White Jennifer, Lukacik Petra, Esser Dirk, Steward Michael, Giddings Naomi, Bright Jeremy R, Fritchley Sarah J, Morgan B Paul, Lea Susan M, Smith Geoffrey P, Smith Richard A G
Adprotech Ltd., Chesterford Research Park, Little Chesterford, Saffron Walden, Essex CB10 1XL, UK.
Protein Sci. 2004 Sep;13(9):2406-15. doi: 10.1110/ps.03455604.
Decay-accelerating factor (DAF, CD55) is a glycophosphatidyl inositol-anchored glycoprotein that regulates the activity of C3 and C5 convertases. In addition to understanding the mechanism of complement inhibition by DAF through structural studies, there is also an interest in the possible therapeutic potential of the molecule. In this report we describe the cloning, expression in Escherichia coli, isolation and membrane-targeting modification of the four short consensus repeat domains of soluble human DAF with an additional C-terminal cysteine residue to permit site-specific modification. The purified refolded recombinant protein was active against both classical and alternative pathway assays of complement activation and had similar biological activity to soluble human DAF expressed in Pichia pastoris. Modification with a membrane-localizing peptide restored cell binding and gave a large increase in antihemolytic potency. These data suggested that the recombinant DAF was correctly folded and suitable for structural studies as well as being the basis for a DAF-derived therapeutic. Crystals of the E. coli-derived protein were obtained and diffracted to 2.2 A, thus permitting the first detailed X-ray crystallography studies on a functionally active human complement regulator protein with direct therapeutic potential.
衰变加速因子（DAF，CD55）是一种糖磷脂酰肌醇锚定糖蛋白，可调节C3和C5转化酶的活性。除了通过结构研究了解DAF抑制补体的机制外，人们还对该分子可能的治疗潜力感兴趣。在本报告中，我们描述了可溶性人DAF四个短共有重复结构域的克隆、在大肠杆菌中的表达、分离以及膜靶向修饰，该结构域带有一个额外的C末端半胱氨酸残基以允许位点特异性修饰。纯化复性后的重组蛋白对补体激活的经典途径和替代途径检测均有活性，并且与在毕赤酵母中表达的可溶性人DAF具有相似的生物学活性。用膜定位肽进行修饰可恢复细胞结合，并大幅提高抗溶血效力。这些数据表明，重组DAF折叠正确，适合进行结构研究，也是基于DAF的治疗药物的基础。获得了大肠杆菌来源蛋白的晶体，其衍射分辨率达到2.2 Å，从而能够首次对具有直接治疗潜力的功能性活性人补体调节蛋白进行详细的X射线晶体学研究。
