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Method for using tobacco mosaic virus to overproduce peptides and proteins

a technology of tmv and tmv, which is applied in the direction of peptides, peptide/protein ingredients, peptide sources, etc., can solve the problems of virus particles being assembled into virus particles, unable to work in a plant system, and tmv vectors developed using a cp gene modified to insert foreign genes usually fail to systemically express foreign genes

Inactive Publication Date: 2003-11-06
THE SCRIPPS RES INST
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  • Abstract
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Benefits of technology

[0019] A method is provided for overexpression in plants of heterologous peptides of from about 5 to 20 amino acids as fusion proteins inserted near, but preferably not at the carboxy terminus of the coat protein of tobacco mosaic virus using a modified cDNA infectious clone of TMV. Despite insertion of the foreign peptide sequence into the viral coat protein, stable virions are provided by this invention so that systemic infections is readily achieved in suitable plants. In some instances, the method utilizes coinfection of the plant with 1) the infectious clone having a modified CP gene, but a wild type movement protein (MP) gene and 2) a second TMV infectious clone having a wild type coat protein gene and a MP gene that has been modified to render the movement protein dysfunctional.

Problems solved by technology

Because the cDNA was unavailable, no work was done in a plant system.
However, the TMV vectors developed using a CP gene modified to insert foreign genes usually fail to systemically express foreign genes, either through failure to produce intact CP for virus particle formation, or through loss of the foreign gene sequence during replication due to RNA recombination (N. Takamatsu, et al., EMBO J., 6:306-311, 1987; N. Takamatsu. et al, FEBS Lett., 269:73-76, 1989; W. O. Dawson, et al., Virology, 172:285-292, 1989).
However, there was no evidence at this point that the protein could be assembled into virus particles except that one of the coat protein fusion sequences led to systemic symptoms.
In addition, it is not known in which tissues or in what quantities MP must accumulate in order for local or long-distance spread of the virus to occur.
However, inoculation of transgenic tobacco plants that expressed a wild-type TMV MP gene resulted in both local and systemic viral infection (Deom et al., supra).
However, plants infected with the viral vector producing this C-terminal modified CP experience only a lethal necrotic (local) reaction.
In addition, in some cases the modifications to the CP interfere with assembly of the recombinant virions in inoculated plants.
Systemic spread of the virions cannot be accomplished without formation of stable virions, and it has been discovered that in some cases systemic spread of the virions containing modified CP could not be accomplished with virus produced from the CP-modified infections clone alone, even though stable capsid formed.
It has been discovered that dual infection of a suitable host plant with both the primary virus containing the modified CP and the helper virus containing the inactivated or modified MP leads to systemic plant infections, and hence to production of large quantities of the heterologous peptides.
The goal is to adjust the ratio to produce sufficient wild type CP to facilitate viral movement through plant cell walls, but not enough that wild type viral particles are formed.

Method used

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  • Method for using tobacco mosaic virus to overproduce peptides and proteins
  • Method for using tobacco mosaic virus to overproduce peptides and proteins
  • Method for using tobacco mosaic virus to overproduce peptides and proteins

Examples

Experimental program
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example 2

Construction of Infectious Clones

[0098] A 270-base pair PstI-EcoRI restriction fragment corresponding to nucleotides 1-270 of the modified TMV sequence was subcloned into the vector pBluescript KS+ (Stratagene, San Diego, Calif.). Sequences in the polylinker between the T7 RNA promoter in the vector and TMV nucleotide number 1 were removed by oligonucleotide-directed deletion mutagenesis utilizing the oligonucleotide pdGTAATACGACTCACTATAGTATTTTTACAACAATTA (SEQ ID NO 9). This placed the T7 promoter region immediately adjacent to the 5' end of the TMV cDNA. The remainder of the TMV genome was reassembled downstream by introducing a SmaI-BamHI (nucleotides 256-3332) restriction fragment, and subsequent a BamHI-KpnI (nucleotides 3333-6396) restriction fragment using standard techniques (Sambrook, supra). Replacements of defective segments of the genome were made by exchanging SmaI-BamHI or BamHI-KpnI restriction gents with those of alternate cDNA clones.

[0099] To produce in vitro transc...

example 3

Isolation of Virion and Component RNAs

[0100] To 8 ml of cold nucleoprotein solution (3 to 7 mg / ml) in 1 mM sodium EDTA buffer was added 0.4 ml of 100 mg / ml SDS. The tube was transferred immediately to a boiling water bath and the contents were stirred until the solution reached 80.degree. C. The tube was transferred to ice water and stirring was continued until the solution temperature dropped below 40.degree. C. After the tube had remained 5 to 10 minutes more on ice, 0.16 ml of 0.4 M Tris, 0.04 M acetic acid was added, followed by 8 ml of liquid phenol that had been equilibrated with 0.05 M Tris, 0.004 M acetic acid. The phases were mixed by shaking for 2-3 minutes and separated by centrifugation. The aqueous phase was extracted a second and, sometimes, a third time. The RNA was precipitated by adding 0.8 ml of 2 M sodium acetate, 0.2 M acetic acid, and 16 ml of cold 95% ethanol. The insoluble material was recovered by centrifugation. The precipitate was washed once with 95% ethan...

example 4

In Vitro Transcription and Inoculation

[0101] Plasmids containing full-length modified TMV cDNA clones were linearized with KpnI, which restricts the plasmid at the 3' end of the TMV cDNA, blunted by removing the 3' overhang with Klenow polymerase, and used for the production of run-off transcripts. Transcription by T7 RNA polymerase (Promega, Madison, Wis.) used the reaction conditions as described by Nielsen and Shapiro (Nucleic Acids Res., 14:5936, 1986), except that the concentrations of ATP, CTP, and UTP were increased to 1 mM each and BSA was added to a final concentration of 100 .mu.g / ml. After incubation, 20 mM sodium phosphate buffer, pH 7.0, was added and the mixture inoculated directly onto leaves dusted with 330 grit carborundum (Fisher Scientific, Pittsburgh, Pa.). Immediately after inoculation, plants were rinsed with water and placed in growth chambers. Plants were observed daily for signs of infection (necrotic local lesions or systemic vein yellowing and mosaic). Acc...

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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.

Description

[0001] This application is a Continuation-in-Part application of U.S. Ser. No. 08 / 192,477, filed Feb. 3, 1994.BACKGROUND OF THE MENTION[0003] 1. Field of the Invention[0004] This invention relates to methods for production of recombinant peptides and proteins. More particularly, this invention relates to techniques for inserting peptides into the coat protein of a virus, particularly for purposes of creating a vaccine.[0005] 2. Description of Related Art[0006] Tobacco mosaic virus (TMV) is a well-characterized plant virus with a single, positive-sense RNA genome of 6395 nucleotides. The sequence of the TMV coat protein was initially derived back in the 1950's, and it was not until 1972 that the structure and roles of the forms of the TMV CP, its assembly and microscopic examination of the polymers were published (Durham, et al. J. Mol. Bio., 67:315-332 and 6:307-314). In 1982 the full genomic sequence of TMV RNA was published, and confirmation of the amino acid sequence of the CP as...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K38/00C07K14/08C07K14/705C07K16/18C12N15/82
CPCA61K38/00C07K14/005C07K14/705C07K16/18C07K2319/00C12N15/8203Y10S977/914C12N15/8242C12N15/8257C12N15/8283C12N2770/00022Y10S977/916C12N15/8216
Inventor FITCHEN, JOHN H.BEACHY, ROGER N.
Owner THE SCRIPPS RES INST
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