Method for preparing modified collagen outside a host cell
View Patent ↗The present invention relates to a method for preparing a modified folded protein, comprising modifying unfolded a-chains comprising a collagenous amino acid sequence and thereafter folding at least part of the modified a-chains into a quaternary protein structure, in particular a helical protein structure. The invention allows for an attractive alternative for making recombinant proteins, such as recombinant collagens and the like, which may be used in a variety of biomedical and other applications.
1. A method for the in vitro production of a modified collagenous protein, comprising
(a) unfolding folded collagen α-chains comprising an association domain to obtain unfolded collagen α-chains;
(b) modifying the unfolded α-chains by adding modifying enzymes; and
(c) folding the modified α-chains into a triple helical structure;
wherein none of steps (a)-(c) is performed intracellularly; and
wherein said association domains comprise one or more interchain crosslinks linking the collagen α-chains together; or
wherein the association domains comprise amino acid sequences that reassociate after unfolding; or
wherein the association domains comprise amino sequences by which the collagen α-chains remain bound to each other during unfolding of the remainder of the collagen α-chains; and
wherein said modifying enzymes modify the amino acid side chains of the α-chains.
2. The method of claim 1 , wherein the modifying is by one or more enzymes selected from the group consisting of prolyl hydroxylases, lysyl hydroxylases, lysyl oxidases, hydroxylysyl-galactosyltransferases and galactosylhydroxylysyl-glucosyltransferases.
3. The method of claim 2 , wherein the modifying comprises hydroxylating proline by a prolyl-3-hydroxylase, or a prolyl-4-hydroxylase or a combination thereof.
4. The method of claim 2 , wherein the modifying comprises hydroxylating lysine by a helical lysyl hydroxylase, or a telopeptide lysyl hydroxylase or a combination thereof.
5. The method of claim 2 , wherein the modifying comprises glycosylating hydroxylysine by a lysyl hydroxylase.
6. The method of claim 2 , wherein the modifying comprises oxidatively deaminating the ε-amino group of lysine or hydroxylysine by a lysyl oxidase.
7. The method of claim 2 , wherein said enzymes are recombinant proteins synthesized by prokaryotic or eukaryotic cells.
8. The method of claim 1 , wherein said interchain crosslinks are Cys-Cys bridges wherein said Cys residues are present in the triple helical part of the α-chain and/or in the telopeptide part of the α-chain and/or in the propeptide part of the α-chain and/or at the start of the α-chain and/or at the end of the α-chain.
9. The method of claim 1 , wherein said amino acid sequences comprise SEQ ID NO:1.
10. The method of claim 1 , wherein said amino acid sequences are present in the triple helical part of the α-chains and/or in the telopeptide part of the α-chain and/or in the propeptide part of the α-chain and/or at the start of the α-chain and/or at the end of the α-chain.
11. The method of claim 1 , wherein said crosslinks are formed by at least two Cys residues per collagen α-chain.