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首页> 外文期刊>Biochemistry >Dissection of the extracellular human interferon gamma receptor alpha-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies.
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Dissection of the extracellular human interferon gamma receptor alpha-chain into two immunoglobulin-like domains. Production in an Escherichia coli thioredoxin gene fusion expression system and recognition by neutralizing antibodies.

机译：将细胞外人干扰素γ受体α链解剖成两个免疫球蛋白样结构域。在大肠杆菌硫氧还蛋白基因融合表达系统中生产并通过中和抗体进行识别。

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摘要

The extracellular interferon gamma receptor alpha-chain (IFN gamma R) is believed to comprise two discrete approximately 110 amino acid immunoglobulin-like domains, perhaps similar to those seen in the crystal structure of the extracellular human growth hormone receptor [De Vos, A. M., Ultsch, M., & Kossiakoff, A. (1992) Science 255, 306-312], a distant relative in the cytokine receptor superfamily. In accord with this idea, we show that these IFN gamma R immunoglobulin-like domains can be produced separately in a soluble form with a native-like fold. The N-terminal domain (residues 1-108), with a Cys105 to Ser105 mutation, was produced at a high level, in a soluble form, as a thioredoxin-interferon gamma receptor fragment fusion protein in the cytoplasm of Escherichia coli. Upon extraction, the receptor Cys60-Cys68 disulfide bond formed spontaneously, to generate a native-like structure directly without the need for refolding. Cleavage of the fusion protein by enterokinase released the receptor fragment (approximately 12 kDa), which was recognized by several neutralizing antibodies with affinities, measured using surface plasmon resonance technology, that were essentially indistinguishable from those seen with the full length extracellular IFN gamma R produced in eukaryotic cells. Circular dichroism and 1D 1H nuclear magnetic resonance spectra indicated that the receptor fragment adopts a folded state, with mainly beta-sheet and reverse turn secondary structure. The second membrane-proximal Ig-like domain of the IFN gamma R (residues 90-229) was produced, albeit less efficiently, and characterized in a similar way. The production of these two independently folded proteins provides experimental support for the two domain organization of the IFN gamma R and opens new avenues for structural studies on these Ig-like molecules by NMR and crystallographic methods.
机译：据信细胞外干扰素γ受体α链（IFN gamma R）包含两个约110个氨基酸的免疫球蛋白样离散域，可能与细胞外人类生长激素受体的晶体结构相似[De Vos，AM， Ultsch，M。和Kossiakoff，A。（1992）Science 255，306-312]，是细胞因子受体超家族的远亲。根据这个想法，我们表明这些IFNγR免疫球蛋白样结构域可以以天然形式折叠的可溶形式分别产生。具有高水平的，具有Cys105至Ser105突变的N末端结构域（残基1-108）以可溶形式作为硫氧还蛋白-干扰素γ受体片段融合蛋白在大肠杆菌的细胞质中产生。提取后，受体Cys60-Cys68二硫键自然形成，无需重新折叠即可直接生成天然结构。肠激酶对融合蛋白的切割释放了受体片段（约12 kDa），该受体片段被几种具有亲和力的中和抗体所识别，使用表面等离振子共振技术测量，与产生的全长细胞外IFN gamma R所观察到的抗体基本上没有区别在真核细胞中。圆二色性和1D 1H核磁共振谱表明受体片段呈折叠状态，主要为β-折叠和反向二级结构。产生了IFNγR的第二膜近端Ig样结构域（残基90-229），尽管效率较低，并且以相似的方式表征。这两个独立折叠的蛋白的产生为IFN gamma R的两个域的组织提供了实验支持，并为通过NMR和晶体学方法对这些Ig样分子进行结构研究开辟了新途径。

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