Acid-labile subunit (ALS) of insulin-like growth factor binding protein complex

Abstract

The acid-labile sub-unit (ALS) of insulin like growth factor binding protein complex in biologically pure form is described. ALS has a molecular weight between 80-115 kd as determined by SDS polyacrylamide gel electrophoresis, run under reducing conditions; and a partial N-terminal amino acid sequence as follows: Gly AspProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla Cys-Ala wherein the first amino acid may be Gly or Ala. Also described are methods of producing ALS, compositions containing the in-vivo IGF protein complex, methods of detecting ALS in body fluids, recombinant nucleic acid sequences encoding ALS, and expression vectors and host cells containing such nucleic acid sequences.

1 . The acid-labile sub-unit (ALS) of insulin like growth factor binding protein complex in biologically pure form. 
     
     
         2 . A composition of matter consisting essentially of the acid-labile sub-unit (ALS) of the insulin like growth factor binding complex. 
     
     
         3 . ALS according to  claim 1  or  2  which has a molecular weight of approximately 80-115 kilodaltons as determined by reducing SDS-polyacrylamide gel electrophoresis. 
     
     
         4 . ALS according to any one of  claims 1  to  3  having the following partial N-terminal amino acid sequence: 
       
         
           
                 
                 
               
                   Gly 
                     
                 
                   AspProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla 
                 
                     
                 
                   Cys-Ala 
                 
             
                
                
                
                
               
            
           
         
       
       wherein said first amino acid may be Gly or Ala. 
     
     
         5 . A method for the preparation of the in-vivo IGF binding protein complex comprising the step of admixing together IGF, the acid-stable IGF binding protein (BP 53) and ALS. 
     
     
         6 . A method according to  claim 5 , wherein the IGF, acid-stable IGF binding protein (BP 53) and ALS are biologically pure. 
     
     
         7 . A composition containing the in-vivo IGF protein complex, prepared according to  claim 5  or  6 , in association with a pharmaceutically or veterinarially acceptable carrier or excipient. 
     
     
         8 . A method for the treatment of wounds comprising applying to the wound or the area adjacent the wound, or administering to the wounded patient, a therapeutically effective amount of a composition as claimed in  claim 7 . 
     
     
         9 . A method for promoting cellular growth in animals and humans comprising administering to a patient a therapeutically effective amount of a composition as claimed in  claim 7 . 
     
     
         10 . A method for the preparation of ALS which comprises the steps of:
 (a) applying a source of ALS to a support matrix having attached thereto IGF bound to or associated with the acid-stable IGF binding protein, whereby the ALS in the applied material binds to the acid stable binding protein and non-bound material is separated from the support matrix; and   (b) selectively eluting and recovering the ALS protein from the IGF protein complex.   
     
     
         11 . A method for the preparation of ALS which comprises the steps of:
 (a) binding IGF to a support matrix;   (b) adding the acid-stable IGF binding protein to the support matrix such that it binds to the IGF;   (c) applying a source of ALS to the support matrix whereby the ALS in the applied material binds to the acid stable binding protein and non-bound material is separated from the support matrix; and   (d) selectively eluting the ALS protein from the IGF protein complex.   
     
     
         12 . A method according to  claim 10  or  11 , wherein the source of ALS is human plasma or Cohn Fraction IV of human plasma. 
     
     
         13 . A method according to  claim 10  or  11 , wherein the source ALS applied to the support matrix is partially purified. 
     
     
         14 . A method according to  claim 13 , wherein the source of ALS is partially purified by fractionation on an ionic resin. 
     
     
         15 . A method according to  claim 14 , wherein the ionic resin is a cation exchange resin. 
     
     
         16 . A method according to any one of  claims 11  to  15 , wherein the eluted ALS from step (d) is further fractionated by HPLC or FPLC. 
     
     
         17 . ALS when prepared according to the method of any one of  claims 10  to  16 . 
     
     
         18 . A method for detecting ALS in a biological fluid comprising the steps of:
 (a) binding proteins of a biological fluid to a support matrix;   (b) reacting the support matrix with antibodies directed against ALS; and   (c) detecting antibody binding.   
     
     
         19 . A method as claimed in  claim 18 , wherein the biological fluid is serum. 
     
     
         20 . A method for detecting the levels of ALS in a body fluid, which comprises fractionating the body fluid on a size fractionation matrix to separate free ALS from the other components of the insulin growth factor binding complex, and thereafter quantitating the levels of ALS in the fractionated sample. 
     
     
         21 . A method according to  claim 20 , wherein the levels of ALS in a body sample are detected by measuring the extent of formation of a reconstituted insulin like growth factor binding protein complex. 
     
     
         22 . A method according to  claim 20  wherein levels of ALS are quantitated by determining the extent of binding of anti-ALS antibodies to ALS in the fractionated sample. 
     
     
         23 . A recombinant nucleic acid sequence encoding the acid-labile sub-unit (ALS) of insulin like growth factor. 
     
     
         24 . A recombinant nucleic acid sequence according to  claim 23 , which encodes ALS, said ALS having the following partial N-terminal amino acid sequence: 
       
         
           
                 
                 
               
                   Gly 
                     
                 
                   AlaProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla 
                 
                     
                 
                   Cys-Ala 
                 
             
                
                
                
                
               
            
           
         
       
       wherein the first amino acid may be Gly or Ala. 
     
     
         25 . An expression vector containing the DNA sequence of  claim 23 . 
     
     
         26 . A host cell, which is a procaryotic or eukaryotic cell, transformed with the expression vector of  claim 25 . 
     
     
         27 . The recombinant nucleic acid sequence of  claim 23  which is cDNA. 
     
     
         28 . ALS when produced by the host cell of  claim 26 . 
     
     
         29 . An antibody reagent capable of binding to ALS. 
     
     
         30 . An antibody reagent according to  claim 29  which is a monoclonal or polyclonal antibody. 
     
     
         31 . An antibody reagent according to  claim 29  or  30  which is labelled with one or more reporter groups. 
     
     
         32 . An antibody reagent according to  claim 31 , wherein the reporter group is selected from fluorescent groups, enzymes or colloidal groups. 
     
     
         33 . A nucleic acid isolate comprising nucleic acid encoding the sequence: 
       
         
           
                 
                 
               
                   Gly 
                     
                 
                   AspProGlyThrProGlyGluAlaGlyGlyProAlaCysProAlaAla 
                 
                     
                 
                   Cys-Ala 
                 
             
                
                
                
                
               
            
           
         
       
       wherein the first amino acid may be Gly or Ala. 
     
     
         34 . A nucleic acid isolate comprising nucleic acid encoding residues 1-5, 2-7, 5-9, 7-11, 8-14, 11-15, 13-17, 3-9, 2-8, 4-10, 6-12, 8-14, 10-16, 12-18, 1-6, 3-9, 5-11, 7-13, 9-15, 11-17, 4-9, 6-11, 8-13, 10-15, or 12-17 of ALS. 
     
     
         35 . The nucleic acid isolate of  claim 33  or  34  further comprising a replicable vector. 
     
     
         36 . The nucleic acid isolate of  claim 33  further comprising nucleic acid encoding a secretion signal sequence ligated to the 5′ end of nucleic acid encoding the sequence: 
       
         
           
                 
                 
               
                   Gly 
                     
                 
                   AspProGlyThrProGlyGluAlaGluGlyProAlaCysProAlaAla 
                 
                     
                 
                   Cys-Ala 
                 
             
                
                
                
                
               
            
           
         
       
       wherein the first amino acid may be Gly or Ala. 
     
     
         37 . A host cell transformed with the isolate of any one of  claims 33  to  36 . 
     
     
         38 . A polypeptide comprising a fragment of ALS comprising a sequence of residues 1-5, 2-7, 5-9, 7-11, 8-14, 11-15, 13-17, 3-9, 2-8, 4-10, 6-12, 8-14, 10-16, 12-18, 1-6, 3-9, 5-11, 7-13, 9-15, 11-17, 4-9, 6-11, 8-13, 10-15, or 12-17 of ALS.