors.lastname: Nagae
authors.affiliation: Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Akemi
authors.initials: A
authors.lastname: Ikeda
authors.affiliation: Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Kana
authors.initials: K
authors.lastname: Morita-Matsumoto
authors.affiliation: Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Adachi
authors.affiliation: Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo, 192-0392, Japan.
authors.forename: Naohito
authors.initials: N
authors.lastname: Ohno
authors.affiliation: Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-0034, Japan. Electronic address: yyoshiki@riken.jp.
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Yamaguchi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002850:Chromatography, Gel; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D037181:Lectins, C-Type / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D012995:Solubility; D013291:Streptococcus / Q000737:chemistry / Q000235:genetics; D000099181:Dectin-1
chemical_list: D001426:Bacterial Proteins; D037181:Lectins, C-Type; D011993:Recombinant Fusion Proteins; D000099181:Dectin-1; C034284:IgG Fc-binding protein, Streptococcus
keywords: Dectin-1; Escherichia coli; GB1 tag; NMR; Solubility enhancement