Beck J T, Marsters S A, Harris R J, Carter P, Ashkenazi A, Chamow S M
Department of Recovery Sciences, Protein Engineering, Genentech, Inc. South San Francisco, CA 94080.
Mol Immunol. 1994 Dec;31(17):1335-44. doi: 10.1016/0161-5890(94)90052-3.
The extracellular portion of the interleukin-1 receptor (IL-1R) is sufficient for high-affinity binding to IL-1; however, the structural basis for binding of the receptor to IL-1 is not known. To produce individual domains of IL-1 receptor for structural studies, we constructed a molecular fusion of IL-1 receptor with immunoglobulin G heavy chain that contains a protease specific sequence joining the two portions of the molecule (IL-1R-G-IgG). We introduced the hexapeptide sequence AAHY:TL (where ":" denotes the scissile bond) at the junction of the IL-1R and IgG regions, for specific cleavage by an H64A variant of subtilisin BPN' (Genenase I), an endoprotease that cleaves selectively at this sequence (Carter et al., (1989) Proteins 6, 240-248). Plasmid DNA encoding the fusion protein was used to transfect human embryonic kidney 293 cells transiently, and secreted IL-1R-G-IgG was purified from cell supernatants by protein A chromatography. The IL-1 receptor's extracellular region was then generated by enzymatic cleavage with Genenase I which was immobilized on controlled-pore glass. Incubation of IL-1R-G-IgG with immobilized Genenase I resulted in specific cleavage at the target site, as confirmed by SDS-PAGE, immunoblotting and direct sequencing of the newly generated termini. The resulting soluble IL-1R was separated from the immunoglobulin Fc cleavage product by re-chromatography on protein A. The purified, soluble IL-1R retained quantitatively the ability to bind to its ligand, IL-1 beta. This approach offers a generic means by which the extracellular region of a given type I transmembrane receptor can be expressed as an immunoadhesin, released enzymatically and then easily purified for crystallographic or ligand binding studies.
白细胞介素-1受体（IL-1R）的胞外部分足以与IL-1进行高亲和力结合；然而，受体与IL-1结合的结构基础尚不清楚。为了产生用于结构研究的IL-1受体的各个结构域，我们构建了IL-1受体与免疫球蛋白G重链的分子融合体，该重链包含连接分子两部分的蛋白酶特异性序列（IL-1R-G-IgG）。我们在IL-1R和IgG区域的连接处引入了六肽序列AAHY:TL（其中“:”表示可裂解键），以便被枯草杆菌蛋白酶BPN'（Genenase I）的H64A变体特异性切割，该内切蛋白酶可选择性地在该序列处切割（Carter等人，（1989年）《蛋白质》6，240 - 248）。编码融合蛋白的质粒DNA用于瞬时转染人胚肾293细胞，分泌的IL-1R-G-IgG通过蛋白A层析从细胞上清液中纯化。然后通过固定在可控孔径玻璃上的Genenase I进行酶切产生IL-1受体的胞外区域。SDS-PAGE、免疫印迹和对新产生末端的直接测序证实，IL-1R-G-IgG与固定化的Genenase I孵育导致在靶位点特异性切割。通过在蛋白A上重新层析将所得的可溶性IL-1R与免疫球蛋白Fc切割产物分离。纯化的可溶性IL-1R在数量上保留了与其配体IL-1β结合的能力。这种方法提供了一种通用手段，通过该手段，给定的I型跨膜受体的胞外区域可以表达为免疫粘附素，通过酶促释放，然后易于纯化以用于晶体学或配体结合研究。
