GENERATION OF SOLUBLE INTERLEUKIN-1 RECEPTOR FROM AN IMMUNOADHESIN BY SPECIFIC CLEAVAGE

被引:14
作者
BECK, JT
MARSTERS, SA
HARRIS, RJ
CARTER, P
ASHKENAZI, A
CHAMOW, SM
机构
[1] GENENTECH INC,DEPT RECOVERY SCI,S SAN FRANCISCO,CA 94080
[2] GENENTECH INC,DEPT MOLEC BIOL,S SAN FRANCISCO,CA 94080
[3] GENENTECH INC,DEPT MED & ANALYT CHEM,S SAN FRANCISCO,CA 94080
[4] GENENTECH INC,DEPT PROT ENGN,S SAN FRANCISCO,CA 94080
关键词
IMMUNOGLOBULIN CHIMERA; CYTOKINE RECEPTOR; PROTEASE; SUBTILISIN;
D O I
10.1016/0161-5890(94)90052-3
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The extracellular portion of the interleukin-l receptor (IL-1R) is sufficient for high-affinity binding to IL-I; 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-I 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-IgC 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-IR 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.
引用
收藏
页码:1335 / 1344
页数:10
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