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Last Updated: March 28, 2024

Claims for Patent: 9,982,270


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Summary for Patent: 9,982,270
Title:Organic compounds
Abstract: Method for heterologous protein production in plant cell plastids comprising introducing into plant cells nucleic acid components that encode heterologous proteins under the control of promoters operative in plastids, vectors, host cells, plants and uses thereof.
Inventor(s): Malcuit; Isabelle (Norwich, GB), Sorokin; Alexander (Norwich, GB)
Assignee: Algentech SAS (Envry, FR)
Application Number:12/601,384
Patent Claims:1. A method of producing at least a heterologous or exogenous polypeptide in a plant or plant cell wherein said method comprises: 1) introducing into the nucleus of the plant cell a DNA construct comprising a plant nuclear promoter operably linked to a group II intron selected from the group consisting of the LtrB intron from Lactococcus lactis and a trnK intron of a plant, wherein the group II intron comprises a plastid transgene cassette inserted in Domain IV of said group II intron operably linked to a primer binding domain (PBD), a) said plastid transgene cassette comprising a left flanking sequence, a plastid specific promoter, a nucleic acid of interest that encodes a heterologous or exogenous polypeptide, a plastid specific terminator, and a right flanking sequence, 2) introducing into the nucleus of the plant cell a second DNA construct comprising a plant nuclear promoter operably linked to a polynucleotide encoding a plastid transit peptide sequence fused to an intron encoded protein selected from the group consisting of LtrA when the introduced intron is LrtB and MatK when the introduced intron is trnK; 3) growing said plant cells of 1) and 2) under conditions expressing the first and second DNA constructs; 4) selecting a plant cell of 3) comprising said plastid transgene cassette integrated into the plastid genome; 5) growing the plant cell of 4) or a plant regenerated therefrom under conditions wherein said plant plastid promoter expresses said heterologous or exogenous polypeptide from said plastid transgene cassette.

2. The method according to claim 1, wherein the nucleic acid sequence that encodes the intron encoded protein is a bacterial LtrA nucleic acid sequence that is codon optimized for expression in plants.

3. The method according to claim 1, wherein the plant plastid is selected from the group consisting of chloroplasts, proplastids, etioplasts, chromoplasts, amyloplasts, leucoplasts and elaioplasts.

4. The method according to claim 3, wherein the plant plastid is a chloroplast.

5. The method according to claim 1, wherein the heterologous or exogenous polypeptide is at least one selected from the group consisting of insulin, preproinsulin, proinsulin, glucagon, interferons, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, fertility hormones, follicle stimulating hormone growth factors, platelet-derived growth factor, granulocyte colony stimulating factor, prolactin, oxytocin, thyroid stimulating hormone, adrenocorticotropic hormone, calcitonin, parathyroid hormone, somatostatin, erythropoietin (EPO), enzymes, hemoglobin, serum albumin, collagen, insect toxic proteins from Bacillus thuringiensis, herbicide resistance proteins, salt-tolerance proteins, and edible vaccines.

6. The method according to claim 1, wherein the plant nuclear promoter is selected from the group consisting of inducible, chemically regulated, constitutive, and tissue specific promoters.

7. The method according to claim 1, wherein the plant plastid promoter is selected from the group consisting of a RNA polymerase promoter, a rpo B promoter element, a atpB promoter element, a clpP promoter element, a 16S rDNA promoter element, a PrbcL promoter, a Prps16 promoter, a Prrn16 promoter, a Prrn-62 promoter, a Pycf2-1577 promoter, a PatpB-289 promoter, a Prps2-152 promoter, a Prps16-107 promoter, a Pycf1-41 promoter, a PatpI-207 promoter, a PclpP-511 promoter, a PclpP-173 promoter, a PaccD-129 promoter, a PaccD-129 promoter of the tobacco accD gene, a PclpP-53 promoter of the clpP gene, a Prrn-62 promoter of the rrn gene, a Prps16-107 promoter of the rps16 gene, a PatpB/E-290 promoter of the tobacco atpB/E gene, and a PrpoB-345 promoter of the rpoB gene.

8. The method according to claim 1, wherein the primer binding site is homologous to the 3'-end of a chloroplast specific tRNA.

9. A polynucleotide construct comprising a plant nuclear promoter operably linked to a group II intron selected from the group consisting of the LtrB intron from Lactococcus lactis and a trnK intron of a plant, wherein the group II intron comprises a plastid transgene cassette inserted in Domain IV of said group II intron operably linked to a primer binding domain (PBD), a) said plastid transgene cassette comprising a left flanking sequence, a plastid specific promoter, a nucleic acid of interest that encodes a heterologous or exogenous polypeptide, a plastid specific terminator, and a right flanking sequence.

10. The isolated polynucleotide according to claim 9, comprising genomic DNA.

11. The isolated polynucleotide according to claim 9, comprising a cDNA component.

12. A polynucleotide construct comprising a plant nuclear promoter operably linked to a polynucleotide encoding a plastid transit peptide sequence fused to an intron encoded protein selected from the group consisting of LtrA and MatK.

13. A host cell containing the isolated polynucleotide according to claim 9.

14. The host cell according to claim 13, wherein the host cell is comprised in a plant, a plant part, a plant propagule, or a plant cell culture.

15. A plant comprising a plant cell, wherein said plant cell is the host cell according to claim 13.

16. The plant according to claim 15, wherein the plant is selected from the group consisting of cotton, rice, oilseed, corn, and soybean.

17. The method according to claim 5, wherein the heterologous or exogenous polypeptide is at least one selected from the group consisting of .alpha.-interferon, .beta.-interferon, .gamma.-interferon, .beta.-glucocerebrosidase, luteinizing hormone, and epidermal growth factor.

18. The method according to claim 1, wherein the heterologous or exogenous polypeptide is a nutritional enhancement protein involved in the biosynthesis of phenolics, starches, sugars, alkaloids, or vitamins.

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