Mutated recombinant collagens

Abstract

The invention provides recombinant procollagen chains having a natural collagen chain separated from one or two propeptide by one or two non-natural site-specific proteolytic agent (e.g., protease) recognition sites. A wide variety of propeptides and site-specific proteolytic agent recognition sites may be used: the selection of particular site-specific proteolytic agent/recognition site pairs is based on the conformation of the resulting procollagen, the availability of the site-specific proteolytic agent, the compatibility of the proteolysis with production of mature collagen, among other factors. Recombinant collagens chains are produced by contacting the subject recombinant procollagen chains with the appropriate site-specific proteolytic agents. Nucleic acids encoding the subject procollagen chains operably linked to transcription regulatory elements are used in vectors and cells for the production of recombinant collagen. Such collagen is used in tissue and cell cultureware and therapeutically, such as in biodegradable surgical materials and for tissue augmentation.

1 . A recombinant procollagen polypeptide chain comprising a natural collagen polypeptide chain, a first propeptide, and a first non-natural site-specific proteolytic agent recognition site, wherein said first non-natural site-specific proteolytic agent recognition site is located between said collagen chain and said first propeptide.  
     
     
         2 . A recombinant procollagen chain according to  claim 1 , wherein said first non-natural site-specific proteolytic agent recognition site is a site-specific protease recognition site.  
     
     
         3 . A recombinant procollagen chain according to  claim 1 , wherein said first non-natural site-specific proteolytic agent recognition site comprises a peptide bond labile to chemical hydrolysis.  
     
     
         4 . A recombinant procollagen chain according to  claim 1 , wherein said first propeptide is a C-terminal propeptide.  
     
     
         5 . A recombinant procollagen chain according to  claim 1 , wherein said first propeptide is a natural procollagen C-terminal propeptide.  
     
     
         6 . A recombinant procollagen chain according to  claim 1 , further comprising a second propeptide and a second non-natural site-specific proteolytic agent recognition site, wherein said second non-natural site-specific proteolytic agent recognition site is located between said collagen chain and said second propeptide.  
     
     
         7 . A recombinant procollagen chain according to  claim 6 , wherein said first and said second non-natural site-specific proteolytic agent recognition sites are different.  
     
     
         8 . A recombinant procollagen chain according to  claim 1 , further comprising a non-natural amino acid sequence between said first site-specific proteolytic agent recognition site and said collagen chain.  
     
     
         9 . A recombinant procollagen chain according to  claim 1 , further comprising a non-natural amino acid sequence between said first site-specific proteolytic agent recognition site and said first propeptide.  
     
     
         10 . A nucleic acid encoding a recombinant procollagen chain according to  claim 1 .  
     
     
         11 . A vector comprising the nucleic acid sequence of  claim 10  operably linked to a transcription regulatory element not naturally linked to said nucleic acid.  
     
     
         12 . A cell comprising a nucleic acid according to  claim 10  operably linked to a transcription regulatory element not naturally linked to said nucleic acid.  
     
     
         13 . A recombinant collagen polypeptide chain comprising a terminal decapeptide having a non-natural amino acid sequence.  
     
     
         14 . A recombinant collagen polypeptide chain produced by contacting a recombinant procollagen chain according to  claim 1  with a first site-specific proteolytic agent capable of selectively cleaving said procollagen chain at said first site-specific proteolytic agent recognition site.  
     
     
         15 . A collagen composition comprising a plurality of recombinant collagen chains according to  claim 14 , wherein said chains are polymerized.  
     
     
         16 . A sterile, nontoxic, biocompatible collagen composition comprising a recombinant collagen chain according to  claim 14 .  
     
     
         17 . A process for the production of a recombinant procollagen polypeptide chain, said process comprising the steps of: 
 culturing a cell according to  claim 12  under conditions suitable for the expression of said nucleic acid; and    recovering said recombinant procollagen chain.    
     
     
         18 . A process for the production of a recombinant collagen polypeptide chain, said process comprising the steps of: 
 contacting a recombinant procollagen chain according to  claim 1  with a site-specific proteolytic agent capable of selectively cleaving said recombinant procollagen chain at said first site-specific proteolytic agent recognition site under conditions wherein said first site-specific proteolytic agent selectively cleaves said procollagen chain at said first site-specific proteolytic agent recognition site, whereby a collagen chain is produced; and recovering said collagen chain.    
     
     
         19 . A method of promoting the growth of cultured cells on a solid substrate or the adherence of cultured cells to a solid substrate, said method comprising contacting said solid substrate with a composition according to  claim 16 .  
     
     
         20 . A method of augmenting localized tissue in a host, said method comprising the step of subcutaneously administering to a host a collagen composition according to  claim 16.