Decorin compositions and use thereof

Abstract

The present invention relates to improved decorin compositions and methods of their production.

1 . A fusion protein comprising a heterologous signal peptide operably linked to a sequence encoding decorin. 
     
     
         2 . The fusion protein of  claim 1 , wherein said signal sequence is an alpha-lactalbumin signal sequence. 
     
     
         3 . The fusion protein of  claim 2 , wherein said alpha lactalbumin sequence is a bovine alpha-lactalbumin signal sequence. 
     
     
         4 . The fusion protein of  claim 1 , wherein said sequence encoding decorin encodes a decorin core protein. 
     
     
         5 . The fusion protein of  claim 4 , wherein said decorin core protein comprises a mutation at position 4 of the mature decorin core protein. 
     
     
         6 . The fusion protein of  claim 5 , wherein said mutation is a serine to alanine mutation. 
     
     
         7 . The fusion protein of  claim 4 , wherein said decorin core protein lacks substantial modification by glycosaminoglycans molecules at position 4 of the mature decorin core protein. 
     
     
         8 . A vector encoding the fusion protein of  claim 1 . 
     
     
         9 . The vector of  claim 8 , wherein said vector comprises a nucleic acid sequence encoding an alpha-lactalbumin signal sequence operably linked to a sequence encoding a decorin core protein. 
     
     
         10 . A host cell comprising the vector of  claim 8 . 
     
     
         11 . A method of producing a non-gagylated decorin core protein comprising:
 providing host cells expressing non-gagylated decorin core protein;   culturing said host cell so that said non-gagylated decorin core protein is produced; and   purifying said non-gagylated decorin core protein.   
     
     
         12 . The method of  claim 11 , wherein said non-gagylated decorin core protein is secreted into medium used to culture said host cells. 
     
     
         13 . The method of  claim 12 , wherein said purifying comprises contacting said culture medium with an ion exchange medium. 
     
     
         14 . The method of  claim 12 , wherein said purifying comprises contacting said culture medium with a hydroxyapatite medium. 
     
     
         15 . The method of  claim 12 , wherein said purifying comprises contacting said culture medium with at least one ion exchange medium and at least one hydroxyapatite medium in any order. 
     
     
         16 . The method of  claim 12 , wherein said purifying comprises:
 contacting said culture medium with a cation exchange medium;   washing said cation exchange medium;   eluting a first decorin-containing eluate from said cation exchange medium;   contacting said first decorin-containing eluate with a hydroxyapatite medium;   washing said hydroxyapatite medium;   eluting a second decorin-containing eluate from said hydroxyapatite medium.   
     
     
         17 . The method of  claim 16 , wherein said cation exchange medium is SP-Sepharose FF. 
     
     
         18 . The method of  claim 16 , wherein said hydroxyapatite medium is ceramic hydroxyapatite type I. 
     
     
         19 . The method of  claim 16 , further comprising further purification of said second decorin-containing eluate with an ion exchange membrane or column. 
     
     
         20 . The method of  claim 19 , wherein said ion exchange membrane is a Q ion exchange membrane. 
     
     
         21 . The method of  claim 16 , further comprising a viral inactivation step. 
     
     
         22 . The method of  claim 21 , wherein said viral inaction step comprises treatment with a surfactant. 
     
     
         23 . The method of  claim 22 , wherein said surfactant is Triton X-100. 
     
     
         24 . The method of  claim 16 , further comprising a viral filtration step. 
     
     
         25 . The method of  claim 16 , further comprising concentrating said decorin. 
     
     
         26 . The method of  claim 11 , wherein said non-gagylated decorin core protein is produced by said host cells at a rate of about greater than 1, 5, or 10 pg/cell/day. 
     
     
         27 . A method for purifying decorin from a decorin-containing medium comprising:
 contacting said decorin-containing medium with a cation exchange medium;   washing said cation exchange medium;   eluting a first decorin-containing eluate from said cation exchange medium;   contacting said at first decorin-containing eluate with a hydroxyapatite medium;   washing said hydroxyapatite medium;   eluting a second decorin-containing eluate from said hydroxyapatite medium;   filtering said second decorin-containing eluate with an ion exchange membrane to provide a decorin-containing filtrate;   treating said filtrate to remove viruses;   concentrating decorin from said filtrate.   
     
     
         28 . A composition comprising a purified decorin core protein comprising a mutation at position 4 of the mature decorin core protein so that said decorin protein is substantially non-gagylated, said composition comprising less than about 100 ng residual host cell protein/mg decorin core protein in said composition and less than about 20 pg residual host cell DNA/mg decorin core protein. 
     
     
         29 . The composition of  claim 28 , said composition comprising less than about 5 ng residual host cell protein/mg decorin core protein in said composition and less than about 5 pg residual host cell DNA/mg decorin core protein. 
     
     
         30 . The composition of  claim 28 , wherein said decorin is formulated as an aqueous solution. 
     
     
         31 . The composition of  claim 30 , wherein said aqueous solution comprises a phosphate buffered solution with a pH of from about 6.5 to about 7.5.