Single alpha chain collagens

Abstract

The invention relates to a method for the production of single alpha chain collagens and an isolated or purified product comprising single alpha chain collagens.

1 . Isolated single alpha chain collagens (SACCs) in aqueous solution wherein said SAC is or has:
 a) a single chain polypeptide existing as an alpha helix and having a molecular weight of approximately 100 kDa;   b) a repeating sub structure of Glycine-X-Y (where X can be any amino acid, and Y is proline or hydroxyproline); and   c) non-helical N-terminal and/or C-terminal ends;   and further wherein said aqueous solution does not include gamma collagen.   
     
     
         2 . The isolated SACCs according to  claim 1  wherein said aqueous solution does not include beta collagen. 
     
     
         3 . The isolated SACCs according to  claim 1 , wherein said aqueous solution contains an amount of SACCs per total collagen protein content of at least 50%, 75%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. 
     
     
         4 . The isolated SACCs according to  claim 1 , wherein said aqueous solution comprises SACCs at a concentration of at least 20% w/v. 
     
     
         5 . The isolated SACCs according to  claim 1 , wherein said SACCs are of mammalian or cnidarian origin. 
     
     
         6 . The isolated SACCs according to  claim 5  wherein said SACCs are of bovine porcine, equine, or jellyfish origin. 
     
     
         7 . The isolated SACCs according to  claim 1 , wherein said SACCs are recombinant or of recombinant origin. 
     
     
         8 . The isolated SACCs according to  claim 1 , wherein each SACC comprises approximately 1000-3500 amino acids. 
     
     
         9 . The isolated SACCs according to  claim 8  wherein each SACC comprises approximately 1000-1500 amino acids. 
     
     
         10 . (canceled) 
     
     
         11 . An electrospinning solution comprising the isolated SACCs according to  claim 1  and a solution suitable for electrospinning. 
     
     
         12 . A method for producing or purifying single alpha chain collagen (SAC) comprising:
 a) obtaining an extraction of collagen in alkaline solution;   b) filtering the extraction of collagen in alkaline solution using at least one first filter membrane to produce a first filter retentate;   c) adjusting the pH of the first filter retentate so that it is acidic, thereby at least partially solubilising the collagen bound by the first filter retentate;   d) filtering the at least partially solubilised first filter retentate using said at least one first filter membrane to produce a second filter retentate;   e) adjusting the pH of the second filter retentate so that it is acidic thereby solubilising the collagen bound by the second filter retentate;   f) filtering the second filter retentate using at least one second filter membrane to produce a first filtrate comprising SACCs; and   g) optionally, filtering the first filtrate using said at least one first filter membrane to produce a second retentate comprising single alpha chain collagen.   
     
     
         13 . The method according to  claim 12 , wherein the at least one first filter membrane has a Nominal Molecular Weight Cut-Off (NMWCO) in the range of 1-100,000. 
     
     
         14 . The method according to  claim 12 , wherein the at least one first filter membrane has a NMWCO of 10,000 or 50,000. 
     
     
         15 . The method according to  claim 12 , wherein the extraction of collagen in alkaline solution in step a) is obtained by treating a collagen-containing sample with alkaline or basic solution to allow cellular matter and other non-collagenous material to be destroyed. 
     
     
         16 . The method according to  claim 12 , wherein in step b) the extraction of collagen in alkaline solution is filtered until a pH of less than 8 is achieved and a first filter retentate is obtained. 
     
     
         17 . The method according to  claim 12 , wherein in step c) the first filter retentate is adjusted to make a solution having an acidic pH of less than 3. 
     
     
         18 . The method according to  claim 12 , wherein in step d) the first filter retentate is filtered until a pH of greater than 6 is achieved. 
     
     
         19 . The method according to  claim 12 , wherein in step b) the first filter retentate is supplemented, before during or after filtering, with deionised water and/or in step d) the second filter retentate is supplemented with deionised water. 
     
     
         20 . The method according to  claim 12 , wherein steps c)-d) are repeated at least once to improve yield and purity. 
     
     
         21 . The method according to  claim 12 , wherein steps c)-d) are performed in a closed circulatory system that either runs constantly or runs constantly during the performance of the said specified steps. 
     
     
         22 . The method according to  claim 12 , wherein said filtering in steps b), d), and f) comprises dialysis. 
     
     
         23 . The method according to  claim 12 , wherein in step e) the second filter retentate is adjusted to make a solution having a pH of less than 3. 
     
     
         24 . The method according to  claim 12 , wherein the at least one second filter membrane is a micro-porous membrane comprising a pore size between about 0.05 to 2 μm and every 0.01 μm integer therebetween. 
     
     
         25 . The method according to  claim 12 , wherein in step f) and/or g) the second filter retentate or the first filtrate is/are filtered until a pH of less than 8 is achieved. 
     
     
         26 . The method according to  claim 12 , wherein said extraction of collagen is obtained from mammalian or cnidarian tissue.