Soluble recombinant alpha beta adhesion receptor

Abstract

The invention relates to a novel purified recombinant α v β 3 adhesion receptor which shows an unimpaired ligand binding activity, and a process for preparing said soluble non-membrane bound receptor in excellent yields by recombinant techniques using a baculovirus-insect cell expression system. The so-synthesized soluble receptor may be used very easily as screening tool for new therapeutic compounds which may inhibit the natural α v β 3 adhesion receptor. Such therapeutic compounds which can be discovered very easily, fast and without health risk by means of the souluble receptors according to the invention may be, for example, RGD peptides or non-peptidic compounds mimicking the natural ligand epitopes. The invention relates, furthermore, to a corresponding process for preparing recombinant full-length α v β 3 adhesion receptor in excellent yields, additionally using detergents to dissolve the membrane bound receptor from the surface of the host cell.

What is claimed is:  
     
         1 . A purified soluble recombinant α v β 3  adhesion receptor which retains a ligand binding activity.  
     
     
         2 . A soluble human α v β 3  adhesion receptor of  claim 1 .  
     
     
         3 . A receptor of  claim 2 , comprising the α v  chain and the β 3  chain of said receptor, wherein each chain is shortened at its C-terminus by a portion containing the transmembrane domain or a portion thereof and the complete cytoplasmic domain.  
     
     
         4 . A receptor of  claim 3 , comprising a truncated α v  chain containing approximately 957 amino acids and a truncated β 3  chain containing approximately 692 amino acids, each starting at the N-terminal of the corresponding mature protein chain.  
     
     
         5 . A human α v β 3  adhesion receptor, obtainable by a process as defined in  claim 7 .  
     
     
         6 . A receptor of  claim 12 , wherein the truncated α v  chain has a molecular weight of 120 kD (±12 kD) and the truncated β 3  chain has a molecular weight of approximately 80 kD (±8 kD), each determined by SDS-PAGE under non-reducing conditions.  
     
     
         7 . A process for preparing a large amount of a highly purified soluble recombinant human α v β 3  adhesion receptor which retains a ligand binding activity, comprising 
 (i) subcloning a first cDNA coding for the α v  chain of said receptor, shortened by a portion comprising at least 52 amino acids calculated from the C-terminus, and a second cDNA coding for the β 3  chain of said receptor, shortened by a portion comprising at least 61 amino acids calculated from the C-terminus, into a baculovirus transfer vector of a baculovirus expression system,  
 (ii) transferring said vector comprising said first and/or second DNA into the genomic DNA of a baculovirus of said expression system,  
 (iii) infecting an insect cell with said complete recombinant baculovirus,  
 (iv) cultivating said infected insect cells in a culture medium whereby said heteromeric truncated α v β 3  receptor is expressed into the medium, and  
 (v) purifying said expressed receptor from the medium by antibody affinity chromatography, wherein the antibody is specific to the human α v β 3  adhesion receptor or its individual component chains.  
 
     
     
         8 . A process of  claim 7 , wherein the first and second cDNA are sub-cloned into the same baculovirus vector.  
     
     
         9 . A process of  claim 7 , wherein the baculovirus expression system is the BacPAK system.  
     
     
         10 . A process of  claim 7 , wherein the insect cells infected are High Five (BTI-TN-5B1-4) cells.  
     
     
         11 . A process of  claim 7 , wherein the specific antibody used for the antibody affinity chromatography is mAb 17E6 produced by a hybridoma cell line having the designation 272-17E6 (DSM ACC2160).  
     
     
         12 . A process of  claim 7 , wherein the first cDNA codes for the α v  chain of said receptor, shortened by a portion encoding approximately 61 amino acids starting at the C-terminus and which corresponds to a mature protein chain containing approximately 957 amino acids, and the second cDNA codes for the β 3  chain of said receptor, shortened by a portion encoding approximately 70 amino acids starting at the C-terminus and which corresponds to a mature protein chain containing approximately 692 amino acids.  
     
     
         13 . A process of  claim 12 , wherein the cDNA of the truncated α v  chain is generated by PCR using the oligonucleotide primers  
       
         
           
                 
               
                     
                 
                   5′-GAC CAG CAT TTA CAG TGA-3′ and 
                 
                     
                 
                   5′-CA CAG GTC TAG ACT ATG GCT GAA TGC CCC AGG-3′, 
                 
                     
                 
             
                
                
                
                
                
               
            
           
         
       
       and the cDNA of the truncated β 3  chain is generated by PCR using the oligonucleotide primers 5′ 
       
         
           
                 
                 
               
                     
                 
                   5′-GCG CGC AAG CTT GCC GCC ACC ATG CGA GCG CGG CCG-3′ and 
                     
                 
                     
                 
                   5′GAT CGA TCT AGA CTA GGT CAG GGC CCT TGG GAC ACT-3′. 
                 
                     
                 
             
                
                
                
                
                
               
            
           
         
       
     
     
         14 . A method of screening a compound for its capability to inhibit an activity of a natural α v β 3  receptor, comprising contacting a soluble α v β 3  receptor of  claim 1  with a compound which may inhibit said α v β 3  receptor, and determining of the receptor activity is inhibited.  
     
     
         15 . A method of  claim 14 , wherein the compound is an RGD-peptide or a non-peptidic analogue.  
     
     
         16 . A method of  claim 15 , wherein the RGD-peptide or a non-peptidic analogue compound is modified by a detectable marker and reacted with said immobilized purified soluble α v β 3  receptor, and the amount of ligand bound to said receptor is measured.  
     
     
         17 . A process for preparing large amounts of a highly purified intact full-length recombinant human α v β 3  adhesion receptor, comprising 
 (i) subcloning a first cDNA coding for the complete a chain of said receptor and a second cDNA coding for the β 3  chain of said receptor into a baculovirus transfer vector of a baculovirus expression system,  
 (ii) transferring said vector comprising said first and/or second DNA into the genomic DNA of a baculovirus of said expression system,  
 (iii) infecting insect cells with said complete recombinant baculovirus,  
 (iv) cultivating said infected insect cells in a culture medium, whereby said receptor is expressed into the cell memembrane,  
 (v) solubilizing said expressed cell membrane-bound receptor with a detergent, and  
 (vi) purifying said solubilized receptor from the cell-free medium by antibody affinity chromatography, wherein the antibody is specific to the human α v β 3  adhesion receptor or its individual component chains.