WO2002006497A2 - Plantes transplastomiques - Google Patents

Plantes transplastomiques Download PDF

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Publication number
WO2002006497A2
WO2002006497A2 PCT/EP2001/008132 EP0108132W WO0206497A2 WO 2002006497 A2 WO2002006497 A2 WO 2002006497A2 EP 0108132 W EP0108132 W EP 0108132W WO 0206497 A2 WO0206497 A2 WO 0206497A2
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Prior art keywords
protein
transplastomic
homotransplastomic
plant
fusion protein
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PCT/EP2001/008132
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English (en)
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WO2002006497A3 (fr
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Vanga Siva Reddy
Leelavathi Sadhu
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International Centre For Genetic Engineering And Biotechnology
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Priority to AU2001281981A priority Critical patent/AU2001281981A1/en
Publication of WO2002006497A2 publication Critical patent/WO2002006497A2/fr
Publication of WO2002006497A3 publication Critical patent/WO2002006497A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/555—Interferons [IFN]
    • C07K14/57—IFN-gamma
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8214—Plastid transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
    • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
    • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • the present invention is in the field of plant biotechnology. It relates more particularly to the recombinant expression of a stable protein in a plastid, to the generation of transplastomic plant cells, plants, seeds and plants of second and further generations, and to the isolation of the thus expressed protein.
  • Plastids are organelles found in plant cells and the cells of photosynthetic algae such as Chlamydamonas.
  • Various kinds of plastids exist and are derived from undifferentiated plastids, termed proplastids.
  • Differentiated plastids include amyloplasts, chromoplasts, chloroplasts, etioplasts and leucoplasts.
  • Chloroplasts are the most common plastids, and are the site of photosynthesis.
  • Each photosynthetic cell contains multiple chloroplasts, typically from 50 to 100.
  • Chloroplasts have their own genome, the plastome, which exists in addition to the main cellular (nuclear) genome, and transcription and translation systems. The latter resemble prokaryotic transcription and translation systems.
  • Each chloroplast contains multiple genome copies, typically from 50 to 100.
  • a plastid genome, referred to as a plastome comprises a double stranded circular DNA molecule
  • transgene expression In the field of biotechnology, the ability to express a foreign gene, referred to as transgene expression, in the organism of choice, is desirable.
  • transgene expression in plants is achieved by the integration of a transgene construct into nuclear DNA. Due to the low copy number of native genes within the nuclear genome, the number of copies of a transgene in a nuclear transformed plant is typically low. Consequently the expression levels achieved by nuclear transformation is typically low. Expression of the transgene may also be affected by other factors, such as the site of transgene integration. This means that the levels of expression achieved by independently derived nuclear transformed plants harbouring the same transgene can be highly variable.
  • Plant zygotes contain nuclear DNA derived from both the female (ova) and male (pollen) gametes, both of which contribute to the characteristics of the mature plant. Therefore, nuclear-encoded transgenes can be spread in the ecosystem by the dispersal of pollen, which contains the male gametes, from plants containing a nuclear transgene and subsequent fertilisation of wild type plants. The dispersal of pollen derived from a nuclear transformed plant, therefore, provides a potential vehicle for the unwanted (lateral) transmission of transgenes into other species.
  • transplastome A transformed plastome is referred to as a transplastome. Due to the existence of multiple plastome copies within each cliloropiast, the copy number of an integrated transgene is high. This leads to a level of expression of a transplastomic gene that is typically higher than for a equivalent transgene integrated into nuclear DNA. Such plants are referred to as transplastomic plants. Plastids are maternally inherited. That is, zygotes derive plastids from the cytoplasm inherited from the female gamete, whereas pollen does not contribute plastids to the zygote. Pollen derived from transplastomic plants does not, therefore, contain the transgene and so transgene transmission to other species is not possible. This is particularly beneficial in view of public fears related to the spread of transgenes and their potential impact on the ecosystem.
  • flanking regions enable the site-specific integration of the transgene construct into plastome by the process of homologous recombination, a process which naturally occurs in plastids. Therefore, the site of transgene integration is more assured in chloroplast- based techniques relying on homologous recombination than in nuclear-based processes. Therefore, more uniform transgene expression results between independently derived transplastomic plants than between independently derived nuclear transformed ones.
  • Staub et al, (2000) discusses the expression of a ubiquitin-somatotropin fusion protein in chloroplasts.
  • WO 00/03017 describes the expression of fusion proteins comprising ubiquitin and GUS and discusses the expression of interferon in the chloroplast.
  • Both documents disclose fusion proteins wherein the protein of interest is fused by its N- terminal to the C-terminal of its fusion protein partner.
  • the fusion partner is cleaved off by endogenous enzymes to leave a 'wild-type' protein of interest in vivo. Cleavage is not accurate and so the N-terminal amino acid of the protein of interest was not predictable. In other cases (e.g.
  • the fusion protein is stable. Staub et al, (2000) and WO 00/03017 teach that fusion proteins accumulate to higher levels than individual proteins of interest because fusion protein partners provide an N-terminal methionine to the immediate translation product fusion and thus enhance translation.
  • transplastomic plants typically express transgenes at higher levels than nuclear-transformed plants, recovery of commercially viable amounts of protein can be problematic.
  • the recombinant protein if needed, must be purified following cost-effective and rapid purification procedures. In plants, rapid purification is important because there are no protease(s) deletion host plants available in which to express recombinant proteins.
  • Rapid purification of recombinant protein can be facilitated by using affinity-based chromatography if the expressed protein is engineered to contain a ligand at the N- or C-terminal ends (Chong et al, 1997; diGuan et al, 1988; Hochuli et al, 1987; Smith and Johnson 1988). Although this approach is commonly used in E. coli, it is rarely used in the purification of recombinant proteins expressed in transgenic plants (Flachmann and Kuhlbrandt 1996; Sugiura 1999).
  • SDS-PAGE SDS-polyacrylamide gel electrophoresis
  • ELISA enzyme linked immunosorbant assay
  • Western blot analysis is time consuming and laborious. If the recombinant protein can be detected by simple and rapid tests, then purification may be achieved much faster. This is an important consideration for highly labile proteins.
  • the Inventors have studied the level of protein expression in nuclear- and plastid-transformed plants. They demonstrated that the expression of human interferon-gamma (IFN- ⁇ ) was very low (0.001% of the total cellular protein) in nuclear transformed plants, whereas a 100 fold increase in IFN- ⁇ expression levels were observed when expressed in the plastid. The inventors compared the plastidic expression of IFN- ⁇ with GUS and found that GUS expression gave levels of accumulation up to 3% of the total cellular protein.
  • IFN- ⁇ human interferon-gamma
  • IFN- ⁇ accumulates to a lower level than GUS in the chloroplast is because IFN- ⁇ has relatively a short half life (4-6 hours) in chloroplasts as compared to a half-life of 48 hours for GUS.
  • This information provides strategies to enhance the accumulation of labile proteins expressed in plastids by decreasing the rate of protein turnover.
  • IFN- ⁇ in chloroplasts could be increased by translationally fusing ifnG (the polynucleotide coding sequence of IFN- ⁇ ) to uidA (the polynucleotide coding sequence of GUS). They have shown that an GUS. FN- ⁇ fusion protein accumulates to about 5% of the total cellular protein. This is an increase of around 500 fold over the accumulation of IFN- ⁇ alone in the cliloropiast and of 5,000 fold over the accumulation of nuclear expressed IFN- ⁇ alone.
  • the inventors have exploited the properties of GUS to identify fractions containing GUS:IFN- ⁇ fusion protein very rapidly during protein purification. This is useful for isolation of labile proteins under conditions employed during purification steps.
  • the inventors have also shown that translational fusion of a purification tag such as a (6x) His-tag at the N- or C-terminal end of the GUS:IFN- ⁇ fusion protein reduces the number of steps involved in purification.
  • a purification tag such as a (6x) His-tag at the N- or C-terminal end of the GUS:IFN- ⁇ fusion protein reduces the number of steps involved in purification.
  • the invention provides a method of producing a protein of interest comprising allowing a polynucleotide fusion construct to be expressed in a plastid thereby to produce a fusion protein comprising the protein of interest, wherein the fusion construct comprises a polynucleotide coding sequence of the protein of interest operably linked to a polynucleotide coding sequence of a fusion protein partner and wherein the fusion protein thus expressed: (a) has a greater half-life than the individually expressed protein of interest;
  • (c) comprises a cleavage site.
  • a method of the invention further comprises obtaining a sample comprising the thus expressed fusion protein, and typically also comprises enriching the thus obtained sample in the fusion protein.
  • a method of the invention may further comprise recovering the fusion protein from a sample by an affinity-based technique that uses a purification tag.
  • a method of the invention further comprises cleaving the fusion protein in vitro to release the protein of interest.
  • the thus released protein of interest may be recovered from the fusion protein partner by an affinity-based technique that uses the purification tag of the fusion protein partner, thereby to recover the protein of interest.
  • the invention also provides:
  • a method of obtaining a transplastomic plastid which method comprises fransfor ing a plastome within a plastid by a fusion construct of the invention
  • a method of obtaining a transplastomic cell which method comprises transforming a plastid within a cell by a method of the invention
  • a method of obtaining a homotransplastomic cell which method comprises obtaining transplastomic cells by a method of the invention and selecting for the presence of the transplastome;
  • - a method of obtaining a first-generation transplastomic or homotransplastomic plant, wherein the method comprises regenerating a transplastomic or homotransplastomic plant cell obtainable by the method of the invention to give a transplastomic or homotransplastomic plant; - a method of obtaining a transplastomic or homotransplastomic plant seed, wherein the method comprises obtaining a transplastomic or homotransplastomic seed from a transplastomic or homotransplastomic plant obtainable by the method of the invention;
  • a method of obtaining a transplastomic or homotransplastomic progeny plant comprising obtaining a second-generation transplastomic or homotransplastomic progeny plant from a first-generation transplastomic or homotransplastomic plant obtainable by a method of the invention, and optionally obtaining transplastomic or homotransplastomic plants of one or more further generations from the second-generation progeny plant thus obtained;
  • transplastomic or homotransplastomic plastid a transplastomic or homotransplastomic plant cell
  • transplastomic or homotransplastomic callus a transplastomic or homotransplastomic first-generation plant, transplastomic or homotransplastomic plant see or progeny plant obtainable by a method of the invention or comprising a polynucleotide fusion construct of the invention;
  • fusion protein or protein of interest obtained by a method of the invention or from a cell or plant of the invention.
  • Fig. 1 Transformation and expression of ifnG in the tobacco nuclear genome.
  • A Map of the vectors pBI121 and pBIIFNG. Double head arrows indicate the size of DNA fragments after the restriction digestion with Pstl (P), Xbal (X) and EcoRl (E).
  • LB and RB represent left and right border sequences of transformed DNA (T-DNA) of Agrobacterium. Dashed arrow indicates the direction and size of the transcript.
  • 35SP CaMV 35S promoter
  • NPTII neomycin phosphotransferase II that confers resistance to kanamycin
  • uidA ⁇ -glucuronidase (GUS) reporter gene.
  • B Southern hybridization of genomic DNA isolated from wild type (1), Nt.
  • Fig. 2 Restriction map of transformation vectors used to express IFN-g in tobacco chloroplasts and the ifnG gene.
  • Vector pVSR326 contained aadA selectable marker that confers resistance to spectinomycin under the control of rice rrn promoter (rrnP) and uidA reporter gene under the regulation of ⁇ cepsbA promoter (psbAP). Double head arrow indicate the size of the DNA fragment expected when digested with Clal (C).
  • the chimeric uidA and aadA genes were flanked by tobacco rbcL and accD gene sequences for site-specific integration into tobacco plastid genome.
  • Dashed arrow indicates the direction and size of the uidA transcript.
  • B Restriction map of vector p326IFNG, partial chloroplast DNA of tobacco (cpDNA) and the transformed tobacco plant (Nt. 326IFNG- 1) plastid DNA. Double head arrows indicate the size of DNA fragments after the restriction digestion with Apal (A) and Clal (C) alone and together. Position of the His- tag (6x (His) and factor Xa site (Fa-Xa) are indicated. Dashed arrow indicates the direction and size of the ifnG transcript. A possible mechanism for site-specific integration of aadA and ifnG through two homologous recombinations (crossed lines) were also shown.
  • C Restriction map of vector p326IFNG, partial chloroplast DNA of tobacco (cpDNA) and the transformed tobacco plant (Nt. 326IFNG- 1) plastid DNA. Double head arrows indicate the size of DNA fragments after the restriction digestion with Apal (A)
  • Dashed arrows indicate the direction and size of the uidA.ifhG and aadA transcripts.
  • Fig. 3 Southern and Northern blot analysis to confirm the stable integration and expression of aadA, ifiiG, uidA and uidA ⁇ fnG genes in tobacco chloroplasts.
  • GUSIFNG (1) and wild type (2) plants was digested with Clal (C), Xhol (Xh), BamHI (B), Ncol Xbal (N+X) and probed with aadA, ifnG, vddA and rbcL-accD gene sequences.
  • D Total RNA isolated from wild type (1), Nt. 326IFNG-1 (2), Nt. 326IFNG-2 (3) plants were separated on agarose gel, blotted on to nylon membrane and hybridized with ifnG probe. Inset at the bottom shows the hybridization of the same blot with 16S rDNA probe as a loading control.
  • E & F Blots containing total RNA from Nt. GUSIFNG-1 (1), Nt. GUSIFNG-2 (2), Nt. 326-37 (3) and wild type (4) plants were hybridized with uidA (E) and ifnG (F) probes.
  • Fig. 4 Analysis and purification of recombinant IFN-g expressed in tobacco chloroplasts. A). Western blot analysis of total soluble proteins from wild type (1) and
  • fusion protein comprising the sequence of a protein of interest.
  • the term protein as used herein refers to peptides and polypeptides both of natural and unnatural sequence. Such proteins can comprise two or more amino acids, although they typically comprise from 20 to 500 amino acid residues.
  • a fusion protein is a single polypeptide comprising at least two contiguous amino acid sequences that are not naturally found joined together. Preferably, the fusion protein will contain three sequences that are not naturally found together, more preferably four, five or more sequences.
  • the sequences represents the sequence of the protein of interest, that is, it has the sequence of a polypeptide that is desirably expressed in a plastid as described below.
  • the fusion protein may contain the sequences of two, three or more polypeptides of interest, which may be the same or different.
  • the fusion protein comprises additional amino acid sequence that provides substantially greater stability to the fusion protein than that enjoyed by the protein of interest alone. Such additional amino acid sequence is herein after referred to as a fusion protein partner.
  • Fusion proteins of the invention may be expressed within plastids by transformation of the plastome with a transforming construct as described below. Fusion proteins of the invention are typically stable.
  • stable means that the fusion protein has a longer half-life than the individual protein of interest, for example the half- life may be 2, 4 or 6 times greater than the individual protein of interest.
  • the increase in half-life is 10-fold, more preferably 12-fold, yet more preferably from 14 to 20-fold.
  • the half-life of proteins can be measured by any method known in the art, for example, by pulse-labelling experiments.
  • the fusion protein accumulates to higher levels than the individual protein of interest when recombinantly expressed in the plastid. Therefore, in a preferred embodiment the fusion protein comprises the amino acid sequence of a protein of interest, fused to another amino acid sequence, the fusion protein partner, which increases the plastidic accumulation of the expressed fusion protein compared to the plastidic accumulation of the individually expressed protein of interest. In a more preferred embodiment the fusion protein increases accumulation by up to 10-fold compared to individually expressed polypeptide of interest. In a yet more preferred embodiment the increase is up to 100-fold, and in an even more preferred embodiment the increase is up to 500-fold. Most preferably the increase is up to 1000-fold. Fusion proteins of the invention are typically substantially resistant to internal cleavage in vivo.
  • substantially resistant to internal cleavage means that at least 50%, more preferably 80%, yet more preferably 95%, most preferably 100% of the degraded plastidic fusion protein will be degraded by the sequential removal of N- or C-terminal amino acids, rather than the cleavage of internal peptide bonds.
  • the protein of interest may have any function.
  • the naturally occurring form is typically an extracellular protein (e.g. a secreted protein), an intracellular protein (e.g. cytosolic, organellar, plastidic, nuclear or membrane protein) or a protein present in the cell membrane.
  • the naturally occurring form may be constitutively expressed or be tissue specific.
  • the protein of interest may be expressed to enable its mass production, and have no particular relation to the biological processes of the plastid, cell or organism in which it is expressed.
  • the protein of interest will be pharmaceutically active, that is it is that the protein will be able to modify a process such as the interaction between proteins, the interaction between a protein and a polynucleotide, the activity of a protein, the expression of a gene or a metabolic pathway in a human or non-human body.
  • It may be any polypeptide known in the art. It may be derived from any organism, preferably from a prokaryote, fungus, plant or animal. Typically the protein of interest may be derived from a human.
  • the protein of interest may perform any function in vivo.
  • It may be a blood protein, such as a clotting protein (e.g. kinogen, prothiOinbin, fibrinogen factor VII, factor VIII or factor IX). It may be an enzyme, such as a catabolic or anabolic enzyme.
  • the enzyme may be a gastro-intestinal enzyme, metabolic (e.g. glycolysis or Krebs cycle) enzyme or a cell signalling enzyme.
  • the enzyme may make, breakdown or modify lipids, fatty acids, glycogen, amino acids, proteins, nucleotides, polynucleofides (e.g. DNA or RNA) or carbohydrate (e.g. sugars), and thus may typically be a protease, lipase or carbohydrase.
  • the enzyme may be a protein modifying enzyme, such as an enzyme that adds or takes chemical moieties from a protein (e.g. a kinase or phosphatase).
  • the protein of interest may be a transport or binding protein (e.g. which binds and/or transports a vitamin, metal ion, amino acid or lipid, such as cholesterol ester transfer protein, phospholipid transfer protein or an HDL binding protein).
  • the protein of interest may be a connective tissue protein (e.g. a collage, elastin or fibronectin), or a muscle protein (e.g. actin, myosin, dystrophin or mini-dystrophin).
  • the protein of interest may be a neuronal, liver, cardiac or adipocyte protein.
  • the protein of interest may be cytotoxic.
  • the protein of interest may be a cytochrome.
  • the protein of interest may be able to cause the replication, growth or differentiation of cells.
  • the protein may be encoded by a development gene (e.g. which is expressed only before birth).
  • the protein may aid transcription or translation or may regulate transcription or translation (e.g. a transcription factor or a protein that binds a transcription factor or polymerase).
  • the protein may be a signalling molecule, such as an intracellular or extracellular signalling molecule (e.g. a hormone).
  • the protein of interest may be an immune system gene, such as an antibody, T cell receptor, MHC molecule, cytokine (e.g IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 10, IL-10, TNF- ⁇ , TNF- ⁇ , TGF- ⁇ ), an interferon (e.g. IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ), chemokine (e.g. MIP-1 , MIP-1 ⁇ , RANTES), an immune receptor (e.g.
  • an antibody such as an antibody, T cell receptor, MHC molecule, cytokine (e.g IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 10, IL-10, TNF- ⁇ , TNF- ⁇ , TGF- ⁇ ), an interferon (e.g. IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ), chemokine (
  • a receptor for a cytokine, interferon or chemokine such as receptor for any of the above-mentioned cytokines, interferons or chemokines
  • a cell surface marker e.g. macrophage, T cell, B cell, NK cell or dendritic cell surfacemarker
  • CD 1, 2, 3, 4, 5, 6, 7, 8, 16, 18, 19, 28, 40, or 45 eg. CD 1, 2, 3, 4, 5, 6, 7, 8, 16, 18, 19, 28, 40, or 45; or a natural ligand thereof
  • the protein of interest may be a trophic factor (e.g. BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF, NT3, T5, HARP) or an apolipoprotein.
  • BDNF e.g. BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF, NT3, T5, HARP
  • the protein of interest may be a tumour suppressor (e.g. p53, Rb, RaplA, DCC or k-rev) or encoded by a suicide gene (thymidine kinase or cytosine deaminase).
  • the protein of interest may be an antibody.
  • Antibodies may be intact molecules, or fragments thereof, such as Fa, F(ab') 2 , Fv, scFv, or single-chain antibodies which are capable of binding to epitopic determinants.
  • the antibody may be humanised, in that, amino acids in the non-antigen binding regions may be replaced to cause the antibody to more closely resemble a human antibody, whilst retaining its original binding activity.
  • the protein of interest may have enzymatic activity.
  • the polypeptide will be useful for industrial purposes.
  • xylanases enzymes involved in plant cell wall modification, can be useful in paper manufacture.
  • the protein of interest may be useful for therapeutic purposes, for example, as anticoagulants, such as hirudin, which may be useful in a method of treatment of the human or animal body, or as an antigenic polypeptide for use as an edible or extractable vaccine.
  • the protein of interest is an interferon.
  • the protein of interest is IFN- ⁇ .
  • IFN- ⁇ also known as immune interferon, mediates many immune responses, for example antiviral, anti-proliferative and several immunoregulatory actions in response to viral and pathogen infections (Pestka and Langers 1987; Lewis et al, 1988, Sen and Lengyel 1992).
  • the human ifnG gene encodes a mature protein of 143 amino acids and is glycosylated at positions Asn 25 and Asn 97 (Gray et al, 1982; James et al, 1995; Rinderl ⁇ iecht et al, 1984).
  • unglycosylated E. co/z ' -derived recombinant IFN- ⁇ shows the same spectrum of biological activities as the natural glycosylated human IFN- ⁇ (Arora and Klianna
  • IFN- ⁇ Recombinant IFN- ⁇ has been extensively tested clinically and used for the treatment of many diseases and disorders, including granulomatous disease (Bemiller et al, 1995; Weening et al, 1995), rheumatoid arthritis (Cannon at al. 1990; Machold et al, 1992) and atopic dermatitis (Hanifin et al, 1993; Ellis et al, 1999).
  • IFN- ⁇ is also useful as an adjuvant in the vaccination of immunocompromised humans (Jaffe and Herberman
  • mice James et al, 1995 and transgenic mice (Dobrovolsky et al, 1993; James et al, 1995;
  • IFN- ⁇ can thus be expressed in any suitable cell system or organism.
  • the protein of interest may function within the plastid.
  • Functions of the protein of interest include herbicide, insecticide or disease resistance.
  • Preferred herbicide resistance genes may be responsible for, for example, tolerance to: Glyphosate (e.g. using an EPSP synthase gene (e.g. EP-A-0 293,358) or a glyphosate oxidoreductase (WO 92/000377) gene); or tolerance to fosametin; a dihalobenzonitrile; glufosinate, e.g. using a phosphinothrycin acetyl transferase (PAT) or glutamine synthase gene (cf.
  • Glyphosate e.g. using an EPSP synthase gene (e.g. EP-A-0 293,358) or a glyphosate oxidoreductase (WO 92/000377) gene
  • fosametin e.g. using a
  • a protoporphyrogen oxidase gene an oxadiazole such as oxadiazon; a cyclic imide such as chlorophthalim; a phenyl pyrazole such as TNP, or a phenopylate or carbamate analogue thereof; spectinomycin e.g using the aadA gene, as exemplified below.
  • Insect resistance may be introduced, for example using genes encoding Bacillus thuringiensis (Bt) toxins. Likewise, genes for disease resistance may be introduced, e.g. as in WO91/02701 or WO95/06128.
  • the protein of interest may have a role in a metabolic pathway, preferably a chloroplastic metabolic pathway, for example the photosynthetic metabolic pathway.
  • a chloroplastic metabolic pathway for example the photosynthetic metabolic pathway.
  • the expression of the encoded protein of interest may alter flux in the pathway, for example the photosynthetic ability of the transformed plant.
  • chlorophyll biosynthesis in lower organisms is light-independent due to the presence of an enzyme that comprises three polypeptides encoded by the chlL, chlN and chlB genes.
  • chlorophyll biosynthesis in angiosperms is dependent on the presence of light and therefore darkness results in low chlorophyll biosynthesis. This problem could be overcome by the generation of a transplastomic angiosperm that accumulates higher levels of the protein products of the chlL, chlN and chlB genes.
  • the ability of the photosynthetic enzyme RUBISCO to fix carbon dioxide varies amongst plants that belong to distinct taxanomic groups such as algae, bryophytes, gymnosperms and angiosperms.
  • the level of carbon dioxide fixation could be modified by transformation of a recipient plastome from one organism with the chloroplastic gene or genes encoding the RUBISCO large subunits from another organism.
  • chloroplastic processes including metabolic and signalling pathways, can be controlled by post-translational protein modification such as glycosylation or phosphorylation.
  • post-translational protein modification such as glycosylation or phosphorylation.
  • These levels could be manipulated in the cliloropiast by transforming a plastome with a gene or genes encoding enzymes responsible for glycosylation, deglycosylation, phosphorylation or dephosphorylation, thus allowing promotion of desirable pathways or inhibition of undesirable pathways.
  • expression of the encoded protein may introduce a new metabolic step or steps to the transformed organism.
  • biodegradable plastics can be produced in the cliloropiast by transforming the plastome with prokaryotic genes known in the art.
  • the genes that control plastid division could be introduced into a plastome to alter the number of plastids within a cell, with concomitant modification in their associated processes, such as photosynthesis. Fusion protein partner
  • the fusion protein partner may comprise any amino acid sequence. Typically the fusion protein partner will be of from 20 to 500, more typically from 50 to 200 amino acids in length. Usually the fusion protein partner will possess a scorable property.
  • a protein with a scorable property is a protein that can be distinguished from other proteins, preferably on the basis of its enzymatic or optical properties. Most preferably the fusion protein comprises the sequence of ⁇ -glucoronidase (GUS), or a fluorescent protein such as green fluorescent protein (GFP), yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP) or a variant thereof.
  • GUS ⁇ -glucoronidase
  • GFP green fluorescent protein
  • YFP yellow fluorescent protein
  • CFP cyan fluorescent protein
  • a variant of a protein with a scorable property will possess substantially the same distinguishing property as the protein from which it was derived.
  • the scorable property is based on the enzymatic activity of the protein then a variant will retain at least 10%, 50%, 80% or 95% of the enzymatic activity of the protein sequence from which it was derived. If the scorable property is based on the optical properties of the protein then a variant will substantially retain the optical property, albeit that the fluorescent or luminescent emissions of the variant may be at a different wavelength to those of the protein sequence from which it was derived.
  • the fusion protein partner may by fused to either end of a polypeptide of interest within the fusion protein, in a preferred embodiment the fusion protein partner is fused to the C-terminal of the polypeptide of interest.
  • a fusion protein of the invention preferably comprises a purification tag which as used herein is an ammo acid sequence that can bind to a target under suitable conditions.
  • suitable conditions will vary dependent on the purification tag and target. Usually the target will be provided by a manufacturer, who will recommend a purification tag and suitable conditions. In other cases, optimal condition can be determined experimentally, e.g. using the "Surface Plasmon Resonance" technique from BIACORE (see http:/www.biacore.com).
  • the target will bind to the purification tag with high specificity.
  • Specificity may be determined by contacting the target with a protein sample under conditions suitable for binding, which sample comprises about 50%> protein with a purification tag, that is, from 40 to 60%, and measuring the percentage of total protein bound to the target that comprises a purification tag. This may be achieved by any method known in the art.
  • a protein comprising a purification tag which binds the target with high specificity will represent at least 50% of the total protein bound to the target, preferably at least 70%, more preferably at least 90%, yet more preferably at least 95%, even more preferably at least 99%, most preferably 100%.
  • the purification tag is preferably capable of facilitating affinity purification of the polypeptide from a sample.
  • capable of facilitating affinity purification means that following an affinity purification step at least 10%>, more preferably at least 50%, yet more preferably at least 80%, yet more preferably 95%, most preferably 100% of the polypeptide comprising a purification tag present in the sample can be substantially isolated from the sample.
  • the substantially isolated polypeptide comprising a purification tag represents at least 50%>, more preferably at least 80%>, most preferably 95%) of the total protein content of the isolate thus produced.
  • a purification tag will comprises at least one but not more than 100 amino acids.
  • a fusion protein of the invention may comprise 1 or more purification tags.
  • the number of purification tags is not more than 5, more typically not more than 2, most typically 1.
  • the purification tag may be positioned anywhere within the fusion protein.
  • the purification tag is part of a fusion protein partner. More preferably the purification tag is retained at a terminus of the fusion partner protein following cleavage of the fusion protein.
  • a purification tag is positioned at the N- or C-terminus of the fusion protein and enables the fusion protein to be purified by affinity based methods. Altematively, a purification tag may only be capable of binding to its target following cleavage of the fusion protein. Thus a purification tag may facilitate the separation of the cleavage products of the fusion protein.
  • the purification sequence is a His-Tag.
  • the His-tag comprises multiple contiguous histidine residues, preferably from 3 to 20, more preferably from 4 to 10 most preferably 6.
  • the His-tag will be positioned at either or both of the N- and C- terminals of the fusion protein.
  • the preferred target for the His-tag is NiNTA, which is available commercially, e.g. from Qiagen (Germany).
  • purification tags include a self-cleaving chitin-based ligand system
  • a fusion protein of the invention usually comprises a cleavage site.
  • a cleavage site is any amino acid sequence that facilitates in vitro cleavage of the fusion protein to release the polypeptide of interest.
  • a cleavage site comprises a sequence in which a peptide bond may be broken by the action of an additional factor, such as another protein.
  • the cleavage site may be of any sequence and any length. Typically the cleavage site is from 2 to 20 amino acids in lengths.
  • the cleavage site is resistant to in vivo cleavage, as defined above.
  • the cleavage site is positioned adjacent to the protein of interest within the fusion protein. Subsequently, in this preferred embodiment, cleavage of the fusion protein at the cleavage site causes the protein of interest to be released from the fusion protein partner.
  • Some cleavage sites may not give accurate and uniform cleavage, and so result in cleavage at slightly different positions within a population of cleaved proteins.
  • fidelity refers to the accuracy of the cleavage at the cleavage site by in vitro mechanisms, that is to say the percentage, in a population that have been subjected to in vitro cleavage, of cleaved proteins with the correct sequence.
  • the percentage at which the protein of interest accurately cleaved can be readily established by the skilled person using techniques well known in the art such as N-terminal sequencing of the protein of interest or the fusion protein partner as appropriate.
  • fidelity of a cleavage site in a fusion protein of the invention will be at least 20%.
  • fidelity will be at least 40%, more preferably at least 50%, 60% or 70%). More preferably fidelity will be at least 80%, even more preferably at least 90%>, yet more preferably at least 95% or 99%, most preferably 100%.
  • a preferred cleavage site for use in fusion proteins of the invention is the factor Xa cleavage site IEGR (Nagai et al, 1985; Quinlan et al, 1989; Wearne, 1990) or a variant thereof that is recognised and cleaved by factor Xa with substantially the same fidelity as IEGR.
  • IEGR factor Xa cleavage site
  • Transforming constructs of the invention may comprise DNA or RNA, preferably DNA. They may also include within them synthetic or modified nucleotides.
  • the invention further provides double stranded polynucleofides comprising a polynucleotide of the invention and its complement.
  • Transforming constructs of the invention may be produced recombinantly, synthetically, or by any means available to those of skill in the art.
  • the constructs are typically provided in isolated and/or purified form.
  • Transforming constructs of the invention typically comprise a sequence encoding a fusion protein of invention.
  • the fransfoiining constructs of the invention may comprise a selectable marker gene i.e. marker genes that allow transformed cells to survive in the presence of agents that kill non-transformed cells.
  • a selectable marker gene may be used in the transforming constructs of the invention.
  • herbicide resistance genes e.g. as defined above
  • coding regions that encode products which provide resistance to aminoglycoside antibiotics may be used as a selectable marker, for example, encoded products that provide resistance to kanomycin, neomycin or chloramphenicol.
  • the encoded polypeptide may cause morphological alterations to cultured transformed cells, such as isopentyltransferase (Kunkel et ⁇ l, 1999).
  • the encoded polypeptide may be a scorable marker, which allows transformed cells to be distinguished from non-transformed cells, generally by alteration of the transformed cell's optical properties. Any scorable marker may be used.
  • Preferred scorable markers include polypeptides which are able to alter the appearance or optical properties of transformed cells, for example: ⁇ -glucoronidase (i.e. the uidA:G ⁇ JS gene); fluorescent proteins such as green fluorescent protein (GFP), yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP); or luminescent proteins such as luciferase or aequorin.
  • Cells with scorable optical differences can be sorted using techniques such as fluorescence activated cell sorting (FACS).
  • the polynucleotide of the invention comprises a selectable marker and a scorable marker, for example, the FLARE-S marker genes which comprise aadA and GFP (Khan and Maliga, 1999).
  • Transforming constructs of the invention typically comprise regulatory regions.
  • a transforming construct will comprise one, more preferably two, yet more preferably three or more regulatory regions, which may be the same or different.
  • a regulatory region may be a promoter, enhancer or terminator.
  • a regulatory region will be operably linked to the coding sequence of the fusion protein and/or the selectable or scorable marker gene.
  • the fransforming construct may also be designed in such a way that, following integration of the construct into the plastome, any or all of the coding sequences of the construct are operably linked to native plastome regulatory regions.
  • Transforming constructs of the invention typically comprise flanking regions that are homologous to regions of the recipient plastome, that is, they define the 5' and 3' ends of the transforming construct.
  • the homologous flanking regions allow insertion of the construct into the recipient plastome by homologous recombination. Methods of measuring nucleic acid homology are discussed in more detail below.
  • the homologous flanking regions comprise sequences at least 80% homologous to regions of the recipient plastome. Preferably the degree of homology will be at least 90%o, most preferably 100%.
  • the homologous flanking regions may be homologous to the same, overlapping, coterminous, or distinct regions of the recipient plastome.
  • the homologous flanking regions may be homologous to any regions of the recipient plastome, preferably to regions comprising a gene, pseudogene or intergenic sequence.
  • a homologous flanking region is homologous to a region of the recipient plastome comprising a gene, it is preferably homologous to regions comprising regulatory regions, coding regions, or intronic regions.
  • Testing of new transforming constructs of the invention may be achieved, for example, by use in transforming a plastome utilising a suitable vector as described below and selection of the cell comprising the transformed plastome. Subsequent generation of a cell culture, callus or transplastomic plant allows fusion protein accumulation and half- life to be determined by methods well known in them art.
  • Variants of regions of the transforming constructs of the invention may be obtained and used in the invention. This may be useful where, for example, sequence alterations can be used to alter homology with endogenous plastomic sequences of the recipient plastome, or to alter the functionality of the sequence within the plastome. Other sequence changes may be desired, for example, in order introduce restriction enzyme recognition sites. Variants may be isolated from natural sources or generated from existing sequences by site directed mutagenesis, synthesis of novel sequences or recombinant techniques.
  • Naturally occurring variants may be obtained by probing cDNA or plastomic libraries with degenerate probes at medium to high stringency (for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C). Alternatively, variants may be obtained using degenerate PCR.
  • medium to high stringency for example 0.03M sodium chloride and 0.03M sodium citrate at from about 50°C to about 60°C.
  • variants may be obtained using degenerate PCR.
  • variants typically have at least 50% homology to the sequence from which they are derived, more typically at least 10%. Preferably variants have at least 90%), more preferably at least 95%, yet more preferably 99%) homology to the sequence from which they are derived.
  • variant refers to a variant of a polynucleotide sequence that is altered by one or more nucleotide residues or of a polypeptide that is altered by one of more amino acid residues, but retains the biological activity of the sequence from which it was derived.
  • variants of regulatory regions such as terminators, enhancers or promoters, will retain the activity of the sequence from which the variant was derived, that is, a promoter variant will retain the ability to initiate transgene expression, an enhancer variant will substantially retain the ability to promote transgene expression, whereas a terminator variant will retain the ability to promote dissociation of RNA polymerase from the transgene and thus terminate transgene expression.
  • Variants of coding sequences will retain the ability to encode a polypeptide having substantially the same biological activity as a polypeptide encoded by a naturally occurring coding sequence.
  • biological activity refers to the binding specificity, enzymatic, structural and immunological properties of the naturally occurring polypeptide.
  • a biologically active variant of a polypeptide will retain substantially the same enzymatic properties as the naturally occurring polypeptide.
  • the biologically active variant of a polypeptide will retain substantially the same binding specificity as the naturally occurring polypeptide.
  • Alterations may include additions, insertions, deletions, substitutions or inversions.
  • addition or insertion refer to a change in the polynucleotide sequence resulting in the addition of one or more nucleotide residues as compared to the naturally occurring molecule.
  • the number of nucleotide additions or insertions will be at most 40, more preferably at most 20, yet more preferably at most
  • deletion refers to a polynucleotide sequence wherein one or more nucleotide residues are absent as compared to the naturally occurring molecule.
  • the number of nucleotide deletions will be at most 40, more preferably at most 20, yet more preferably at most 10, and most preferably at most 5. Fragments are therefore generated by deletion of residues from polynucleofides of the invention.
  • substitution refers to the replacement of one or more nucleotide residues by different residues.
  • the number of nucleotide substitutions will be at most 40, more preferably at most 20, yet more preferably at most 10, and most preferably at most 5.
  • inversion refers to a polynucleotide sequence wherein a contiguous region within the sequence is reversed in orientation relative to the remaining molecule.
  • the number of contiguous regions of sequence inverted will be 4, more preferably 3, yet more preferably 2, most preferably 1.
  • variants of polypeptides of the invention can be generated by additions, insertions, deletions, substitutions or inversions of amino acid residues.
  • the same preferred embodiments apply to polypeptide variants in respect of the number of amino acid residues added, inserted, deleted, substituted or inverted as for the number of nucleotide changes for polnucleotide variants as described above.
  • the PILEUP and BLAST algorithms can be used to line up sequences (for example as described in Altschul, 1993; Altschul etal, 1990). Many different settings are possible for such programs. According to the invention, the default settings may be used.
  • the BLAST algorithm is suitable for determining sequence similarity and it is described in Altschul et al, 1990.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, 1990).
  • HSPs high scoring sequence pair
  • T is referred to as the neighbourhood word score threshold (Altschul et al, 1990).
  • These initial neighbourhood word hits act as seeds for initiating searches to find HSPs containing them.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a fused gene or cDNA if the smallest sum probability in comparison of the test nucleic acid to a fused nucleic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • the invention provides a method of making transforming polynucleotides of the invention by introducing a transforming polynucleotide of the invention into a replicable vector, introducing the vector into a compatible host cell, and cultivating the host cell under conditions which bring about replication of the vector.
  • the vector may be recovered from the host cell. Suitable host cells are described below in connection with expression vectors. Bacterial cells, especially E. coli are preferred.
  • the vectors may be for example, plasmid, cosmid, virus or phage vectors provided with an origin of replication.
  • the vectors may contain one or more selectable marker genes, for example antibiotic ampicillin resistance genes. These will generally be operably linked to regulatory sequences capable of securing their expression in the host cell, as described herein for the coding sequences of the invention.
  • the vector may contain one scorable marker, as described above, preferably interrupted by the integration of the transforming polynucleotide, to allow cells transformed by recombinant vectors (i.e. comprising the transforming polynucleotide) to be discriminated from those transformed with non-recombinant vector.
  • Plastids suitable for use in this invention may be derived from any organism that has plastids, preferably a multicellular organism that has plastids. They may be derived from any cell type and may be of any differentiated or undifferentiated state. Such states include undifferentiated proplastid, amyloplast, chromoplast, chloroplast, etioplast, leucoplast. Preferably, the plastid will be a chloroplast.
  • Plastids comprise their own genome, herein referred to as a plastome.
  • individual plastids comprise multiple plastomes, more typically from 5 to 500, most typically from 50 to 100.
  • a recipient plastome is one that may be transformed with a transforming polynucleotide of the invention, as described below.
  • Recipient plastomes for use in the invention may be isolated from natural sources, or may be artificially generated by techniques known in the art, such as recombinant techniques, random mutagenesis, site directed mutagenesis, or other alterations. Alterations may include additions, insertions, deletions, substitutions or inversions.
  • transplastome a recipient plastome transformed with a transforming polynucleotide according to the invention.
  • Plastids comprising a transplastome are referred to as transplastomic.
  • Plastids wherein all plastomes are identical, or substantially identical, transplastomes are referred to as homotransplastomic.
  • the plastomes of plastids are substantially identical if they all comprise the coding region of the transforming polynucleotide of the invention, and preferably any associated regulatory sequences, or at least enough of the coding regulatory sequences to secure expression of the coding sequence.
  • Cells containing plastids are homotransplastomic if all the plastids in the cell are homotransplastomic.
  • Plants, plant parts and seeds are homotransplastomic if all of their cells are homotransplastomic.
  • Suitable sources of recipient plastome include plants, for example, spermatophytes, pteridophytes (ferns, clubmosses, horsetails), bryophytes (liverworts and mosses), and algae.
  • the recipient plastome will be a plastome of a multicellular organism, usually a spermatophyte.
  • the plastome may be a plastome of any gymnosperm or an angiosperm. Suitable gymnosperms include conifers (such as pines, larches, firs, spruces and cedars), cycads, yews and ginkgos.
  • the recipient plastome is an angiosperm plastome and is of a monocotyledonous or dicotyledonous plant, preferably a crop plant.
  • Preferred dicotyledonous crop plants include tomato; potato; sugarbeet cassava; cruciferous crops, including oilseed rape; linseed; tobacco; sunflower; fibre crops such as cotton; and leguminous crops such as peas, beans, especially soybean, and alfalfa.
  • Tobacco is particularly preferred.
  • Preferred monocotyledonous plants include graminaceous plants such as wheat, maize, rice, oats, barley, rye, sorghum, friticale and sugar cane. Rice is particularly preferred.
  • plastomes and regions of transforming construct for use in the methods of the invention may be recombinant or entirely synthetic in origin. Further scope of the invention
  • the transforming polynucleotide is adapted for use in the transformation of other organelles comprising genomes, such as, mitochondria.
  • organelles comprising genomes, such as, mitochondria.
  • the skilled person will readily appreciate that the methods described herein are equally applicable to the production of transgenic mitochondrial genomes, transgenic mitochondria, cells, calli, plants and seeds comprising stable transgenic mitochondria. Furthermore, the production of stable transgenic mitochondria is possible in all eukaryotic organisms.
  • the cell used for transformation may be from any suitable organism (see above list) and may be in any form.
  • it may be an isolated cell, e.g. a protoplast or single cell organism, or it may be part of a plant tissue, e.g, a callus, for example a solid or liquid callus culture, or a tissue excised from a plant, or it may be part of a whole plant.
  • the cell may, for example, be part of an embryo, or a meristem, e.g. an apical meristem of a shoot.
  • the cell is a cell containing chloroplasts, e.g. a leaf or stem cell, most preferably a leaf cell derived from the abaxial side of the leaf. Transformation may thus give rise to a chimeric tissue or plant in which some cells are transgenic and some are not.
  • the polynucleotide may be inserted by any method known in the art, such as recombinant techniques, random insertion, or site directed integration.
  • the method of polynucleotide insertion is site directed integration, more preferably by the process of homologous recombination.
  • the transforming polynucleotide may be inserted into an isolated plastome or an in vivo plastome within a plastid.
  • the plastid used may be in vivo or ex vivo. Insertion of the transforming polynucleotide is preferably performed by transformation of an in vivo plastid.
  • the plastid is within a cell, though it may be in isolated form.
  • Cell transformation may be achieved by any suitable transformation method, for example the transformation techniques described herein.
  • Preferred transformation techniques include electroporation of plant protoplasts (Taylor and Walbot, 1985), PEG- based procedures (Golds et al, 1993), microinjection (Neuhas et al, 1987; Po ⁇ rykus et al, 1985), injection by galinstan expansion femtosyringe (Knoblauch et al, 1999) and particle bombardment (Boynton et al, 1988; Svab et al, 1990; Svab and Maliga 1993; US-A-5,451,513; US-A-5,545,817; US-A-5,545,818; US-A-5,576,198; US-A- 5,866,421). Particle bombardment is particularly preferred.
  • Homotransplastomic (see above) plastids, cells, plants, seeds, plant parts, plant tissues are preferred.
  • Cells generated by the transformation techniques discussed above will typically be present in chimeric tissues, and thus will be surrounded by other non-transformed cells.
  • transplastomic plastids will typically contain multiple copies of untransformed plastomes.
  • homotransplastomic cells that is, cells in which all plastids are homotransplastomic, in that all genomes within those plastids comprise the transforming polynucleotide of the invention, it is necessary to undergo rounds of screening.
  • selectable or scorable marker coding region as defined above, in the integrated polynucleotide.
  • Preferred selectable markers include the aadA gene or the NPTII gene.
  • Homotransplastomic cells can be generated by mutiple rounds of screening of the primary transformed cells for the presence of the selectable or scorable marker. Preferably, at least one round of screening is used, more preferably at least two rounds, most preferably three rounds or more. Typically the homotransplastomic nature of the thus generated cells are ascertained. Homotransplastomicity can be assayed by analysis of isolated plastomic DNA by Southern analysis or by performing polymerase chain reaction amplification. These techniques are suitably sensitive such that the presence of a single untransformed plastome could be detected.
  • Transplastomic or homotransplastomic cells may be regenerated into a transgenic plant by techniques known in the art. These may involve the use of plant growth substances such as auxins, giberellins and/or cytokinins to stimulate the growth and/or division of the transplastomic or homotransplastomic cell. Similarly, techniques such as somatic embryogenesis and meristem culture may be used. Regeneration techniques are well known in the art and examples can be found in, e.g.
  • one step is the formation of a callus, i.e. a plant tissue comprising expanding and/or dividing cells.
  • a callus i.e. a plant tissue comprising expanding and/or dividing cells.
  • Such calli are a further aspect of the invention as are other types of plant cell cultures and plant parts.
  • the invention provides transplastomic or homotransplastomic plant tissues and parts, including embryos, meristems, seeds, shoots, roots, stems, leaves and flower parts. These may be chimeric in the sense that some of their cells are transplastomic or homotransplastomic and some are not. Similarly they may be chimeric in the sense that all cells are transplastomic but only some are homotransplastomic.
  • Regeneration procedures will typically involve the selection of transplastomic and/or homotransplastomic cells by means of marker genes, as discussed above.
  • the regeneration step gives rise to a first generation transplastomic or homotransplastomic plant.
  • the invention also provides methods of obtaining transplastomic or homotransplastomic plants of further generations from this first generation plant. These are known as progeny transplastomic or homotransplastomic plants. Progeny plants of second, third, fourth, fifth, sixth and further generations may be obtained from the first generation transplastomic or homotransplastomic plant by any means known in the art.
  • the invention provides a method of obtaining a transplastomic or homotransplastomic progeny plant comprising obtaining a second-generation transplastomic or homotransplastomic progeny plant from a first-generation transplastomic or homotransplastomic plant of the invention, and optionally obtaining transplastomic or homotransplastomic plants of one or more further generations from the second-generation progeny plant thus obtained.
  • Such progeny plants are desirable because the first generation plant may not have all the characteristics required for cultivation.
  • a plant of a taxon that is easy to transform and regenerate may be chosen. It may therefore be necessary to introduce further characteristics in one or more subsequent generations of progeny plants before a transplastomic or homotransplastomic plant more suitable for cultivation is produced.
  • Progeny plants may be produced from their predecessors of earlier generations by any known technique.
  • progeny plants may be produced by:
  • transplastomic or homotransplastomic seed from a transplastomic or homotransplastomic plant of the invention belonging to a previous generation, then obtaining a transplastomic or homotransplastomic progeny plant of the invention belonging to a new generation by growing up the transplastomic or homotransplastomic seed;
  • transplastomic or homotransplastomic plant of the invention belonging to a previous generation to give a transplastomic or homotransplastomic progeny plant of the invention belonging to a new generation;
  • transplastomic or homotransplastomic progeny plant of the invention belonging to a new generation crossing a first-generation transplastomic or homotransplastomic plant of the invention belonging to a previous generation with another compatible plant to give a transplastomic or homotransplastomic progeny plant of the invention belonging to a new generation; and optionally
  • transplastomic or homotransplastomic progeny plants of one or more further generations from the progeny plant thus obtained are obtained.
  • clonal propagation and sexual propagation may be used at different points in a process that gives rise to a transplastomic or homotransplastomic plant suitable for cultivation.
  • repetitive back-crossing with a plant taxon with agronomically desirable characteristics may be undertaken.
  • Further steps of removing cells from a plant and regenerating new plants therefrom may also be carried out.
  • further desirable characteristics may be introduced by transforming the cells, plant tissues, plants or seeds, at any suitable stage in the above process, to introduce desirable coding sequences other than the transforming construct of the invention.
  • This may be carried out by conventional breeding techniques, e.g. fertilizing a transplastomic or homotransplastomic plant of the invention with pollen from a plant with the desired additional characteristic.
  • the characteristic can be added by further transformation of the plant obtained by the method of the invention, using the techniques described herein for further plastomic transformation, or by nuclear transformation using techniques well known in the art such as electroporation of plant protoplasts, transformation by Agrobacterium tumefaciens or particle bombardment. Particle bombardment is particularly preferred for nuclear transformation of monocot cells.
  • different transgenes are linked to different selectable of scorable markers to allow selection for both the presence of further transgenes. Selection, regeneration and breeding techniques for nuclear transformed plants are known in the art. Techniques along the lines of those described may be used.
  • the invention also provides methods of obtaining crop products by harvesting, and optionally processing further, transplastomic or homotransplastomic cells, calli, plants or seeds of the invention.
  • crop product is meant any useful product obtainable from a crop plant.
  • Such a product may be obtainable directly by harvesting or indirectly, by harvesting and further processing.
  • Directly obtainable products include: grains, e.g. grains of monocotyledonous species, preferably graminaceous species, for example wheat, oats, rye, rice, maize, sorghum, friticale, especially wheat; other seeds; shoots, especially tubers, such as potato tubers; fruit; and other plant parts, for example as defined herein.
  • such a product may be obtainable indirectly, by harvesting and further processing.
  • products obtainable by further processing are: flour; oil; rubber; beverages such as juices and fermented and/or distilled alcoholic beverages; food products made from directly obtained or further processed material, e.g. bread made from flour or margarine made from oil; tobacco and tobacco products such as cigarettes and cigars; fibres, e.g. cotton, linen, flax and hemp fibres and textile items made therefrom; paper or timber derived from woody plants.
  • the fusion protein may be recovered by any method available in the art. Typically, the total protein content of the cell or organism is extracted by a technique known in the art to provide a crude protein sample.
  • a crude protem sample is enriched in the fusion protein of the invention, that is to say, the amount of fusion protein as a percentage of the total protein content of the enriched sample is substantially greater than that in the crude protein sample.
  • the sample will comprises at least at least 20%), preferably at least 50%), more preferably at least 80%, even more preferably at least 90%, most preferably at least 95% of the fusion protein of the invention.
  • Enrichment can be performed by any method known in the art. Usually the protein is initially fractionated. Fractionation can be performed using any method known in the art and typically separates proteins based on their physical properties, for example, their size, mass, hydrophobicity or hydrophilicity. Fractionation thus results in the separation of the total protein extract into a number of discreet fractions. The fraction (or fractions) containing the fusion protein can then be identified and further purification limited the fraction (or fractions) so identified. Identification can be performed by any method known in the art, although in a preferred embodiment, the fusion protein comprises a scorable property to allow rapid identification of the fraction.
  • the fusion protein comprises the sequence of GUS, and the fraction (or fractions) containing the fusion protein can thus be detected by histochemical assays and measured by fluorometric assays (Gallagher, 1992). Fractionation may be performed any number of times, and each fractionation may separate the proteins based on the same or different properties as the previous fractionation(s). Typically, fractionation is performed no more than 5 times, more typically no more than 3 times, most typically once. Following fractionation, the fusion protein can be further purified from the fraction (or fractions) identified as containing the fusion protein by any method known in the art.
  • Fusion proteins can be recovered from a crude protein sample or in a preferred embodiment from an enriched sample. Recovery as used herein means that the protein targeted for recovery is substantially separated from the other proteins. Typically following recovery the recovered sample will comprise, as a percentage of the total protein content of the recovered sample, at least 50%>, preferably at least 80%>, more preferably at least 90%>, yet more preferably at least 95%>, even more preferably at least 99%>, most preferably 100% of the fusion protein of the invention.
  • the fusion protein recovered by affinity based methods suitable to the sequence of the fusion protein and any purification tag present therein.
  • the fusion protein comprises a His-tag and affinity purification is performed using a Ni- NTA agarose column under suitable conditions.
  • affinity purification is performed using a Ni- NTA agarose column under suitable conditions.
  • affinity based methods are equally suitable, such as a self- cleaving chitin-based ligand system (Chong et al, 1997) a sequence capable of binding maltose (di Guan et al, 1988) a nickel-based ligand system (Houchuli et al, 1987) and a glutathione S-transferase based system (Smith & Johnson, 1988).
  • Fusion proteins can be processed in order to release the protein of interest, typically by cleavage of the fusion protein. Cleavage may be performed by any method known in the art, and the method chosen will be particular to the cleavage sequence (or sequences) present in the fusion protein.
  • the cleavage sequence is an Fa- Xa site, IEGR, and is cleaved by incubation with factor Xa (Nagai, 1985; Quinlan, 1989; Wearne 1990).
  • the resultant cleavage mixture comprises the protein of interest and polypeptide fragments representing the rest of the fusion protein, in particular the fusion protein partner.
  • the protein of interest can then be recovered by any method known in the art, for example either affinity based methods or fractionation.
  • the fusion protein partner comprises a purification tag.
  • the protein of interest may be recovered from the resultant cleavage mixture by applying the mixture to an affinity column that binds and retains the fusion protein partner due to its purification tag, and allows the elution of the protein of interest. The elute thus represents the recovered protein of interest.
  • the purity and identity of the recovered protein of interest can then be verified by any method known in the art, for example, by western blotting, by sequencing, or by assaying the putative properties, enzymatic or otherwise, of the polypeptide.
  • any of the proteins of the invention in any form or in association with any other agent discussed above is included in the term 'agent' below.
  • An effective non-toxic amount of such an agent may be given to a human or non-human patient in need thereof.
  • the condition of a patient suffering from a disease can therefore be improved by administration of such an agent.
  • the agent may be administered prophylactically to an individual who does not have a disease in order to prevent the individual developing the disease.
  • the invention provides the agent for use in a method of treating the human or animal body by therapy.
  • the invention provides the use of the agent in the manufacture of a medicament for treating the disease.
  • the invention provides a method of treating an individual comprising administering the agent to the individual.
  • the agent is typically administered by any standard technique used for administration, such as by injection.
  • the same or a different agent of the invention can be given.
  • the subject is given 1 , 2, 3 or more separate administrations, each of which is separated by at least 12 hours, 1 day, 2, days, 7 days, 14 days, 1 month or more.
  • the agent may be in the form of a pharmaceutical composition which comprises the agent and a pharmaceutically acceptable carrier or diluent.
  • Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline.
  • the composition is formulated for parenteral, intravenous, intramuscular, subcutaneous, transdermal, intradermal, oral, intranasal, intravaginal, or intrarectal administration.
  • a suitable dose of the agent may be determined according to various parameters, especially according to the substance used; the age, weight and condition of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient.
  • a suitable dose may however be from lO ⁇ g to lOg, for example from 100 ⁇ g to lg of the agent. These values may represent the total amount administered in the complete treatment regimen or may represent each separate administration in the regimen.
  • weights are given in grams (g), milligrams (mg) or micrograms ( ⁇ g), all temperatures are given in degrees centigrade (°C), concentrations are given as molar (M), millimolar (mM) or micromolar (mM), nanomolar ( ⁇ M), picomolar (pM) and volumes are given in litres (L), millilitres (ml), microlitres ( ⁇ l), unless otherwise indicated.
  • Example 1 Expression vectors for ifnG in chloroplasts
  • PCR Polymerase chain reaction
  • the plastid transformation vector, pVSR326 (Fig. 2A), was constructed using the rrn an ⁇ psbA promoters and 3' untranslated regions of psbA and rbcL genes from rice plastome primary clones (Hiratsuka et ⁇ l, 1988).
  • the selectable ⁇ dA and reporter uidA genes were cloned from pUC-atpX-AAD (Goldsclimidt-Clermont 1991) and pGUSN358-S (Clontech) plasmids, respectively.
  • the tobacco plastid genome sequences spanning rbcL- ⁇ ccD genes (Shinozaki et ⁇ l, 1986) were used for site specific integration of chimeric ⁇ dA and uidA genes into plastid DNA.
  • p326IFNG was a derivative of vector pVSR326.
  • the uidA was replaced with a multiple cloning site through the insertion of SR45 (SEQ ID NO: 45) and SR46 (SEQ ID NO: 46) primers that were complimentary to each other at the Bglll and Sad sites to create pVSRIFNGl .
  • (6x) His-tag was introduced using SR47 (SEQ ID NO: 47) and SR48 (SEQ ID NO: 48) primers that were partially complimentary to each other at Ncol and Apal sites to create pVSRIFNG2.
  • the ifnG coding region (Fig. 2C) was PCR amplified from an E.
  • coli expression vector pPLIFNG Wang et al. 1992
  • SR49 SEQ ID NO: 49
  • SR50 SEQ ID NO: 50
  • telomere sequence For the construction of pGUSIFNG, the uidA coding region, devoid of stop codon, was PCR amplified from pGUSN358 ⁇ S (Farrell and Beachy 1990) using SR51 (SEQ ID NO: 51) and SR52 (SEQ ID NO: 52) primers and cloned into vector pQE30 (Qiagen) at BamHI and Kpnl sites to create pQEGUS.
  • the ifnG coding region was PCR amplified from pPLIFNG using SR53 (SEQ ID NO: 53) and SR54 (SEQ ID NO: 54) primers and cloned into vector pQE31 (Qiagen) at the Pstl site to create pQE31IFNG.
  • the uidA along with T7 promoter and (6x) His-tag was released from pQEGUS as BamHI and Smal fragment and cloned into pQE31IFNG digested with Kpnl (end filled) and BamHI to create vector pQEGUSIFNG.
  • the uidA:ifnG fusion gene was PCR amplified from pQEGUSIFNG using SR47 and SR55 (SEQ ID NO: 55), digested with Ncol and cloned into Ncol and Sad (end filled) digested pVSR326 to create vector pGUSIFNG.
  • the vector pBIIFNG was created by cloning PCR amplified ifnG from pPLIFNG using SR56 (SEQ ID NO: 56) and SR57 (SEQ ID NO: 57) primers into vector pBI121 (Clonetech) at Xbal site.
  • the ifnG was transformed into tobacco nucleus or plastid genome to express it as an individual or as a fusion protein.
  • a binary vector pBIIFNG derived from pBI121 was used for the nuclear expression (Fig. 1 A).
  • the coding region of ifnG was transcriptionally fused to a reporter uidA gene (GUS) in pBI121 vector.
  • GUS reporter uidA gene
  • Both uidA and ifnG have their own translation initiation (ATG) and termination (TGA) codons and both the genes were under the transcription control of the same CaMV 35S promoter.
  • the p326IFNG was obtained from pVSR326 by replacing uidA with that of ifnG (Fig. 2B).
  • the complete nucleotide and the deduced amino acid sequences along with features incorporated to express and purify the IFN-g were presented in Fig. 2C.
  • a (6x) His-tag was added in frame at the N-terminal end of IFN-g to purify recombinant protein using an Ni-NTA column.
  • a protease site, IEGR, recognized by factor Xa was introduced in-between the His-tag and IFN-g to allow cleavage of the His-tag from the recombinant IFN-g after purification.
  • the ifnG was translationally fused at the C-tenninal end of uidA (Fig. 2D).
  • the (6x) His-tag was added at the N-terminal end of GUS:IFN-g and a factor Xa recognition site was introduced at the fusion junction (Fig. 2D).
  • the IFN-g released upon the cleavage of fusion protein by factor Xa will thus contain the same amino acid sequence as that of mature IFN-g produced in the human body.
  • transcripts from uidA, ifnG, uidAvemhG and aadA genes a possible mechanism for transgene integration into the tobacco plastome and the size of DNA fragments from restriction digestion with relevant enzymes is shown in Fig. 2A-D.
  • the Agrobacterium mediated transformation method was followed for nuclear transformation of tobacco with pBI121 and pBIIFNG binary vectors under kanamycin selection. Particle bombardment of leaf tissue was used for chloroplast transformation under spectinomycin selection using DNA of vectors pVSR326, p326IFNG and pGUSIFNG. Tobacco (Nicotiana tabacum cv. Petit Havana) was transformed using particle delivery system PDS1000 (BioRad) according to the method described by (Svab and Maliga 1993).
  • RMOP medium Svab and Maliga 1993
  • a modified MS medium containing 0.1 mg/1 thiamine, 100 mg/1 inositol, 3% sucrose, 1 mg/1 BA and 0.1 mg/1 NAA, 0.6% agar, pH 5.8.
  • Transformed shoots were selected on RMOP medium containing 500 mg/1 spectinomycin dihydrochloride.
  • Three additional cycles of regeneration on spectinomycin (500 mg/1) containing RMOP medium was carried out to obtain homotransplastomic plastid containing plants (Svab and Maliga 1993).
  • the Agrobacterium strain LB A 4404 containing vector pBIIFNG/pBI121 was used for nuclear transformation following a leaf disc method (Horsch 1985).
  • the selectable aadA is expected to express and confer resistance to spectinomycin only when it entered the chloroplasts due to the specificity of the rrn promoter.
  • homotransplastomic lines were established by repeating regeneration process three times from the leaf tissues of primary transformants under spectinomycin selection. Unless and otherwise mentioned, Nt. BI121-1, Nt. BIIFNG- 1/2, Nt. VSR326-37, Nt. 326IFNG-1/2 and Nt.
  • the membranes were UV crosslinked and then probed with 32p labeled psbA (SEQ ID NO: 39), 16S rRNA (SEQ ID NO: 32), uidA (SEQ ID NO: 33), aadA (SEQ ID NO: 34) and targeting sequence (SEQ ID NO: 35) amplified using SRI 7 (SEQ ID NO: 17) - SRI 8 (SEQ ID NO: 18), SR19 (SEQ ID NO:19) - SR20 (SEQ ID NO: 20), SR41 (SEQ ID NO: 41) - SR42 (SEQ ID NO: 42), SR43 (SEQ ID NO: 43) - SR44 (SEQ ID NO: 44) AND SR12 (SEQ ID NO: 12) - SR13 (SEQ ID NO: 13) primer pairs respectively. Standard procedures were followed for hybridization (Sambrook et al, 1989) and membranes were subjected to autoradiography.
  • FIG. 3B the size of the fragments that hybridized to the aadA and ifnG in Nt. 326IFNG-1 and Nt. 326IFNG-2 plants were in agreement with the predicted size DNA fragments when transgenes are integrated into the plastid genome site- specifically.
  • Southern hybridization confirmed the stable integration of uidA ⁇ fnG into Nt. GUSIFNG-1 plastome (Fig. 3C).
  • Example 4 Transcription of chimeric uidA, ifnG and uidA: ifnG genes
  • Hybridization using uidA probe confirmed transcription of uidA and uidA fnG inNt. 326-37 andNt. GUS:IFNG-1 plants, respectively (Fig. 3E and F).
  • the uidA transcript levels in Nt. 326-37 were comparable with the uidA: ifnG transcript levels in Nt. GUS:IFNG-1, indicating that the fusion of ifnG to uidA had no adverse effect on fusion gene transcription.
  • Reprobing the same blot with ifnG reconfirmed the presence of 2.3 kb fusion transcript in the Nt. GUSIFNG-1 (Fig. 3F, lanes 1 and 2).
  • the RNA sample from Nt. VSR326-37 (Fig.
  • total protein was labeled by incubating the leaf discs in 10 ml of MS medium containing 3.5 mCi labeled amino acid mix (S-35 Express, NEN/DuPont) and incubated at 25°C under 4,000 lux light. After one hour, leaf discs were thoroughly washed with MS medium and continued d e incubation for 96 hours. At various defined intervals, leaf discs were quickly frozen in liquid nitrogen and protein extracted, immunoprecipitated (Pineiro et al, 1999) with anti-His antibodies (Qiagen), separated on SDS-PAGE, transferred onto nitrocellulose membrane and subjected to autoradiography. The signal intensity was quantified using ID Image analysis software (Kodak).
  • the estimated levels of IFN-g and GUS was found to be 0.01% and 3% of the total cellular protein in the leaf extracts of Nt. 326IFNG-1 and Nt. 326-37 plants, respectively.
  • the leaf discs from Nt. VSR326-37 and Nt. 326IFNG-1 plants were pulse labeled with S-35 (Methionine and Cysteine) to analyze the half life of recombinant IFN-g and GUS proteins in the chloroplasts.
  • S-35 Methionine and Cysteine
  • GUS:IFN-g-l plant in buffer A 50 mM Tris-HCl pH 7.0, 5 mM DTT, 1 mM Na2EDTA, 0.1% SDS, 0.1% Triton X-100, one protease inhibitor cocktail tablet per each 50 ml of buffer
  • buffer A 50 mM Tris-HCl pH 7.0, 5 mM DTT, 1 mM Na2EDTA, 0.1% SDS, 0.1% Triton X-100, one protease inhibitor cocktail tablet per each 50 ml of buffer
  • the column was washed with 5 vol buffer B (buffer A, 50 mM NaCl) and the bound proteins were eluted with 50-500 mM NaCl gradient in buffer A.
  • the GUS positive fractions, eluted between 100-200 mM NaCl were pooled and directly loaded on to a 15 ml Ni-NTA agarose column.
  • the column was washed with 4 vol of buffer C (100 mM potassium phosphate buffer pH 8, 20 mM imidazole) and eluted with a 20-250 mM imidazole gradient in buffer C.
  • the fractions with peak GUS activity were dialyzed against buffer D and fusion protein was cleaved (in 4 mg batches) by the incubation with factor Xa.
  • the biotinilated factor Xa cleavage and removal kit (Boeringer Mannheim) was used to separate the IFN-g from the fusion protein as per the supplier instructions.
  • the human lung carcinoma cells precultured for 24 h in the presence or absence of rh-IFN-g were challenged with 104 PFU of encephalomyocarditis (EMC) virus.
  • EMC encephalomyocarditis
  • One unit of antiviral activity was defined as the amount of rh-IFN-g required to produce equivalent antiviral activity expressed by 1 U of the (NIH IFN-g reference standard (Gg 23-901-530)).
  • One unit is defined as the amount of activity required to release one ⁇ mole of MU from MUG in one minute at 37°C.
  • the crude extract was loaded on to DE-52 column and the bound proteins were eluted with 0-1.0 M salt gradient. These fractions were tested for the presence of GUS:IFN-g fusion protein by a GUS assay.
  • the colorless X-GlcU substrate containing solution turned to blue color in less than 5 min when the fractions that contained highest amounts of GUS:IFN-g fusion protein were assayed.
  • the activity of GUS was detected by histochemical assay and measured by fluoromefric assay (Gallagher 1992).
  • the fractions containing GUS:IFN-g fusion protein were identified using a histochemical X-GlcU substrate.
  • the colorless X-GlcU substrate produced a visually detectable blue indigo dye upon the addition of a small aliquot of fractions that contained GUS:IFN-g protein when incubated at 37°C. Protein concentration was determined with the Bradford reagent (BioRad) using BSA as standard.
  • IFN-g The antiviral activity of recombinant IFN-g was assayed following the standard procedures (Lewis 1988) using human lung carcinoma (A 549) cells and encephalomyocarditis (EMC) viras.
  • EMC encephalomyocarditis
  • the expression levels of IFN-g and GUS were quantified by comparing with E. coli derived IFN-g (Boeringer Mannheim) and GUS protein (Sigma) standards, respectively, using ELISA method.
  • the GUS-positive factions were loaded onto an Ni-NTA column directly and the bound protein was eluted with 0-250 mM imidazole gradient. All the fractions were subjected to GUS assay and analyzed on SDS-PAGE (Fig. 4D). Using this procedure, we were able to obtain about 18 mg of fusion protein (75% recovery) starting from 50 grams of fresh leaf material.
  • the IFN-g was separated from the GUS using a biotinilated factor Xa cleavage and purification procedure. After the cleavage, the biotinilated factor Xa was removed using strepatavidin beads and the protein was passed through a second Ni- NTA column to remove the His-tag containing GUS protein.
  • the IFN-g present in the flow through from the second Ni-NTA column was purified on an S-sepharose column with an estimated recovery of 70% protein.
  • the recombinant IFN-g was found to be highly pure, as judged by commassie blue staining (data not shown), and cross reacted with anti-IFN-g antibodies (Fig. 4E, lane 2).
  • a 24 h pretreatment of human lung carcinomas (A 549) with 25 pg of the purified IFN-g offered complete protection against the infection of by EMC virus (Fig. 4F), whereas the untreated cells were completely infected (Fig. 4G).
  • IFN- ⁇ The expression of IFN- ⁇ was very low (0.001%) in the nuclear transformed plants despite of the fact that the ifnG was placed under the regulation of a strong and constitutive CaMV 35S promoter. Low expression of foreign genes is not uncommon in transgenic plants (Goddijin and Pen 1995). Although it is difficult to compare the expression levels of various proteins expressed in the nuclear transformed plants due to variations in the promoters used, copy number of integrated gene, site of integration and methods followed for protein extraction, the expression levels are generally low, especially, when compared to microbial expression systems (Goddijin and Pen 1995).
  • the ifnG was cloned into plastid transformation vector p326IFNG and transformed into tobacco chloroplasts. Although there was 100 fold increase in the IFN- ⁇ expression levels in the plastid transformed Nt. 326IFNG-1 plant when compared to nuclear transformed Nt. BIIFNG-1 plant, these expression levels are 200-300 fold low when compared to GUS expressed under the same psbA promoter and integrated into the plastid genome at the same site. One of the reasons for such low levels of IFN- ⁇ expression could be due to the lack of efficient transcription/mRNA stability and/or fast degradation of the recombinant protein.
  • the Northern hybridization analysis revealed efficient transcription of ifnG in Nt.
  • 326IFNG- 1 plant at a level that is comparable with that of uidA transcription in Nt. 326-37 plant expressed under the same promoter, ruling out the possibility of low levels of transcription or mRNA stability as a cause for low ifnG expression.
  • the pulse labeling experiments have shown that the IFN- ⁇ has relatively a short half life (4-6 hours) in chloroplasts as compared to 48 hours for GUS suggesting that the rapid degradation of IFN- ⁇ was the reason for such a low accumulation.
  • the ifnG was translationally fused to high expressing uidA in chloroplasts and integrated again into tobacco chloroplast genome.
  • the Nt. GUSIFNG-1 plants about 5% of the total cellular protein were found to be of GUS:IFN- ⁇ fusion protein.
  • the 5.0% expression levels achieved through GUS-fusion strategy are exceptionally significant. Therefore, GUS fusion offers an attractive way to increase the low expressing proteins/peptides in transgenic plants.
  • GUS fusion in transgenic tobacco plants.
  • One key advantage of GUS fusion system is its ability to accept N- and C-terminal fusions without any loss of its activity (Jefferson et al, 1989). This ability of GUS has been widely exploited for detecting sub-cellularly targeted proteins and in tracking the virus movement in plants. In the present study, we have exploited this property during the protein purification to identify fractions containing GUS:IFN- ⁇ fusion protein very rapidly using a simple and inexpensive GUS assay (Gallagher 1992).
  • GUS fusion system combined with chloroplast transformation described here offers key advantages in increasing the yields of poorly accumulating proteins and reduce purification time considerably. It should be mentioned here that, so far, GUS is the safest and most commonly used reporter in transgenic plants (Gilissen et ⁇ Z, 1998).
  • GUSIFNG-1 plant leaf extracts in a single step using Ni-NTA column was futile and yielded only 10% of the total estimated protein. This might be due to the presence of a vast pool of free histidine and other substances in the crude extract that might be competing directly for Ni-NTA binding.
  • the GUS:IFN- ⁇ fusion protein was purified to near homogeneity by a two column purification process involving DE 52 followed by Ni-NTA column. Biotinilated factor Xa cleavage and removal procedure was adapted to separate IFN- ⁇ from the GUS fusion partner and the cleaved-IFN- ⁇ was purified further by S-sepharose column.
  • the purified recombinant human IFN- ⁇ offered complete protection against the infection of human lung carcinomas (A 549) by EMC virus suggesting that IFN- ⁇ expressed in chloroplasts folded correctly and retained it's biological activity at a level that is comparable to E. coli derived r-IFN- ⁇ .
  • His-tag In the present study, as a test case, we have used His-tag.
  • Plant derived proteins with high level of purity may be more readily acceptable than the similar products obtained from bacteria and transgenic animals due to possible contamination by human pathogens (Miele 1997).
  • There are a number of small proteins/peptides that are highly useful in the pharmaceutical industry if made available in bulk and at a low-cost (Ellis 1996; Goddijin and Pen 1995; Krebbers and Vandekerckhove 1990; Rudolph 1999). Initially, it may be easy to couple such production systems with the existing and well organized floriculture industry (Miele 1997). Floriculture, a multi-billion dollar industry spreading all over the world, utilizes weather controlled greenhouses for the production of quality cut flowers.
  • the greenhouse grown plants being relatively free from pests and pathogens when compared to the open field cultivated plants may offer certain advantages and qualify better in case of stringent quality control tests imposed by various national/international health agencies/organizations (Miele 1997).
  • floriculture only a fraction of the plant biomass (flowers) is harvested and the majority of biomass consisting of mostly leaf material go waste.
  • the extension of chloroplast transformation in conjunction with fusion protein and affinity based purification strategies to floricultural crops can result in the addition of substantial value to the crop without any additional inputs and convert the so far unutilized plant biomass as a raw material for new industrial applications benefiting both farmer and industry enormous while providing better health for centuries.

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Abstract

L'invention porte sur un procédé de production d'une protéine d'intérêt consistant à faire exprimer un polynucléotide produit d'assemblage de fusion dans un plastide de manière à produire une protéine de fusion consistant dans la protéine d'intérêt. Le produit d'assemblage de fusion de la protéine d'intérêt comporte une séquence de codage de polynucléotide de la protéine d'intérêt fonctionnellement liée à la séquence de codage de polynucléotide d'une protéine de fusion partenaire. La protéine de fusion ainsi exprimée: a une demi-vie supérieure à celle d'une protéine d'intérêt exprimée individuellement, est résistante aux clivages internes in vivo, et comporte un site de clivage.
PCT/EP2001/008132 2000-07-14 2001-07-13 Plantes transplastomiques WO2002006497A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1682664A2 (fr) * 2003-11-14 2006-07-26 SemBioSys Genetics Inc. Procedes pour la production d'apolipoproteines dans des plantes transgeniques
WO2010018251A1 (fr) * 2008-07-31 2010-02-18 Fundación Para El Desarrollo De La Investigación En Genómica Y Proteómica Méthode pour améliorer l'expression des protéines dans des chloroplastes
EP2215247A1 (fr) * 2007-11-13 2010-08-11 The Scripps Research Institute Production de fusion anticorps-toxine cytotoxique dans une algue eucaryotique
US20210330780A1 (en) * 2018-11-15 2021-10-28 Bioapplications Inc. Recombinant vector for expressing virus-like particles in plant and method for preparation of vaccine composition containing circovirus-like particles by using same

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003887A1 (fr) * 1987-10-20 1989-05-05 Plant Genetic Systems N.V. Procede de production de peptides biologiquement actifs par l'expression de genes de proteines de graines de stockage modifies, dans des plantes transgeniques
WO1993021320A1 (fr) * 1991-02-22 1993-10-28 University Technologies International, Inc. Proteines de corps huileux utilisees en tant que vecteurs de peptides de grande valeur dans des plantes
EP0573767A1 (fr) * 1992-04-28 1993-12-15 Nihon Nohyaku Co., Ltd. Procédé de préparation d'un gène étranger ou son produit dans les cellules de plante
WO1998021348A1 (fr) * 1996-11-12 1998-05-22 Battelle Memorial Institute Procede de production facteurs de croissance humain a partir de plantes entieres ou de cultures de cellules vegetales
US5877402A (en) * 1990-05-01 1999-03-02 Rutgers, The State University Of New Jersey DNA constructs and methods for stably transforming plastids of multicellular plants and expressing recombinant proteins therein
WO1999010513A1 (fr) * 1997-08-07 1999-03-04 Auburn University Vecteurs universels d'integration et d'expression de chloroplastes, plantes transformees et produits obtenus
US5939288A (en) * 1995-06-07 1999-08-17 Iowa State University Research Foundation, Inc. Plant secretory signal peptides and nectarins
WO1999066026A2 (fr) * 1998-06-15 1999-12-23 John Innes Centre Procedes et moyens d'expression de polypeptides de mammiferes dans des plantes monocotyledones
WO1999067401A2 (fr) * 1998-06-22 1999-12-29 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Bioreacteur de plantes cultivees non alimentaires
WO2000003012A2 (fr) * 1998-07-10 2000-01-20 Calgene Llc Expression de peptides eukaryotes dans les plastes de vegetaux
WO2000007431A1 (fr) * 1998-08-03 2000-02-17 Rutgers, The State University Of New Jersey Elements de regulation de traduction pour expression de proteine de niveau eleve dans les plastes de plantes superieures et leurs procedes d'utilisation
WO2000071701A1 (fr) * 1999-05-24 2000-11-30 New England Biolabs, Inc. Procede de generation de genes non transferables separes capables d'exprimer un produit proteique actif
WO2001042441A2 (fr) * 1999-12-08 2001-06-14 International Centre For Genetic Engineering And Biotechnology Transformation de plaste

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06125782A (ja) * 1992-10-15 1994-05-10 Toagosei Chem Ind Co Ltd 外来ペプチド発現方法および形質転換植物
US6271444B1 (en) * 1998-07-10 2001-08-07 Calgene Llc Enhancer elements for increased translation in plant plastids
CN1330718A (zh) * 1998-10-07 2002-01-09 辛根塔参与股份公司 植物中的治疗活性蛋白质
US6515206B1 (en) * 1998-12-23 2003-02-04 Calgene Llc Plastid transformation of Brassica

Patent Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003887A1 (fr) * 1987-10-20 1989-05-05 Plant Genetic Systems N.V. Procede de production de peptides biologiquement actifs par l'expression de genes de proteines de graines de stockage modifies, dans des plantes transgeniques
US5877402A (en) * 1990-05-01 1999-03-02 Rutgers, The State University Of New Jersey DNA constructs and methods for stably transforming plastids of multicellular plants and expressing recombinant proteins therein
WO1993021320A1 (fr) * 1991-02-22 1993-10-28 University Technologies International, Inc. Proteines de corps huileux utilisees en tant que vecteurs de peptides de grande valeur dans des plantes
EP0573767A1 (fr) * 1992-04-28 1993-12-15 Nihon Nohyaku Co., Ltd. Procédé de préparation d'un gène étranger ou son produit dans les cellules de plante
US5939288A (en) * 1995-06-07 1999-08-17 Iowa State University Research Foundation, Inc. Plant secretory signal peptides and nectarins
WO1998021348A1 (fr) * 1996-11-12 1998-05-22 Battelle Memorial Institute Procede de production facteurs de croissance humain a partir de plantes entieres ou de cultures de cellules vegetales
WO1999010513A1 (fr) * 1997-08-07 1999-03-04 Auburn University Vecteurs universels d'integration et d'expression de chloroplastes, plantes transformees et produits obtenus
WO1999066026A2 (fr) * 1998-06-15 1999-12-23 John Innes Centre Procedes et moyens d'expression de polypeptides de mammiferes dans des plantes monocotyledones
WO1999067401A2 (fr) * 1998-06-22 1999-12-29 Her Majesty The Queen In Right Of Canada As Represented By The Minister Of Agriculture And Agri-Food Canada Bioreacteur de plantes cultivees non alimentaires
WO2000003012A2 (fr) * 1998-07-10 2000-01-20 Calgene Llc Expression de peptides eukaryotes dans les plastes de vegetaux
WO2000007431A1 (fr) * 1998-08-03 2000-02-17 Rutgers, The State University Of New Jersey Elements de regulation de traduction pour expression de proteine de niveau eleve dans les plastes de plantes superieures et leurs procedes d'utilisation
WO2000071701A1 (fr) * 1999-05-24 2000-11-30 New England Biolabs, Inc. Procede de generation de genes non transferables separes capables d'exprimer un produit proteique actif
WO2001042441A2 (fr) * 1999-12-08 2001-06-14 International Centre For Genetic Engineering And Biotechnology Transformation de plaste

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE BIOSIS [Online] BIOSCIENCES INFORMATION SERVICE, PHILADELPHIA, PA, US; January 1998 (1998-01) OKAMOTO MASAJI ET AL: "Enhanced expression of an antimicrobial peptide sarcotoxin IA by GUS fusion in transgenic tobacco plants." Database accession no. PREV199800126718 XP002193857 & PLANT AND CELL PHYSIOLOGY, vol. 39, no. 1, January 1998 (1998-01), pages 57-63, ISSN: 0032-0781 *
DATABASE WPI Section Ch, Week 199423 Derwent Publications Ltd., London, GB; Class C06, AN 1994-187941 XP002193858 & JP 06 125782 A (TOA GOSEI CHEM IND LTD), 10 May 1994 (1994-05-10) *
FLACHMANN RALF ET AL: "Crystallization and identification of an assembly defect of recombinant antenna complexes produced in transgenic tobacco plants." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES, vol. 93, no. 25, 1996, pages 14966-14971, XP002193856 1996 ISSN: 0027-8424 *
ROSE ALAN B ET AL: "Introns act post-transcriptionally to increase expression of the Arabidopsis thaliana tryptophan pathway gene PAT1." PLANT JOURNAL, vol. 11, no. 3, 1997, pages 455-464, XP000891346 ISSN: 0960-7412 *
STAUB J M ET AL: "HIGH-YIELD PRODUCTION OF A HUMAN THERAPEUTIC PROTEIN IN TOBACCO CHLOROPLASTS" NATURE BIOTECHNOLOGY, NATURE PUBLISHING, US, vol. 18, no. 3, 2000, pages 333-338, XP000887246 ISSN: 1087-0156 *

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1682664A2 (fr) * 2003-11-14 2006-07-26 SemBioSys Genetics Inc. Procedes pour la production d'apolipoproteines dans des plantes transgeniques
EP1682664A4 (fr) * 2003-11-14 2008-04-02 Sembiosys Genetics Inc Procedes pour la production d'apolipoproteines dans des plantes transgeniques
AU2004289720B2 (en) * 2003-11-14 2011-02-03 Sembiosys Genetics Inc. Methods for the production of apolipoproteins in transgenic plants
EP2215247A1 (fr) * 2007-11-13 2010-08-11 The Scripps Research Institute Production de fusion anticorps-toxine cytotoxique dans une algue eucaryotique
EP2215247A4 (fr) * 2007-11-13 2010-11-10 Scripps Research Inst Production de fusion anticorps-toxine cytotoxique dans une algue eucaryotique
AU2008321026B2 (en) * 2007-11-13 2014-01-16 The Scripps Research Institute Production of cytotoxic antibody-toxin fusion in eukaryotic algae
WO2010018251A1 (fr) * 2008-07-31 2010-02-18 Fundación Para El Desarrollo De La Investigación En Genómica Y Proteómica Méthode pour améliorer l'expression des protéines dans des chloroplastes
ES2336754A1 (es) * 2008-07-31 2010-04-15 Plant Bioproducts, S.L Metodo para mejorar la expresion de proteinas en cloroplastos.
US20210330780A1 (en) * 2018-11-15 2021-10-28 Bioapplications Inc. Recombinant vector for expressing virus-like particles in plant and method for preparation of vaccine composition containing circovirus-like particles by using same

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