Materials and methods for preparing dimeric growth factors

Abstract

Proteins consisting of, from amino to carboxyl terminus, a first PDGF-D growth factor domain polypeptide, a linker polypeptide, and a second PDGF-D growth factor domain polypeptide, and materials and methods for making the proteins are disclosed. Each of the first and second PDGF-D growth factor domain polypeptides consists of a sequence of amino acid residues as shown in SEQ ID NO:2 or SEQ ID NO:4 from amino acid x to amino acid y, wherein x is an integer from 246 to 258, inclusive, and y is an integer from 365-370, inclusive. The linker polypeptide consists of from 11-40 amino acid residues. The proteins can be used to stimulate the production of bone and/or connective tissue in both humans and non-human animals.

1 . An expression vector comprising a DNA segment, the DNA segment consisting of the following operably linked elements:
 (a) a transcription promoter;   (b) a coding sequence encoding a fusion protein consisting of, from amino terminus to carboxyl terminus:
 a first PDGF-D growth factor domain polypeptide; 
 a linker polypeptide; and 
 a second PDGF-D growth factor domain polypeptide, 
   wherein each of said first and second PDGF-D growth factor domain polypeptides consists of a sequence of amino acid residues as shown in SEQ ID NO:2 or SEQ ID NO:4 from amino acid x to amino acid y, wherein x is an integer from 246 to 258, inclusive, and y is an integer from 365 to 370, inclusive; and wherein said linker polypeptide consists of from 11-40 amino acid residues; and   (c) a transcription terminator.   
     
     
         2 . An expression vector comprising a DNA segment, the DNA segment consisting of the following operably linked elements:
 (a) a transcription promoter;   (b) a secretory signal sequence encoding a secretory peptide;   (c) a coding sequence encoding a fusion protein consisting of, from amino terminus to carboxyl terminus:
 a first PDGF-D growth factor domain polypeptide; 
 a linker polypeptide; and 
 a second PDGF-D growth factor domain polypeptide, 
   wherein each of said first and second PDGF-D growth factor domain polypeptides consists of a sequence of amino acid residues as shown in SEQ ID NO:2 or SEQ ID NO:4 from amino acid x to amino acid y, wherein x is an integer from 246 to 258, inclusive, and y is an integer from 365 to 370, inclusive; and wherein said linker polypeptide consists of from 11-40 amino acid residues; and   (c) a transcription terminator.   
     
     
         3 . The expression vector of  claim 1  or  2  wherein y is 370. 
     
     
         4 . The expression vector of  claim 1  or  2  wherein x is 246, 248, or 250. 
     
     
         5 . The expression vector of  claim 1  or  2  wherein x is 250 and y is 370. 
     
     
         6 . The expression vector of  claim 1  or  2  wherein the linker polypeptide consists of from 12 to 20 amino acid residues. 
     
     
         7 . The expression vector of  claim 1  or  2  wherein the linker polypeptide consists of from 14 to 16 amino acid residues. 
     
     
         8 . The expression vector of  claim 1  or  2  wherein the linker polypeptide does not contain Lys or Arg. 
     
     
         9 . The expression vector of  claim 1  or  2  wherein the linker polypeptide does not contain Cys. 
     
     
         10 . The expression vector of  claim 1  or  2  wherein the linker polypeptide does not contain Pro. 
     
     
         11 . The expression vector of  claim 1  or  2  wherein the linker polypeptide comprises a proteolytic cleavage site. 
     
     
         12 . The expression vector of  claim 10  wherein the cleavage site is a plasmin cleavage site, a thrombin cleavage site, or a factor Xa cleavage site. 
     
     
         13 . The expression vector of  claim 1  or  2  wherein the linker polypeptide comprises the amino acid sequence Ser-Gly-Ser-Gly-Ser-Ser-Gly-Ser-Gly-Ser (SEQ ID NO:33) and a proteolytic cleavage site. 
     
     
         14 . The expression vector of  claim 2  wherein the secretory peptide consists of residues 1 to 18 of SEQ ID NO: 2. 
     
     
         15 . A cultured cell comprising the expression vector of  claim 1  or  2