Production of peptides in plants as N-terminal viral coat protein fusions

• US 20020061309A1
• Filed: 03/29/2001
• Published: 05/23/2002
• Est. Priority Date: 03/08/2000
• Status: Abandoned Application

• Current Assignee: Cynthia Gross, Gregory P. Pogue, John A. Lindbo, Stephen J. Garger
• Original Assignee: Cynthia Gross, Gregory P. Pogue, John A. Lindbo, Stephen J. Garger
• Inventors: Pogue, Gregory P., Lindbo, John A., Garger, Stephen J., Gross, Cynthia
• Application Number:
  US09/823,936
• Publication Number:
  US 20020061309A1
• Time in Patent Office: Days
• US Class Current: 424/184.1
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/015 Hemosporidia antigens, e.g. Plasmodium antigens
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/445 Plasmodium
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C12N 15/8203 Virus mediated transformation
• CPC Class Codes: C12N 15/8257 for the production of primary gene products, e.g. pharmaceutical products, interferon
• CPC Class Codes: C12N 15/8258 for the production of oral vaccines (antigens) or immunoglobulins
• CPC Class Codes: C12N 2770/00022 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention relates to foreign peptide sequences fused to the N-terminal of plant viral structural proteins and a method of their production. Fusion proteins are economically synthesized in plants at high levels by biologically contained tobamoviruses. The foreign peptide sequences can be cleaved from the fusion proteins by proteolytic enzymes or chemical reagents. The foreign peptide sequences of the invention have many uses. Such uses include use as antigens for inducing the production of antibodies having desired binding properties, e.g., protective antibodies, for use as vaccine antigens for the induction of protective immunity, including immunity against parasitic infections, for use as a protein involved in hormonal activity, or for use as a protein involved in immunoregulatory activity.

Claims

1. A polynucleotide encoding a fusion protein, wherein the fusion protein comprises a protein of interest linked to the N-terminus of a plant viral coat protein via a linking element, wherein the linking element is capable of being cleaved by a chemical reagent, wherein the linking element comprises a methionine and the chemical reagent is cyanogen bromide.

2. The polynucleotide according to claim 1 wherein the fusion protein is capable of being expressed in a plant or a plant cell.

3. The polynucleotide according to claim 1 wherein the protein of interest is an antigen.

4. The polynucleotide according to claim 1 wherein the plant viral coat protein is obtained from a single-stranded plus-sense RNA virus.

5. The polynucleotide according to claim 4 wherein the single-stranded plus-sense RNA virus is a tobacco mosaic virus.

6. The polynucleotide according to claim 1 wherein the protein of interest is a vaccine.

7. The polynucleotide according to claim 1 wherein the protein of interest is more than 15 amino acids long.

8. The polynucleotide according to claim 1 wherein the fusion protein is capable of being expressed and the plant viral coat protein is capable of being expressed in a form that is not linked to the protein of interest.

9. A recombinant viral nucleic acid comprising a polynucleotide according to claim 1.

10. A recombinant virus particle comprising a recombinant viral nucleic acid according to claim 9.

11. A recombinant plant virus wherein the plant viral coat protein is encoded by the polynucleotide according to claim 1.

12. A plant cell comprising the polynucleotide according to claim 1.

13. A plant cell comprising the recombinant viral nucleic acid according to claim 9.

14. A plant cell comprising the recombinant virus particle according to claim 10.

15. A plant cell comprising the recombinant plant virus according to claim 11.

16. A plant comprising the polynucleotide according to claim 1.

17. A plant comprising the recombinant viral nucleic acid according to claim 9.

18. A plant comprising the recombinant virus particle according to claim 10.

19. A plant comprising the recombinant plant virus according to claim 11.

20. A method for synthesizing a protein of interest, comprising the steps of:

(a) contacting a plant or a plant cell with a recombinant plant virus nucleic acid comprising a polynucleotide encoding a fusion protein, wherein the fusion protein comprises a protein of interest linked to the N-terminus of a plant viral coat protein via a linking element capable of cleavage by a cyanogen bromide, wherein the linking element comprises a methionine,

(b) growing the plant or the plant cell under conditions such that the fusion protein is expressed, and

(c) reacting the linking element with a chemical reagent such that at least one covalent bond between the protein of interest and the plant viral coat protein is broken.

21. The method according to claim 20 further comprising the step of:

(d) purifying the protein of interest from the plant viral coat protein.

23. The method according to claim 20 further comprising the step of:

(d) purifying the fusion protein from the plant or the plant cell, wherein step (d) is prior (c).