Method for using tobacco mosaic virus to overproduce peptides and proteins
AssigneeThe Scripps Research Institute
InventorsJohn H. Fitchen, Roger N. Beachy
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Abstract
The invention describes compositions and methods of use in which an infectious modified Tobacco Mosaic Virus (TMV) virion comprising a coat protein (CP) or a movement protein (MP) gene is replaced with a nuclear inclusion protease (NIa) expression cassette for the expression of a heterologous peptide in a tobacco mosaic virus (TMV) host plant.
Claims52
A method for producing a heterologous peptide in a plant comprising:
- a) infecting a suitable plant with a CP modified infectious clone of TMV having a wild type MP gene and a modified CP gene comprising a nucleotide sequence encoding a heterogeneous peptide;
- b) culturing the plant under conditions to foster plant growth;
- c) recovering the heterologous peptide from the leaves of the plant.
A method for producing an immune response stimulating peptide in a plant comprising:
- a) infecting a suitable plant with a CP modified infectious clone of TMV having a wild type MP gene and a modified CP gene comprising a nucleotide sequence encoding an immune response stimulating peptide;
- b) culturing the plant under conditions to foster plant growth;
- c) recovering the heterologous peptide from the leaves of the plant.
A method for producing a heterologous peptide in a plant comprising:
- a) infecting a suitable plant with a CP modified infectious clone of TMV comprising a NIa based cassette vector comprising:
- (1) a nucleotide sequence encoding a nuclear inclusion protein from tobacco etch virus and flanking self-cleavage sequences therefor, and
- (2) nucleotide sequences encoding heterologous peptides flanking the self-cleavage sites;
- b) culturing the plant under conditions to foster plant growth;
- and
- c) recovering the heterologous peptide from the leaves of the plant.
A nucleotide sequence encoding a modified infectious clone of TMV comprising a heterologous DNA sequence encoding a peptide of from about 5 to 20 amino acids wherein the heterologous DNA is inserted immediately after the codon encoding amino acid 154 of the wild type coat protein of TMV.
A nucleotide sequence encoding a modified infectious clone of TMV comprising a nucleotide sequence encoding a wild type coat protein and a dysfunctional movement protein.
A modified TMV virion comprising a heterologous peptide sequence of from about 5 to 20 amino acids inserted immediately following amino acid 154 of the wild type TMV coat protein.
A nucleotide sequence encoding a modified infectious clone of TMV comprising a heterologous DNA sequence encoding a wild type CP and a NIa based cassette filter comprising DNA encoding:
- a) the nuclear inclusion (NIa) protease,
- b) multiple restriction endonuclease sites;
- and
- c) self-cleavage sites for the protease, wherein the self-cleavage sites flank the protease and each restriction site, except at the termini of the nucleotide sequence;
- wherein the cassette is inserted in the place of the nucleotide sequence encoding the wild type movement protein thereof.
Dependencies are inferred from the claim text.