Dulal Hari Prasad, Nagae Masamichi, Ikeda Akemi, Morita-Matsumoto Kana, Adachi Yoshiyuki, Ohno Naohito, Yamaguchi Yoshiki
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.
Structural Glycobiology Team, Systems Glycobiology Research Group, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Protein Expr Purif. 2016 Jul;123:97-104. doi: 10.1016/j.pep.2016.04.002. Epub 2016 Apr 5.
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.
Dectin-1是一种针对β(1-3)-葡聚糖的C型凝集素样模式识别受体。它通过与真菌细胞壁上常见的β-葡聚糖结合，在抵御真菌入侵中发挥关键作用。为了通过核磁共振（NMR）探究其配体结合机制，我们使用pCold载体在大肠杆菌中表达重组小鼠Dectin-1 C型凝集素样结构域（CTLD）并进行了纯化。然而，由于其固有的低溶解性和低细菌表达量，无法获得NMR分析所需的高浓度Dectin-1 CTLD。在本研究中，我们尝试通过密码子优化和融合GB1标签（链球菌蛋白G的B1结构域）来提高Dectin-1 CTLD的表达量和溶解性。GB1被插入到人和小鼠Dectin-1 CTLD的N末端（NT）或C末端以及两端。仅通过NT-GB1融合的小鼠Dectin-1获得了纯单体样品。小鼠Dectin-1 CTLD的表达量为0.9±0.2 mg/L培养物，密码子优化后的小鼠Dectin-1 CTLD产量为1.4±0.2 mg/L，标签融合结构域为7.1±0.3 mg/L。该标签还将溶解度从0.1 mM提高到了1.4 mM。重组蛋白正确折叠，呈单体状态，并特异性结合β-葡聚糖海带多糖。这些结果表明，将GB1融合到小鼠Dectin-1结构域的N末端可有效提高产量和溶解性，保留天然结构，且融合位点至关重要。
