Method for producing hemin proteins using plant cells, resulting proteins and products containing same
View Patent ↗A method for producing haemin proteins by (i) inserting into plant cells one or more nucleic acid molecules that each comprise at least one sequence coding for a protein component of an animal haemin protein capable of reversibly binding oxygen, or for a variant or portion of said protein component, and optionally a sequence coding for a selection agent; (ii) selecting cells containing nucleic acid coding for the protein component of the haemin protein; (iii) optionally propagating the transformed cells either in a culture or by regenerating whole transgenic or chimeric plants; and (iv) recovering and optionally purifying a haemin protein that includes a complex consisting of the protein or proteins coded for by said nucleic acid and at least one iron-containing porphyritic nucleus, or a plurality of such complexes.
1. A recombinant hemin protein having the capacity to reversibly bind oxygen, comprising at least one iron-containing porphyrin nucleus, of plant origin, and a protein component comprising at least one polypeptide chain selected from the group consisting on hemoglobin, myoglobin, cytochromes, peroxidases, and catalases of animal origin.
2. The recombinant protein according to claim 1 , wherein the at least one iron-containing porphyrin nucleus is iron-containing protoporphyrin IX, or a protoporphyrin differing from protoporphyrin IX in the nature of the side chains, carried by the β atoms of pyrrole rings.
3. The recombinant protein according to claim 1 , wherein the protein component comprises at least one α and/or β-globin polypeptide chain, or variants of said polypeptide chain, wherein the variant of the α chain has at least 90% homology with an α chain having an amino acid sequence of SEQ ID NO:31 and the variant of the β chain has at least 90% homology with a β chain having an amino acid sequence of SEQ ID NO:33, and the hemin protein is capable of binding oxygen reversibly.
4. The recombinant protein according to claim 3 , wherein the α or β-globin chain, or variants of the said polypeptide chain, further comprises a chloroplast targeting signal, a mitochondrial targeting signal, or a N-terminal signal peptide in combination with a signal responsible for retaining a protein in the endoplasmic reticulum or a N-terminal signal peptide in combination with a vacuolar targeting signal.
5. The recombinant protein according to claim 3 , wherein each α and/or β-globin polypeptide chain lacks an NH 2 terminal methionine.
6. The recombinant hemin protein according to claim 1 , wherein the protein component comprises at least four polypeptide chains of α and/or β-globin or variants of the said polypeptide chain, each said polypeptide chain being bound to an iron-containing protoporphyrin nucleus, wherein the variant of the α chain has at least 90% homology with an α chain having an amino acid sequence of SEQ ID NO:31 and the variant of the β chain has at least 90% homology with a β chain having an amino acid sequence of SEQ ID NO:33, and the hemin protein is capable of binding oxygen reversibly.
7. The recombinant protein according to claim 6 , wherein the protein component comprises 2 α-globin chains and 2 β globin chains, or variants of the said polypeptide chain.
8. The recombinant protein according to claim 1 , wherein said protein binds oxygen with an affinity of between 7 and 40 mm Hg.
9. A pharmaceutical product comprising one or more recombinant hemin protein(s) according to claim 1 in association with a physiologically acceptable excipient.
10. A recombinant hemin protein having the capacity to reversibly bind oxygen, comprising at least one iron-containing porphyrin nucleus of plant origin, and a protein component comprising at least one polypeptide chain selected from the group consisting of hemoglobin, myoglobin, and cytochromes of animal origin.
11. The recombinant protein according to claim 10 , wherein the at least one iron-containing porphyrin nucleus is iron-containing protoporphyrin IX, or a protoporphyrin differing from protoporphyrin IX in the nature of the side chains, carried by the β atoms of pyrrole rings.
12. The recombinant protein according to claim 10 , wherein the protein component comprises at least one α and/or β-globin polypeptide chain, or variants of said polypeptide chain, wherein the variant of the α chain has at least 90% homology with an α chain having an amino acid sequence of SEQ ID NO:31 and the variant of the β chain has at least 90% homology with a β chain having an amino acid sequence of SEQ ID NO:33, and the hemin protein is capable of binding oxygen reversibly.
13. The recombinant protein according to claim 12 , wherein the α or β-globin chain, or variants of the said polypeptide chain, further comprises a chloroplast targeting signal, a mitochondrial targeting signal, or a N-terminal signal peptide in combination with a signal responsible for retaining a protein in the endoplasmic reticulum or a N-terminal signal peptide in combination with a vacuolar targeting signal.
14. The recombinant protein according to claim 12 , wherein each α and/or β-globin polypeptide chain lacks an NH 2 terminal methionine.
15. The recombinant hemin protein according to claim 10 , wherein the protein component comprises at least four polypeptide chains of α and/or β-globin or variants of said polypeptide chain, each said polypeptide chain being bound to an iron-containing protoporphyrin nucleus, wherein the variant of the α chain has at least 90% homology with an α chain having an amino acid sequence of SEQ ID NO:31 and the variant of the β chain has at least 90% homology with a β chain having an amino acid sequence of SEQ ID NO:33, and the hemin protein is capable of binding oxygen reversibly.
16. The recombinant protein according to claim 15 , wherein the protein component comprises 2 α-globin chains and 2 β chains, or variants of the said polypeptide chain.
17. The recombinant protein according to claim 10 , wherein said protein binds oxygen with an affinity of between 7 and 40 mm Hg.
18. A pharmaceutical product comprising one or more recombinant hemin protein(s) according to claim 10 in association with a physiologically acceptable excipient.
19. The recombinant protein according to claim 10 , wherein the protein component comprises at least one α and/or β-globin polypeptide chain, or variants of said polypeptide chain, wherein the variant of the α chain has a heme binding domain and the variant of the β chain has a heme binding domain and the hemin protein is capable of binding oxygen reversibly.