Enhancing recombinant hemoglobin production by co-expression with alpha hemoglobin stabilizing protein
View Patent ↗The present disclosure relates to the use of AHSP to stabilize the α subunit of rHb. AHSP may be co-expressed with the hemoglobin genes. AHSP stabilization may be used to increase the production of intact rHb in various systems, such as E. coli , other microorganisms, or animal erythroid cells. This intact rHb may then be used as part of a blood substitute product.
1. A method of producing a recombinant hemoglobin α subunit comprising co-expressing a nucleic acid encoding the recombinant hemoglobin α subunit, a nucleic acid encoding a recombinant α hemoglobin stabilizing protein, and a recombinant heme transport gene product in an Escherichia coli or a non- Escherichia coli microorganism cell.
2. A method according to claim 1 , wherein the recombinant hemoglobin α subunit is a human recombinant hemoglobin α subunit and the recombinant α hemoglobin stabilizing protein is a human recombinant α hemoglobin stabilizing protein.
3. A method according to claim 1 , wherein the nucleic acid encoding the α hemoglobin stabilizing protein is located in a plasmid.
4. A method according to claim 1 , wherein the nucleic acid encoding the recombinant hemoglobin α subunit is located in a plasmid.
5. A method according to claim 1 , wherein the nucleic acid encoding the heme transport gene product is located in a plasmid.
6. A method according to claim 1 , wherein the nucleic acid encoding the recombinant α hemoglobin stabilizing protein, the recombinant hemoglobin α subunit, and the recombinant heme transport gene product are located in a single plasmid.
7. A recombinant hemoglobin α subunit production cell comprising:
a nucleic acid encoding a recombinant hemoglobin α subunit;
a nucleic acid encoding a recombinant α hemoglobin stabilizing protein; and
a nucleic acid encoding a recombinant heme transport gene,
wherein the recombinant α hemoglobin stabilizing protein stabilizes the recombinant hemoglobin α subunit; and
wherein the cell is a Escherichia coli or a non- Escherichia coli microorganism cell.
8. A cell according to claim 7 , wherein the recombinant hemoglobin α subunit is a human recombinant hemoglobin α subunit and the recombinant α hemoglobin stabilizing protein is a human recombinant α hemoglobin stabilizing protein.
9. A cell according to claim 7 further comprising a plasmid comprising the nucleic acid encoding the recombinant α hemoglobin stabilizing protein.
10. A cell according to claim 7 , further comprising a plasmid comprising the nucleic acid encoding the recombinant hemoglobin α subunit.
11. A cell according to claim 7 , further comprising a plasmid comprising the nucleic acid encoding the recombinant heme transport gene product.
12. A cell according to claim 7 , further comprising a single plasmid comprising the nucleic acid encoding the recombinant α hemoglobin stabilizing protein, the recombinant hemoglobin α subunit, and the recombinant heme transport protein.
13. A system for recombinant hemoglobin subunit production comprising:
an Escherichia coli or non- Escherichia coli microorganism production cell;
a nucleic acid encoding a recombinant hemoglobin α subunit;
a nucleic acid encoding an α hemoglobin stabilizing protein; and
a nucleic acid encoding a recombinant heme transport gene product,
wherein the nucleic acid encoding the recombinant hemoglobin α subunit and the nucleic acid encoding the recombinant α hemoglobin stabilizing protein are co-expressed and the recombinant α hemoglobin stabilizing protein stabilizes at least one recombinant hemoglobin α subunit.
14. A system according to claim 13 , wherein the recombinant hemoglobin α subunit is a human recombinant hemoglobin α subunit and the recombinant α hemoglobin stabilizing protein is a human recombinant α hemoglobin stabilizing protein.
15. A system according to claim 13 , further comprising a plasmid comprising the nucleic acid encoding the recombinant α hemoglobin stabilizing protein.
16. A system according to claim 13 , further comprising a plasmid comprising the nucleic acid encoding the recombinant hemoglobin α subunit.
17. A system according to claim 13 , further comprising a plasmid comprising the nucleic acid encoding the recombinant heme transport gene product.
18. A system according to claim 13 , further comprising a single plasmid comprising the nucleic acid encoding the recombinant hemoglobin α subunit, the nucleic acid encoding the recombinant α hemoglobin stabilizing protein, and the nucleic acid encoding the recombinant heme transport gene product.
19. The system according to claim 13 , further comprising a nucleic acid encoding a recombinant hemoglobin β subunit.
20. A system according to claim 13 , wherein the system produces recombinant hemoglobin that may be used as part of a blood substitute product.