Collagen 7 and related methods
View Patent ↗Disclosed are methods of making collagen 7, or functional fragments thereof, as well as collagen 7, and functional fragments thereof produced by such methods, nucleic acids encoding collagen 7, and functional fragments thereof, as well as vectors and host cells comprising such nucleic acids.
1. A method of making human collagen 7, or a functional fragment of human collagen 7, comprising:
providing a cell, which comprises an exogenously introduced nucleic acid that encodes human collagen 7, or a functional fragment thereof, wherein said cell is recombinantly manipulated to express one or more polypeptides that increase expression of human collagen 7, or a functional fragment thereof, and wherein the one or more polypeptides comprises prolidase; and
culturing said cell under conditions sufficient for the production of human collagen 7, or the functional fragment of human collagen 7, and prolidase, thereby making human collagen 7, or the functional fragment thereof.
2. The method of claim 1 , wherein said cell is genetically manipulated to express a glycosyl transferase.
3. The method of claim 1 , wherein said cell comprises an exogenously introduced nucleic acid that encodes prolidase.
4. The method of claim 2 , wherein said cell comprises an exogenously introduced nucleic acid that encodes the glycosyl transferase.
5. The method of claim 1 , wherein said cell comprises an expression vector that comprises a sequence that encodes human collagen 7.
6. The method of claim 1 , further comprising recovering human collagen 7, or functional fragment thereof, from said cultured cell.
7. The method of claim 1 , wherein at least 30, 40, 50, 60, 70, 80, 90 or 95% of said human collagen 7, or functional fragment thereof, is incorporated into homotrimers.
8. The method of claim 1 , wherein at least 30, 40, 50, 60, 70, 80, 90 or 95% of said human collagen 7, or functional fragment thereof, is incorporated into hexamers.
9. The method of claim 1 , wherein the exogenously introduced nucleic acid that encodes human collagen 7, or the functional fragment thereof, is a high glycine codon-optimized nucleic acid sequence.
10. The method of claim 1 , wherein the cell is a fibroblast.
11. A cell comprising:
a first expression vector comprising a first nucleic acid sequence that encodes human collagen 7 or a functional fragment thereof,
a second expression vector comprising a second nucleic acid sequence that encodes one or more polypeptides that increase expression of human collagen 7, wherein said one or more polypeptides comprises prolidase, and
optionally a third expression vector comprising a third nucleic acid sequence that encodes glycosyl transferase.
12. The cell of claim 11 , which is in a cell culture.
13. A cell comprising:
a vector comprising a first nucleic acid sequence that encodes human collagen 7 or a functional fragment thereof, a second nucleic acid sequence that encodes prolidase, and optionally a third nucleic acid sequence that encodes glycosyl transferase.
14. A method of making a cell suitable for expressing human collagen 7, or a functional fragment thereof, comprising
recombinantly manipulating said cell to express recombinant human collagen 7, or the functional fragment thereof; and
recombinantly manipulating said cell to express one or more polypeptides that increase expression of human collagen 7, wherein the one or more polypeptides comprises recombinant prolidase;
thereby making a cell suitable for expressing recombinant human collagen 7, or the functional fragment thereof.
15. The method of claim 14 , further comprising recombinantly manipulating said cell to express recombinant glycosyl transferase.