Compositions and methods for preparing factor Xa and derivatives
The present disclosure relates to protein sequences which can be used to generate factor Xa proteins and derivatives thereof. The protein sequences include a factor Xa light chain portion, a heavy chain catalytic domain portion, and an activation peptide C-terminal to the heavy chain catalytic domain portion.
1. A protein comprising the amino acid sequence of formula (I):
LC - L 1- HC - L 2- AP (I),
wherein:
LC comprises the amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:13,
L1 is a peptide linker comprising a protease recognition site,
HC comprises the amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO:11,
L2 is absent or is a peptide linker that cannot be processed by the protease recognizing the protease recognition site in L1, and
AP is an activation peptide comprising the amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 12; and
wherein, when the L1 is processed by the protease, a two-chain polypeptide comprising the LC and the HC on separate chains is produced, and the two-chain polypeptide binds to a factor Xa (fXa) inhibitor.
2. The protein of claim 1 , wherein the protease is furin.
3. The protein of claim 1 , wherein the L1 comprises the amino acid sequence of RKR or RKRRKR (SEQ ID NO:7).
4. The protein of claim 1 , wherein the L2 is 0-50 amino acid residues in length.
5. The protein of claim 4 , wherein at least 50% of the amino acid residues of L2 is Gly or Ser.
6. The protein of claim 1 , wherein the activation peptide comprises a glycosylation site.
7. The protein of claim 1 , wherein the activation peptide comprises the amino acid sequence of SEQ ID NO: 12.
8. The protein of claim 1 , wherein the protein further comprises human serum albumin (HSA) at the N-terminus of the formula (I).
9. The protein of claim 8 , wherein the LC does not comprise amino acid residues 1-45 of SEQ ID NO:2.
10. The protein of claim 1 , wherein the produced two-chain polypeptide has reduced capacity to assemble into a prothrombin complex as compared to a wild-type fXa.
11. The protein of claim 1 , wherein the LC comprises the amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:6.
12. The protein of claim 1 , wherein the produced two-chain polypeptide has reduced catalytic activity as compared to a wild-type fXa.
13. The protein of claim 1 , wherein the HC comprises the amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:10.
14. The protein of claim 1 , wherein the HC comprises the amino acid sequence of SEQ ID NO:11.