Generation of soluble interleukin-1 receptor from an immunoadhesin by specific cleavage.
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.
Beck
J T
JT
Department of Recovery Sciences, Protein Engineering, Genentech, Inc. South San Francisco, CA 94080.
Marsters
S A
SA
Harris
R J
RJ
Carter
P
P
Ashkenazi
A
A
Chamow
S M
SM
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000737:chemistry*; D008969:Molecular Sequence Data; D017472:Receptors, Interleukin-1 / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D013381:Subtilisins; D014162:Transfection / Q000235:genetics
D015815:Cell Adhesion Molecules; D007074:Immunoglobulin G; D017472:Receptors, Interleukin-1; D011993:Recombinant Fusion Proteins; D013381:Subtilisins