c端加工对人γ干扰素活性的影响。

Drug design and delivery Pub Date : 1989-05-01
T Arakawa, M A Narachi, Y R Hsu, R R Everett, P H Lai, E N Fish
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引用次数: 0

摘要

从大肠杆菌中获得的同质重组人干扰素γ (ifn - γ)用机械裂解的大肠杆菌细胞制备的含有蛋白酶的部分处理。所得产物的聚丙烯酰胺凝胶电泳显示,两个主要成分的分子量小于完整的ifn - γ。在7 M尿素的存在下,通过离子交换色谱纯化了这些蛋白,发现它们具有完整的ifn - γ n端序列,这表明它们是由ifn - γ的c端裂解产生的。氨基酸分析表明,其中一个缺失ifn - γ的4个c端残基,另一个缺失15个。缺乏4个c端残基的物种在使用脑心炎治疗的HeLa或T98G细胞进行抗病毒和生长抑制试验以及使用巨噬细胞样U937细胞进行巨噬细胞激活试验时,其活性与ifn - γ几乎没有区别。缺乏15个c末端残基的物种在这些实验中的活性明显降低,并且对ifn - γ细胞表面受体的结合亲和力降低。这些观察结果定义了对ifn - γ的生物活性很重要的c端残基——这些信息对于设计具有增强或改变生物活性的ifn - γ类似物应该是有用的。
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The effect of C-terminal processing on the activity of human interferon-gamma.

Homogeneous recombinant human interferon-gamma (IFN-gamma) obtained from Escherichia coli (E. coli) was treated with a protease-containing fraction prepared from mechanically lysed E. coli cells. Polyacrylamide gel electrophoresis of the resulting product revealed two major components of molecular weight less than that of intact IFN-gamma. These were purified by ion exchange chromatography in the presence of 7 M urea and shown to have intact IFN-gamma N-terminal sequences, suggesting that they resulted via C-terminal cleavages of IFN-gamma. Amino acid analysis indicated that 4 C-terminal residues of IFN-gamma were lacking in one, and 15 in the other. The species lacking 4 C-terminal residues had activities virtually indistinguishable from those of IFN-gamma in antiviral and growth inhibitory assays using Encepharomyocarditis-treated HeLa or T98G cells and in a macrophage activation assay using macrophage-like U937 cells. The species lacking 15 C-terminal residues had markedly decreased activities in each of these assays, and had decreased binding affinity for IFN-gamma cell surface receptors. These observations define the C-terminal residues important for IFN-gamma's biological activity--information which should be useful in designing analogs of IFN-gamma with enhanced or altered biological activities.

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