Letter to the Editor:: Sequential resonance assignments of the extracellular ligand binding domain of the human TGF-β type II receptor
Letter to the Editor:: Sequential resonance assignments of the extracellular ligand binding domain of the human TGF-β type II receptor
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DOI：
10.1023/a:1026775321886
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发表时间：
2000-12-01
影响因子：
2.7
通讯作者：
Freedberg, DI
中科院分区：
文献类型：
--
作者：
Hinck, AP;Walker, KP;Freedberg, DI
方法和结果将人TβR2的胞外配体结合结构域的编码区插入质粒pET32a(Novagen)中。该构建体缺乏前 14 个残基的编码区，并在第 19 位包含天冬酰胺至丙氨酸的取代。后者对于消除与该位置天然存在的天冬酰胺脱酰胺相关的问题非常重要（Lin 等人，1992）。重组可溶性TβR2，本文称为sTβR2，是通过在大肠杆菌菌株BL21(DE3)中过表达该蛋白质并通过从不溶性级分中分离该蛋白质而获得的。然后对蛋白质进行针对 20 mM 乙酸（pH 3.55）的重复透析，然后通过将酸溶性蛋白质缓慢转移至等体积或更大体积的含有 200 mM Tris-Cl、2 mM 还原型谷胱甘肽、0.5 mM 氧化型谷胱甘肽、pH 8.0 的缓冲液中来折叠。使用阴离子交换层析将正确折叠的蛋白质与错误折叠的蛋白质和其他杂质分离，并发现其表现出与培养的小鼠骨髓瘤细胞系（R&D Systems）中产生的全长人 sTβR2 结构域相同的 TGF-β 结合特性（Qian 等人，1996）。通过在含有 0.1% 15NH4Cl 和 0.3% 均匀 13C 标记的 D-葡萄糖的 M9 基本培养基上培养细胞，制备了 sTβR2 结构域的同位素富集形式，用于 NMR 光谱。
Methods and resultsThe coding region for the extracellular ligand binding domain of the human TβR2 was inserted into plasmid pET32a (Novagen). This construct lacked the coding region for the first 14 residues and included an asparagine-to alanine-substitution at position 19. The latter was important in eliminating problems associated with deamidation of the naturally occurring asparagine at this position (Lin et al., 1992). Recombinant soluble TβR2, herein designated sTβR2, was obtained by overexpressing the protein in E. coli strain BL21 (DE3), and by isolating the protein from the insoluble fraction. The protein was then subjected to repeated dialysis against 20 mM acetic acid (pH 3.55), and then folded by slowly transferring the acid-soluble protein to an equal or greater volume of buffer containing 200 mM Tris-Cl, 2 mM reduced glutathione, 0.5 mM oxidized glutathione, pH 8.0. The properly folded protein was separated from misfolded protein and other impurities using anion-exchange chromatography, and was found to exhibit identical TGF-β binding properties relative to that of the full-length human sTβR2 domain as produced in a cultured mouse myeloma cell line (R&D Systems)(Qian et al., 1996). Isotopically enriched forms of the sTβR2 domain were prepared for NMR spectroscopy by culturing the cells on M9 minimal medium containing 0.1% 15NH4Cl and 0.3% uniformly 13C-labeled D-glucose.