title: Cleavable CD40Ig fusion proteins and the binding to sgp39.
abstract: Recombinant immunoglobulin (Ig) fusion proteins of cell surface and intracellular proteins have wide applications. For example, fusion proteins have been used in the isolation, identification and study of ligands and the effects of binding or blocking a receptor-ligand pair, either in vivo or in vitro. For some applications, removal of the immunoglobulin Fc region is advantageous. We have developed two vectors for the expression of Ig fusion proteins that contain recognition sequences for protease cleavage using thrombin. In one vector, the sequence encoding the thrombin cleavage site is located at the junction of the DNA fragment encoding the protein or protein fragment to be studied and the hinge and constant regions of the immunoglobulin, allowing the generation of a monomeric form of the protein of interest. In the second vector, the sequence encoding the thrombin cleavage site is located between the sequences encoding the hinge and constant regions of the immunoglobulin, allowing for the generation of covalent dimers of the recombinant protein without the constant Fc domains. We have used these vectors to produce the constructs encoding two forms of the extracellular domain of CD40, CD40ThrIg and CD40HinThrIg, allowing production of a monomeric and dimeric form of recombinant CD40. Cleavage is efficient and complete. Following cleavage, there was no detectable binding of the monomeric form of CD40 to a soluble form of gp39, the ligand of CD40, while the dimeric form was able to bind. These vectors have been constructed to allow facile substitution with other sequences to generate cleavable forms of other proteins of interest.
authors.affiliation: Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle, WA 98121, USA.
authors.forename: D
authors.initials: D
authors.lastname: Hollenbaugh
authors.forename: J
authors.initials: J
authors.lastname: Douthwright
authors.forename: V
authors.initials: V
authors.lastname: McDonald
authors.forename: A
authors.initials: A
authors.lastname: Aruffo
mesh_terms: D001483:Base Sequence; D019013:CD40 Antigens / Q000737:chemistry* / Q000235:genetics; D023201:CD40 Ligand; D002460:Cell Line; D005822:Genetic Vectors / Q000276:immunology; D006801:Humans; D007136:Immunoglobulins / Q000737:chemistry* / Q000235:genetics; D008024:Ligands; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000276:immunology
chemical_list: D019013:CD40 Antigens; D007136:Immunoglobulins; D008024:Ligands; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D023201:CD40 Ligand