Expression of triple-helical collagen-like products in e.coli

Abstract

Recombinant bacterial triple-helical collagen-like proteins comprising two or more repetitive sequences of Gly-Xaa-Yaa yielding high-stability polymeric constructs without the need for post-translational modifications and which may incorporate one or more functional domains of biological or structural importance. The polymers are capable of high-yield production for a variety of applications

We claim: 
     
         1 . A recombinant bacterial collagen-like protein structure which is stable at temperatures between 35° C. and 40° C. in either its native form or after chemical crosslinking, comprising a formula:
   [(Gly-Xaa-Yaa) m -(insert) n ] p    
 where m is between about 1 to 200 and (Gly-Xaa-Yaa) m  represents a triple helical domain with Xaa and Yaa being independently any natural or unnatural imino or amino acid for each repeat unit, wherein the insert is comprised of about 1 to 50 of any imino or amino acids, with n being 0 or 1, and p being any number from about 2 to about 10 wherein the value of n is unique for each repeat and at least one insert is provided in the collagen-like protein. 
 
     
     
         2 . The recombinantly expressed protein of  claim 1 , wherein at least one triple helical domain has a proline content of greater than 19%. 
     
     
         3 . The recombinantly expressed protein of  claim 2 , wherein at least one triple helical domain has a proline content of between 19% and 40%. 
     
     
         4 . The recombinantly expressed protein of  claim 1 , wherein at least one triple helical domain has a concentration of charged amino acids of greater than 14%. 
     
     
         5 . The recombinantly expressed protein of  claim 4 , wherein at least one triple helical domain has a concentration of charged amino acids of between 14-35%. 
     
     
         6 . The recombinantly expressed protein of  claim 1 , wherein the triple helical domain(s) are independently selected from the group consisting of SEQ ID NOS: 7-11 and combinations thereof. 
     
     
         7 . The recombinantly expressed protein of  claim 1 , further comprising a non-collagenous domain bound at either an amino terminus end or a carboxy terminus end of the collagen-like protein, which facilitates protein folding of the triple helical domains. 
     
     
         8 . The recombinantly expressed protein of  claim 7 , wherein the non-collagenous domain is selected from the group consisting of SEQ ID NO.: 47, SEQ ID NO.: 48, SEQ ID NO.: 49 and SEQ ID NO.: 51 and combinations thereof. 
     
     
         9 . The recombinantly expressed protein of  claim 7 , wherein the non-collagenous domain is SEQ ID NO.: 47 and is bound to the protein at the amino terminus end of the triple helical domain. 
     
     
         10 . The recombinantly expressed protein of  claim 7 , wherein the non-collagenous domain is SEQ ID NO.: 51 and is bound to the protein at the carboxy terminus end of the triple helical domain. 
     
     
         11 . The recombinantly expressed protein of  claim 7 , wherein the non-collagenous domain is selected from the group consisting of a foldon, a coiled coil sequence, and a C-propeptide. 
     
     
         12 . The recombinantly expressed protein of  claim 1 , wherein an insert sequence includes at least one integrin binding site. 
     
     
         13 . The recombinantly expressed protein of  claim 12 , wherein the integrin binding site is selected from the group consisting of SEQ ID NOS: 15, SEQ ID NO.: 18 and combinations thereof. 
     
     
         14 . The recombinantly expressed protein of  claim 1 , wherein an insert sequence includes at least one DDR2 domain. 
     
     
         15 . The recombinantly expressed protein of  claim 14 , wherein the DDR2 domain is SEQ ID NO.: 78. 
     
     
         16 . The recombinantly expressed protein of  claim 1 , wherein the insert sequence includes at least one integrin binding site and at least one DDR2 domain. 
     
     
         17 . The recombinantly expressed protein of  claim 1 , wherein the insert sequence includes at least one matrix metalloproteinase cleavage site. 
     
     
         18 . The recombinantly expressed protein of  claim 17 , wherein the matrix metalloproteinase cleavage site is selected from the group consisting of SEQ ID NOS: 19-28, SEQ ID NO.: 62, SEQ ID NOS.: 67-75, fragments thereof, and combinations thereof. 
     
     
         19 . The recombinantly expressed protein of  claim 1 , wherein the insert sequence includes at least one non-collagen natural break having a peptide sequence spaced between two glycine residues. 
     
     
         20 . The recombinantly expressed protein of  claim 19 , wherein the non-collagen natural break is selected from the group consisting of SEQ ID NOS.: 12-14, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 50, SEQ ID NO.: 65, and combinations thereof. 
     
     
         21 . The recombinantly expressed protein of  claim 1 , wherein the triple-helical domain aggregates at neutral pH. 
     
     
         22 . A recombinant bacterial collagen-like protein structure which is stable at mammalian bodily temperatures comprising a formula:
   [(Gly-Xaa-Yaa) m -(insert) n ] p      where m is between about 1 to 200 and (Gly-Xaa-Yaa) m  represents a triple helical domain with Xaa and Yaa being independently any natural or unnatural imino or amino acid for each repeat unit, wherein the insert is comprised of about 1 to 50 of any imino or amino acids, with n being 0 or 1, and p being any number from about 2 to about 10 wherein the value of n is unique for each repeat and at least one insert is provided in the collagen-like protein; and
 a non-collagenous domain bound to the protein at either or both an amino terminus end or a carboxy terminus end, which facilitates protein folding of the triple helical domain. 
   
     
     
         23 . The recombinantly expressed protein of  claim 22 , wherein at least one triple helical domain has a Proline content of greater than 19%. 
     
     
         24 . The recombinantly expressed protein of  claim 23 , wherein at least one triple helical domain has a Proline content of between 19% and 40%. 
     
     
         25 . The recombinantly expressed protein of  claim 22 , wherein at least one triple helical domain has a concentration of charged amino acids of greater than 14%. 
     
     
         26 . The recombinantly expressed protein of  claim 22 , wherein at least one triple helical domain has a concentration of charged amino acids of between 14-35%. 
     
     
         27 . The recombinantly expressed protein of  claim 26 , wherein the triple helical domain(s) are stable at temperatures between 35° C. and 40° C. either in its native form or after chemical crossing linking. 
     
     
         28 . The recombinantly expressed protein of  claim 22 , wherein the triple helical domain(s) are independently selected from the group consisting of SEQ ID NOS: 7-11 and combinations thereof. 
     
     
         29 . The recombinantly expressed protein of  claim 22 , wherein the non-collagenous domain is selected from the group consisting of SEQ ID NOS 47, SEQ ID NO.: 48, SEQ ID NO.: 49, SEQ ID NO.: 51 and combinations thereof. 
     
     
         30 . The recombinantly expressed protein of  claim 22 , wherein the non-collagenous domain is SEQ ID NO.: 47 and is bound to the protein at the amino terminus end of the triple helical domain. 
     
     
         31 . The recombinantly expressed protein of  claim 22 , wherein the non-collagenous domain is SEQ ID NO.: 51 and is bound to the protein at the carboxy terminus end of the triple helical domain. 
     
     
         32 . The recombinantly expressed protein of  claim 22 , wherein the non-collagenous domain is selected from the group consisting of a foldon, a coiled coil sequence, and a C-propeptide. 
     
     
         33 . The recombinantly expressed protein of  claim 22 , wherein an insert sequence includes at least one integrin binding site. 
     
     
         34 . The recombinantly expressed protein of  claim 33 , wherein the integrin binding site is selected from the group consisting of SEQ ID NOS: 15, SEQ ID NO.: 18 and combinations thereof. 
     
     
         35 . The recombinantly expressed protein of  claim 22 , wherein an insert sequence includes at least one DDR2 domain. 
     
     
         36 . The recombinantly expressed protein of  claim 35 , wherein the DDR2 domain is SEQ ID NO.: 78. 
     
     
         37 . The recombinantly expressed protein of  claim 22 , wherein the insert sequence includes at least one integrin binding site and at least one DDR2 domain. 
     
     
         38 . The recombinantly expressed protein of  claim 22 , wherein the insert sequence includes at least one matrix metalloproteinase cleavage site. 
     
     
         39 . The recombinantly expressed protein of  claim 38 , wherein the matrix metalloproteinase cleavage site is selected from the group consisting of SEQ ID NOS: 19-28, SEQ ID NO.: 62, SEQ ID NOS.: 67-75, fragments thereof, and combinations thereof. 
     
     
         40 . The recombinantly expressed protein of  claim 22 , wherein the insert sequence includes at least one non-collagen natural break having a peptide sequence spaced between two glycine residues. 
     
     
         41 . The recombinantly expressed protein of  claim 40 , wherein the non-collagen natural break is selected from the group consisting of SEQ ID NOS.: 12-14, SEQ ID NO.: 16, SEQ ID NO.: 17, SEQ ID NO.: 50, SEQ ID NO.: 65, and combinations thereof. 
     
     
         42 . The recombinantly expressed protein of  claim 22 , wherein the triple-helical domain aggregates at neutral pH. 
     
     
         43 . A biological aggregate for use in a biomedical product comprising:
 a recombinant bacterial collagen-like protein structure which is stable at mammalian bodily temperatures comprising a formula
   [(Gly-Xaa-Yaa) m -(insert) n ] p    
   where m is between about 1 to 200 and (Gly-Xaa-Yaa) m  represents a triple helical domain with Xaa and Yaa being independently any natural or unnatural imino or amino acid for each repeat unit, wherein the insert is comprised of about 1 to 50 of any imino or amino acids, with n being 0 or 1, and p being any number from about 2 to about 10 wherein the value of n is unique for each repeat and at least one insert is provided in the collagen-like protein; and
 optionally a non-collagenous domain bound to the protein at either or both an amino terminus end or a carboxy terminus end, which facilitates protein folding of the triple helical domain. 
   
     
     
         44 . A method of producing a recombinant collagen-like protein comprising
 (a) isolating 2 or more nucleic acid sequences each encoding separate triple helical domains formed from repeat sub-units of the formula:
   (Gly-Xaa-Yaa) m    
   
       where m is between about 1 to 200 and Xaa and Yaa are independently for each repeat unit any natural or unnatural imino or amino acid for each repeat unit, with the proviso that neither Xaa nor Yaa is a hydroxyproline,
 (b) inserting the two or more nucleic acid sequences encoding helical domains into a single nucleic acid vector; 
 (c) optionally inserting a non-collagen sequence encoding 1 to about 50 amino acids between each nucleic acid sequence encoding the helicial domains; 
 (d) optionally inserting a non-collagenous domain nucleic acid sequence at either or both an amino terminus end or a carboxy terminus end of the triple helical domain, which facilitates protein folding of the triple helical domain upon expression; 
 (e) optionally inserting a sequence tag; 
 (f) expressing the nucleic acid sequence within a micro-organism; and 
 (g) isolating the expressed nucleic acid sequence. 
 
     
     
         45 . The method of  claim 44  wherein the nucleic acid vector is a cold-shock vector. 
     
     
         46 . The method of  claim 44  wherein the nucleic acid sequence is expressed within the micro-organism at temperatures below 37° C. 
     
     
         47 . The method of  claim 44  wherein the nucleic acid sequence is expressed within the micro-organism at a temperature of about 15° C.