Nishihira J, Kuriyama T, Nishino H, Ishibashi T, Sakai M, Nishi S
Central Research Institute, Hokkaido University School of Medicine.
Biochem Mol Biol Int. 1993 Dec;31(5):841-50.
We cloned cDNA of human macrophage migration inhibitory factor (MIF) from a T-cell line encoding 116 amino acid residues and expressed it in E. coli. MIF specifically bound glutathione (dissociation constant = 600 microM), and the protein was effectively purified by an S-hexylglutathione Sepharose affinity column. MIF was homogeneous on SDS-PAGE and the molecular weight was calculated as 12.5 kDa. The molecular weight was in excellent agreement with that of the primary structure assumed from the cDNA. On the other hand, the molecular weight in the native form calculated by Sephadex G-100 gel chromatography was 24.3 kDa, which suggested that MIF formed a dimeric structure. To further confirm the native molecular weight, we carried out analytical ultracentrifugation. This precise hydrodynamic analysis revealed that the native molecular weight was 24.8 kDa, which confirmed that MIF existed as a homodimeric form.
我们从一个编码116个氨基酸残基的T细胞系中克隆了人类巨噬细胞移动抑制因子（MIF）的cDNA，并在大肠杆菌中进行表达。MIF特异性结合谷胱甘肽（解离常数 = 600 microM），该蛋白通过S-己基谷胱甘肽琼脂糖亲和柱有效纯化。MIF在SDS-PAGE上呈均一性，计算出的分子量为12.5 kDa。该分子量与从cDNA推导的一级结构的分子量非常吻合。另一方面，通过葡聚糖凝胶G-100凝胶色谱法计算出的天然形式的分子量为24.3 kDa，这表明MIF形成了二聚体结构。为了进一步确认天然分子量，我们进行了分析超速离心。这种精确的流体动力学分析表明天然分子量为24.8 kDa，证实MIF以同源二聚体形式存在。
