Monaco L, Bond H M, Howell K E, Cortese R
European Molecular Biology Laboratory, Heidelberg, FRG.
EMBO J. 1987 Nov;6(11):3253-60. doi: 10.1002/j.1460-2075.1987.tb02643.x.
We have constructed a plasmid, pLM8, containing the coding sequence of the mature human apoA-1 fused to the coding sequence of the IgG-binding domains of protein A (PA) from Staphylococcus aureus. The hybrid gene is transcribed in Escherichia coli under the control of a heat-sensitive repressor, leading to the synthesis of large amounts of hybrid protein (apoA-1--PA). The hybrid protein was purified by denaturation with urea and alkali, renaturation and affinity chromatography on an IgG Sepharose column. ApoA-1--PA is soluble and has an Mr of 316 kd, as determined by gel filtration. This is five times the monomer size of 62 kd, predicted from the sequence and found by SDS-PAGE analysis. Cell surface binding activity of the hybrid protein was tested using two different cell types (J774 macrophages and Fao hepatocytes) and compared to human high density lipoprotein (HDL). High-affinity binding was found for both ligands in both cell lines (Kd = 3.4 X 10(-8)M in Fao cells, 4.9 X 10(-8) M in J774 cells for apoA-1--PA and 3.0 X 10(-8) M in Fao cells, 2.8 X 10(-8) M in J774 cells for HDL), with approximately 2 X 10(5) high-affinity binding sites per cell. ApoA-1--PA and HDL effectively competed with each other for binding to the cell surface. Additionally, they both bound to a 110-kd polypeptide on a ligand blot, identifying an HDL receptor. The binding parameters of HDL were very similar to those of apoA-1--PA.(ABSTRACT TRUNCATED AT 250 WORDS)
我们构建了一个质粒pLM8，它包含成熟人载脂蛋白A-1的编码序列，该序列与来自金黄色葡萄球菌的蛋白A（PA）的IgG结合结构域的编码序列融合。杂合基因在大肠杆菌中由热敏感阻遏物控制转录，从而大量合成杂合蛋白（载脂蛋白A-1-PA）。杂合蛋白通过用尿素和碱变性、复性以及在IgG琼脂糖柱上进行亲和层析来纯化。通过凝胶过滤测定，载脂蛋白A-1-PA是可溶的，其分子量为316kd。这是根据序列预测并经SDS-PAGE分析得出的62kd单体大小的五倍。使用两种不同的细胞类型（J774巨噬细胞和Fao肝细胞）测试了杂合蛋白的细胞表面结合活性，并与人类高密度脂蛋白（HDL）进行比较。在两种细胞系中均发现两种配体具有高亲和力结合（在Fao细胞中，载脂蛋白A-1-PA的Kd = 3.4×10⁻⁸M，在J774细胞中为4.9×10⁻⁸M；对于HDL，在Fao细胞中为3.0×10⁻⁸M，在J774细胞中为2.8×10⁻⁸M），每个细胞约有2×10⁵个高亲和力结合位点。载脂蛋白A-1-PA和HDL在细胞表面结合方面有效地相互竞争。此外，它们都在配体印迹上与一条110kd的多肽结合，从而鉴定出一种HDL受体。HDL的结合参数与载脂蛋白A-1-PA的非常相似。（摘要截短至250字）
