GB2364705A - Fusion protein expression in plastids - Google Patents

Abstract

A method of producing a protein of interest by expression of a polynucleotide encoding a fusion protein in a plastid. The protein of interest is linked to a fusion protein partner wherein the fusion protein has a greater half-life than the individually expressed protein of interest, is resistant to internal cleavage in vivo and comprises a cleavage site, preferably IEGR. Methods of protein purification, plastid transformation to create transplastomic plastids and plant propagation are also claimed. Also claimed is the use of a polynucleotide encoding a fusion partner to increase the stability of a recombinantly expressed protein of interest in a plastid.

Description

2364705 TRANSPLASTOMIC PLANTS

FIELD OF THE INVENTION

5 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 farther generations, and to the isolation of the thus expressed protein.

10 BACKGROUND OF THE INVENTION

Plastids Plastids are organelles found in plant cells and the cells of photosynthetic algae such as 15 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 5 0 to 100. Chloroplasts have their own genome, the plastome, which 20 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 0 to 100. A plastid genome, referred to as a plastome, comprises a double stranded circular DNA molecule.

25 Nuclear transformation In the field of biotechnology, the ability to express a foreign gene, referred to as transgene expression, in the organism of choice, is desirable. Typically, transgene expression in plants is achieved by the integration of a transgene construct into nuclear

30 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.

I 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 5 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 10 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. There is currently considerable concern about this, especially over the possible transmission of herbicide/insecticide/ 15 disease resistance traits from transgenic crops (typically cereals) to weedy relatives growing around the crop fields, leading to the possibility of resistant weeds (so-called 'super-weeds') which are hard to eliminate because of their resistant traits (Daniell, 1999).

20 Chloroplast transformation Many of the disadvantages of nuclear transformation can be avoided by targeting transgene integration to the plastome. A transfonned plastome is referred to as a transplastome. Due to the existence of multiple plastome copies within each chloroplast, 25 the copy number of an integrated transgene is high. This leads to a level of expression of a transplastornic gene that is typically higher than for an 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 30 zygote. Pollen derived from transplastomic, plants does not, therefore, contain the transgene and so transgene transmission to other species is not possible. This is 2 particularly beneficial in view of public fears related to the spread of transgenes and their potential impact on the ecosystem.

Foreign DNA has previously been introduced into chloroplasts using a biolistic. method 5 (Boynton et al, 198 8; 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) and a PEG- based procedure (Golds et al, 1993). Typically, the transgene in a chloroplast transformation vector is flanked by DNA regions homologous to regions of the plastome. These flanking regions enable the site-specific integration of the transgene construct into 10 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 20 terminal to the C-terminal of its fusion protein partner. In the case of ubiquitin fusions, 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-ten-nin-al amino acid of the protein of interest was not predictable, In other cases (e.g. GUS fusion) the fusion protein is stable. Staub et al, (2000) and WO 00/03017 teach that fusion proteins 25 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.

Although transplastomic plants typically express transgenes at higher levels than 30 nuclear-transformed plants, recovery of commercially viable amounts of protein can be problematic. For a commercially viable biofarming, the recombinant protein, if needed, 3 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 5 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).

Identification of recombinant protein-containing fractions by conventional methods such as SDS-polyacrylamide gel electrophoresis (SDS- PAGE), enzyme linked immunosorbant assay (ELISA) and Western blot analysis is time consuming and laborious. If the recombinant protein can be detected by simple and rapid tests, then 15 purification may be achieved much faster. This is an important consideration for highly labile proteins.

SUMMARY OF THE INVENTION

20 Against this background 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-,y) was very low (0.00 1% of the total cellular protein) in nuclear transformed plants, whereas a 100 fold increase in IFN-7 expression levels were observed when expressed in the plastid. The inventors compared the plastidic

25 expression of IFN-y with GUS and found that GUS expression gave levels of accumulation up to 3% of the total cellular protein. By comparing the protein turnover rate of IFN-y and GUS, the inventors have demonstrated that the reason that IFN-y accumulates to a lower level than GUS in the chloroplast is because IFN-y has relatively a short half life (4-6 hours) in chloroplasts as compared to a half-life of 48 hours for 30 GUS. This information provides strategies to enhance the accumulation of labile proteins expressed in plastids by decreasing the rate of protein turnover.

4 The inventors demonstrated that accumulation of IFN--y in chloroplasts could be increased by translationally fusing ifnG (the polynucleotide coding sequence of IFN-y) to uidA (the polynucleotide coding sequence of GUS). They have shown that an GUS:IFN-y fusion protein accumulates to about 5% of the total cellular protein, This 5 is an increase of around 500 fold over the accumulation of IFN-y alone in the chloroplast and of 5,000 fold over the accumulation of nuclear expressed IFN-y alone.

Furthermore, the inventors have exploited the properties of GUS to identify fractions containing GUS:IFN-y fusion protein very rapidly during protein purification. This is 10 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-y fusion protein reduces the number of steps involved in purification. By engineering the protein to comprise a cleavage site sensitive to cleavage methods such as biotinilated factor Xa, the inventors 15 have also shown that IFN-,y can be separated from the GUS fusion partner and retain its biological activity at a level comparable to E. coli derived r-IFN-7 and thus transplastornically expressed fusion proteins can be cleaved with high fidelity.

Accordingly the invention provides a method of producing a protein of interest 20 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; (b) is resistant to internal cleavage in vivo; and (c) comprises a cleavage site.

Usually, 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. Preferably, a method of the invention further comprises 5 cleaving the fusion protein in vitro to release the protein of interest. Optionally, 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.

10 The invention also provides:

- a method of obtaining a transplastomic plastid, which method comprises transforming a plastome within a plastid by a fusion construct of the invention; 15 - 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 20 presence of the transplastome; - a method of obtaining a first-generation transplastornic or homotransplastomic plant, wherein the method comprises regenerating a transplastomic or homotransplastomic plant cell obtainable by the method of the invention to give a 25 transplastomic or homotransplastomic plant; - a method of obtaining a transplastomic or homotransplastomic plant seed, wherein the method comprises obtaining a transplastornic or homotransplastomic seed from a transplastomic or homotransplastomic plant obtainable by the method of the 30 invention; 6 - a method of obtaining a transplastomic or homotransplastomic progeny plant comprising obtaining a second-generation transplastornic or hornotransplastomic progeny plant from a first-generation transplastomic or homotransplastomic plant obtainable by a method of the invention, and optionally obtaining transplastomic or 5 homotransplastomic plants of one or more further generations from the second-generation progeny plant thus obtained; - the use of a polymicleotide encoding a fusion protein partner to increase the stability of a recombinantly expressed protein of interest in a plastid; 10 - the use of a polynucleotide fusion construct of the invention to increase the stability of a recombinantly expressed protein of interest in a plastid; a transplastome, a transplastomic or homotransplastomic plastid, transplastomic or 15 homotransplastomic plant cell, transplastornic or homotransplastomic callus, a transplastomic or homotransplastomic first-generation plant, transplastornic or homotransplastomic plant see or progeny plant obtainable by a method of the invention or comprising a polynucleotide fusion construct of the invention; 20 - a fusion protein or protein of interest obtained by a method of the invention or from a cell or plant of the invention.

- a method of obtaining a crop product comprising harvesting a crop product from a cell or plant of the invention and optionally further processing the harvested product; 25 and - a crop product obtainable by a method of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1: Transformation and expression of i fnG in the tobacco nuclear genome. A). Map 7 of the vectors p131121 and pBIIFNG. Double head arrows indicate the size of DNA fragments after the restriction digestion with Pstl (P), Xbal (X) and EcoRI (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:

5 CaMV 35S promoter, NPTII: neomycin phosphotransferase 11 that confers resistance to kanamycin, uidA: P-glucuronidase (GUS) reporter gene. B). Southern hybridization of genomic DNA isolated from wild type (1), Nt. 121-1 (2), Nt. BlIFNG-l (3), Nt.

BlIFNG-2 (4) plants was digested with Pstl + EcoRl (P+E), Xbal (X) and probed with ifnG and uidA radiolabeled probes. Q. RT-PCR analysis of RNA from wild type (1 &3) 10 and Nt. BIIFNG-1 (2 & 4) plants using ifnG-uidA gene specific primers (I & 2) and ifnG gene specific primers (3 & 4).

Fig. 2: Restriction map of transformation vectors used to express IFN-g in tobacco chloroplasts and the ifnG gene. A). Vector pVSR326 contained aadA selectable marker 15 that confers resistance to spectinomycin under the control of rice rrn promoter (rrnP) and uicL4 reporter gene under the regulation of rice psbA promoter (psbAP) . Double head arrow indicate the size of the DNA fragment expected when digested with Clal (Q. The chimeric uid,4 and aadA genes were flanked by tobacco rbcL and accD gene sequences for site-specific integration into tobacco plastid genome. Dashed arrow indicates the 20 direction and size of the uicL4 transcript. B), Restriction map of vector p3)261FNG, partial chloroplast DNA of tobacco (cpDNA) and the transformed tobacco plant (Nt. 3261FNG 1) plastid DNA. Double head arrows indicate the size of DNA fragments after the restriction digestion with ApaI (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 25 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. Q. Nucleic acid and deduced amino acid sequence of ifnG used to express recombinant IFN-g in tobacco chloroplasts. Sequences coding for His-tag, factor Xa and various restriction enzymes are also indicated. D). Restriction map of vector 30 pGUSIFNG, partial chloroplast DNA of tobacco (cpDNA) and the transformed tobacco plant (Nt. GUSIFNG-I) plastid DNA. Double head arrows indicate the size of DNA 8 fragments after the restriction digestion with Xhol (Xh), Clal (C), BamHI (B), XbaI (X) alone or in combination. Position of the His-tag (6x (His) and factor Xa site (Fa-Xa) are indicated. Dashed arrows indicate the direction and size of the uidk- ffinG and aadA transcripts. A possible mechanism for site-specific integration of Oad,4 and uidA:ifnG 5 through two homologous recombinations (crossed lines) were also shown.

Fig. 3: Southern and Northern blot analysis to confirm the stable integration and expression of aadA, ifnG, uidA and uidA:ifnG genes in tobacco chloroplasts. A).

Genomic DNA isolated from wild type (1) and six independently transformed (2-7) 10 plants was digested with Clal and hybridized with rbcl-accD targeting DNA probe. B).

Genomic DNA isolated from Nt. _3261FNG-1 (1), Nt. 3261FNG-2 (2) and wild type (3) plants was digested with Clal (C), ApaI + XbaI (A+X), Apal (A) enzymes and probed with aadA and ifnG gene probes. Q. Genomic DNA isolated from Nt. GUSIFNG (1) and wild type (2) plants was digested with ClaI (C), Xhol (Xh), BamHI (B), Ncol Xbal 15 (N+X) and probed with aad,4, ifnG, uid,4 and rbcL-accD gene sequences. D). Total RNA isolated from wild type (1), Nt. 3261FNG- 1 (2), Nt. 3 261FNG-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.

20 GUSIFNG-2 (2), Nt. 326-37 (3) and wild type (4) plants were hybridized with uid,4 (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 25 Nt. 3 261FNG- 1 (2) plants were probed with anti-His and anti-IFN-g antibodies. Proteins were partially purified on Ni-NTA column and 10 tg of protein was loaded in each lane.

Arrow indicates the presence of an expected size protein. B). Western blot analysis of total soluble proteins from Nt. GUSIFNG- 1 (1), Nt. 326-27 (2) and wild type (3) plants were probed with anti-GUS antibodies, About 10 [tg protein from crude extracts was 30 loaded in each lane. Arrows indicate the presence of expected size GUS and GUS:IFN-g fusion proteins. Q.Estimation of half-life for GUS and IFN-g proteins expressed in 9 tobacco chloroplasts. From a defined intervals, pulse labeled proteins were in-imunoprecipitated with anti-GUS or anti-IFN-g, separated on SDSPAGE and exposed to autoradiography. The intensity of the signal was quantified and data plotted as graph. Arrows indicate half-life of both the proteins. D). The SDS-PAGE analysis of Ni-NTA 5 fractions showing highly purified single band of 85 kDa protein corresponding to the size of GUS:IFN-g fusion protein, as judged by commassie blue stain. Activity of the GUS is correlated with the amount of the protein present in the fractions. E). Western blot showing the cross reactivity of Ni-NTA purified GUS:IFN-g fusion protein (1) and recombinant IFN-g separated from the GUS fusion partner and purified further using - 10 Sepharose column (2) with anti-IFN-g antibodies. F). Antiviral activity of the purified recombinant IFN-g. The human lung carcinoma cells precultured for 24 h in the presence (top) or absence (bottom) of rh-IFN-g were challenged with 10' PFU of encephalornyocarditis (EMQ virus. A 24 h pretreatment of rh-IFN-g offered complete protection whereas the untreated cell were infected and disintegrated.

DETAILED DESCRIPTION OF THE INVENTION

Fusion Proteins 20 Methods and uses of the invention provide for the increased accumulation of a protein of interest by expression of a 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. Typically, at least one of the sequences represents the sequence 30 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 ftision protein may contain the sequences of two, three or more polypeptides of interest, which may be the same or different, Typically 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 5 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. In this context 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 10 interest. Preferably, 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.

Therefore, the fusion protein accumulates to higher levels than the individual protein of 15 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 20 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 10046 Id, and in an even more preferred embodiment the increase is up to 500-fold. Most preferably the increase is up to 1000-fold.

25 Fusion proteins of the invention are typically substantially resistant to internal cleavage in vivo, Put another way, although the fusion protein may be turned over by endogenous protein degradation pathways, it is not typically sensitive to cleavage by in vivo mechanisms to specificially release the individual protein of interest. In this context, substantially resistant to internal cleavage means that at least 50%, more 30 preferably 80%, yet more preferably 95%, most preferably 100% of the degraded plastidic fusion protein will be degraded by the sequential removal of Nor C 11 terminal amino acids, rather than the cleavage of internal peptide bonds.

The Protein of Interest 5 The protein of interest may have any function. The naturally occurring forrn 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. Preferably 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 15 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 20 be a blood protein, such as a clotting protein (e.g. kinogen, prothrombin, 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, 25 polynucleotides (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), 3 30 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 12 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 5 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 10 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 15 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-a, TNF-P, TGF-P), an interferon (e.g. IFN-a, IFN-P, IFN-y), chemokine (e.g. MIP- I a, MIP- I P, RANTES), an immune receptor (e.g. 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 20 cell or dendritic cell surfacemarker)(eg. CD 1, 2, 3, 4, 5, 6, 7, 8,, 16, 18,, 19, 28, 40, or 45; or a natural ligand thereof) or a complement gene.

The protein of interest may be atrophic factor (e.g. BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, VEGF, NT3, T5, HAR-P) or an apolipoprotein. The protein of interest 25 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, arnino 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.

13 The protein of interest may have enzymatic activity. Typically the polypeptide will be useful for industrial purposes. For example, xylanases, enzymes involved in plant cell wall modification, can be useful in paper manufacture. Alternatively, the protein of interest may be useful for therapeutic purposes, for example, as anticoagulants, such as 5 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.

In a preferred embodiment the protein of interest is an interferon. In ayet more preferred embodiment the protein of interest is IFN-y. IFN-y, also kmown as immune 10 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 and Asn 97 (Gray et al, 1982; James et al, 1995; Rinderknecht et al, 1984) .

15 However, unglycosylated E. coli-derived recombinant IFN-7 shows the same spectrum of biological activities as the natural glycosylated human IFN-T (Arora and Khanna 1996; Vega et at. 1990; Vileek 1990). The threedimensional structure of human IFN-T has been determined using E coli expressed recombinant protein (Ealick et al, 1991). Recombinant IFN-y has been extensively tested clinically and used for the treatment of 20 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; IIlis et al, 1999). IFN-'Y is also useful as an adjuvant in the vaccination of immunocompromised humans (Jaffe and Herberman 1988). In view of these applications and for the purposes of various biochemical 25 studies, human IFN-y has been recombinantly expressed in a variety of hosts such as E coli (Gray et al, 1982; Vega et al, 1990; Arora and Khanna 1996) monkey COS-7 cells (Gray et al, 1982), Chinese hamster ovary cells (James et al, 1995), SP9 insect cells (James et al, 1995) and transgenic mice (Dobrovolsky et al, 1993; James et al, 1995; Lagutin et al, 1999). IFN-y can thus be expressed in any suitable cell system or 30 organism.

14 Alternatively, 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 5 (WO 92/000377) gene); or tolerance to fosametin; a dihalobenzonitrile; glufosinate, e.g.

using a phosphinothrycin acetyl transferase (PAT) or glutamine synthase gene (cf. EP A-0 242,236); asulam, e.g. using a dihydropteroate synthase gene (EP-A-0 369,367); or a sulphonylurea, e.g. using an ALS gene); diphenyl ethers such as acifluorfen or oxyfluorfen, e.g. using a protoporphyrogen oxidase gene); an oxadiazole such as 10 oxadiazon; a cyclic imide such as chlorophthalim; a phenyl pyrazole such as TNP, or a phenopylate or carbarnate analogue thereof, spectinomycin e.g using the aadA gene, as exemplified below.

Insect resistance may be introduced, for example using genes encoding Bacillus 15 thuringiensis (Bt) toxins. Likewise, genes for disease resistance may be introduced, e.g.

as in W091/02701 or W095/06128.

The protein of interest may have a role in a metabolic pathway, preferably a chloroplastic metabolic pathway, for example the photosynthetic metabolic pathway.

20 Typically the expression of the encoded protein of interest may alter flux in the pathway, for example the photosynthetic ability of the transformed plant.

For example, chlorophyll biosynthesis in lower organisms is lightindependent due to the presence of an enzyme that comprises three polypeptides encoded by the chIL, chIN 25 and chIB genes. However 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 chIL, chIN and chIB genes.

30 Furthermore, 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.

Additionally, many of the chloroplastic processes, including metabolic and signalling pathways, can be controlled by post-translational protein modification such as glycosylation or phosphorylation. These levels could be manipulated in the chloroplast by transforming a plastome with a gene or genes encoding enzymes responsible for 10 glycosylation, deglycosylation, phosphorylation or dephosphorylation, thus allowing promotion of desirable pathways or inhibition of undesirable pathways.

Alternatively, expression of the encoded protein may introduce a new metabolic step or steps to the transformed organism. In one example of this, biodegradable plastics can 15 be produced in the chloroplast by transforming the plastome with prokaryotic genes known in the art. As another example, 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.

20 Fusion protein partne 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.

25 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 proper-ties. Most preferably the fusion protein comprises the sequence of 0-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 In this context 30 a variant of a protein with a scorable property will possess substantially the same distinguishing property as the protein from which it was derived. Thus, if the 16 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, 5 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.

Whilst the invention anticipates that the fusion protein partner may by fused to either end of a polypeptide of interest within the fusion protein, in a preferred embodiment 10 the fusion protein partner is fused to the C-terminal of the polypeptide of interest.

Purification Ta A fusion protein of the invention preferably comprises a purification tag which as 15 used herein is an amino acid sequence that can bind to a target under suitable conditions. The skilled person will appreciate that 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 20 "Surface Plasmon Resonance" technique from BIACORE (see http:/www. biacore.com).

Typically 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 25 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, 30 preferably at least 70%, more preferably at least 90%, yet more preferably at least 95%, even more preferably at least 99%, most preferably 100%.

17 Where a polypeptide comprises a purification tag then the purification tag is preferably capable of facilitating affinity purification of the polypeptide from a sample. In this context, capable of facilitating affinity purification means that following an affinity purification step at least 10%, more preferably at least 50%, yet 5 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. In a preferred embodiment 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 10 produced.

Typically a purification tag will comprises at least one but not more than 100 amino acids. A fusion protein of the invention may comprise I or more purification tags.

Typically, the number of purification tags is not more than 5, more typically not 15 more than 2, most typically 1.

The purification tag may be positioned anywhere within the fusion protein. In a preferred embodiment the purification tag is part of a fusion protein partner. More preferably the purification tag is retained at a ten-ninus of the fusion partner protein 20 following cleavage of the fusion protein. Usually 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. Alternatively, 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 25 protein.

In a preferred embodiment, the purification sequence is a His-Tag. Typically the His-tag comprises multiple contiguous histidine residues, preferably from 3 to 20, more preferably from 4 to 10 most preferably 6. Typically the His-tag will be 30 positioned at either or both of the Nand C- terminals of the fusion protein. The preferred target for the His-tag is NiNTA, which is available commercially, e.g. from 18 Qiagen (Germany). Other preferred purification tags include a self- cleaving chitinbased 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). 5 Cleavage Site 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 10 release the polypeptide of interest. Put another way, 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.

15 In a preferred embodiment the cleavage site is resistant to in vivo cleavage, as defined above. In another preferred embodiment, 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. However, in fusion proteins of the invention it can be important to provide for accurate and uniform cleavage of the fusion protein. Put another way, it can be 25 important for the protein of interest to be released with substantially no additions or deletions to the desired amino acid sequence, since the biological activity of the thus released protein of interest may be affected.

To ensure that the protein of interest thus released has the desired sequence and 30 activity, typically the fidelity of the cleavage will be high. In this context, fidelity refers to the accuracy of the cleavage at the cleavage site by in vitro mechanisms, that 19 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 5 interest or the fusion protein partner as appropriate.

Usually the fidelity of a cleavage site in a fusion protein of the invention will be at least 20%. Preferably 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 10 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 Z-- variant thereof that is recognised and cleaved by factor Xa with substantially the 15 same fidelity as IEGR.

Transforming Constructs Transforming constructs of the invention may comprise DNA or RNA, preferably DNA.

20 They may also include within them synthetic or modified nucleotides. The invention further provides double stranded polynucleotides comprising a polynucleotide of the invention and its complement.

Transforming constructs of the invention may be produced recombinantly, synthetically, 25 or by any means available to those of skill in the art. The constructs are typically provided in isolated and/or purified form.

Although in general the techniques mentioned herein are well known in the art, reference may be made in particular to Sambrook et al, 1989, Molecular Cloning: a laboratory 30 manual.

Transforming constructs of the invention typically comprise a sequence encoding a fusion protein of invention.

The transforming constructs of the invention may comprise a selectable marker gene i.e.

5 marker genes that allow transformed cells to survive in the presence of agents that kill non-transformed cells. Any selectable marker gene may be used in the transforming constructs of the invention. Typically, herbicide resistance genes, e.g. as defined above, may be used as selectable markers. Alternatively, coding regions that encode products which provide resistance to aminoglycoside antibiotics may be used as a selectable 10 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 al, 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 15 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: P-glucoronidase (i.e. the uidA:GUS 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.

20 Cells with scorable optical differences can be sorted using techniques such as fluorescence activated cell sorting (FACS). In a preferred embodiment, the polynucleotide of the invention comprises a selectable marker ald a scorable marker, for example, the FLARE-S marker genes which comprise aad,4 and GFP (Khan and Maliga, 1999).

Transforming constructs of the invention typically comprise regulatory regions.

Preferably 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. Usually a regulatory 30 region will be operably linked to the coding sequence of the fusion protein and / or the selectable or scorable marker gene. The transforming construct may also be designed in 21 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.

5 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 transfon-ning 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.

Typically 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%, most preferably 100%. The homologous flanking regions may be homologous to the same, overlapping, coterminous, or distinct regions of the recipient 15 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. When 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- 25 life to be determined by methods well known in them art.

Variants Variants of regions of the transforming constructs of the invention, in particular, 30 promoter, enhancer, terminator regions and coding regions, may be obtained and used in the invention. This may be useful where, for example, sequence alterations can be 22 used to alter homology with endogenous plastornic 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 5 sequences by site directed mutagenesis, synthesis of novel sequences or recombinant tecliniques. Naturally occurring variants may be obtained by probing cDNA or plastornic 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.

Typically variants have at least 50% homology to the sequence from which they are derived, more typically at least 70%. Preferably variants have at least 90%, more preferably at least 95%, yet more preferably 99% homology to the sequence from which they are derived.

The term variant, as used herein, 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. In this context, variants of regulatory regions, such as terminators, 20 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.

25 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. In this context, biological activity refers to the binding specificity, enzymatic, structural and immunological properties of the naturally occurring polypeptide. In a preferred embodiment a biologically active variant of a 30 polypeptide will retain substantially the same enzymatic properties as the naturally occurring polypeptide. In another preferred embodiment the biologically active variant 23 of a polypeptide will retain substantially the same binding specificity as the naturally occurring polypeptide.

Alterations may include additions, insertions, deletions, substitutions or inversions. The 5 terms addition or insertion, as used herein, 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. Preferably the number of nucleotide additions or insertions will be at most 40, more preferably at most 20, yet more preferably at most 10, and most preferably at most 5. The term deletion, as used herein, refers to a 10 polynucleotide sequence wherein one or more nucleotide residues are absent as compared to the naturally occurring molecule. Preferably 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 polynucleotides of the invention, The term substitution, as used herein, 15 refers to the replacement of one or more nucleotide residues by different residues.

Preferably 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. The term inversion, as used herein, refers to a polynucleotide sequence wherein a contiguous region within the sequence is reversed in orientation relative to the remaining molecule.

20 Preferably the number of contiguous regions of sequence inverted will be 4, more preferably 3), yet more preferably 2, most preferably 1. The skilled person will understand that 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 25 acid residues added, inserted, deleted, substituted or inverted as for the number of nucleotide changes for polnucleotide variants as described above.

Homolo 30 Methods of measuring nucleic acid homology are well known in the art. For example the UWGCG Package provides the BESTFIT program which can be used to calculate 24 homology (Devereux et al, 1984) e.g. in the context of whether sequences are homologous or heterologous for the purposes of the invention.

Similarly, the PILEUP and BLAST algorithms can be used to line up sequences (for 5 example as described in Altschul, 1993; Altschul et al, 1990). Many different settings are possible for such programs. According to the invention, the default settings may be used.

In more detail, the BLAST algorithm is suitable for determining sequence similarity and 10 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.govo. 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 15 of the same length in a database sequence. 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 20 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 seqiwnce is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the 25 BLOSUM62 scoring matrix (see Henikoff and Henikoff 1992) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

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 30 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. For example, 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 0.0 1, and most preferably less than about 0. 00 1.

Cloning Vectors To facilitate cloning of the transforming polynucleotide of the invention, it can be incorporated into recombinant replicable vectors. Such vectors may be used to replicate 10 the nucleic acid in a compatible host cell. Thus in a further embodiment, 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 15 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, 20 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 sequence s 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.

25 comprising the transforming polynucleotide) to be discriminated from those transformed with non-recombinant vector.

Mastids 30 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 26 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.

5 Plastids comprise their own genome, herein referred to as a plastome. Typically individual plastids comprise multiple plastomes, more typically from 5 to 500, most typically from 5 0 to 100. Herein, 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 10 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.

Herein, a recipient plastome transformed with a transformingpolynucleotide according 15 to the invention is referred to as a transplastome. Plastids comprising a transplastome are referred to as transplastomic. Plastids wherein all plastomes are identical, or substantially identical, transplastomes are referred to as homotransplastomic. In this context, the plastomes of plastids are substantially identical if they all comprise the coding region of the transtorming polymicleotide ot tile invention, and preferably any 20 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 homotranspla7stomic. Plants, plant parts and seeds are homotransplastomic if all of their cells are homotransplastomic.

25 Recipient plastomes Suitable sources of recipient plastome include plants, for example, spermatophytes, pteridophytes (fems, clubmosses, horsetails), bryophytes (liverworts and mosses), and algae. Typically the recipient plastome will be a plastome of a multicellular organism, 30 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 27 and cedars), cycads, yews and ginkgos. More typically 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 5 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, triticale and sugar cane. Rice is particularly preferred. Alternatively, plastomes and regions of transforming construct for use in the methods of the invention may be recombinant or 10 entirely synthetic in origin.

Further scope of the invention In a further embodiment of the invention, the transforming polynucleotide is adapted for 15 use in the transformation of other 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 20 in all eukaryotic organisms.

Cells for transformation The cell used for transformation may be from any suitable organism (see above list) and 25 may be in any form. For example, 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.

It may, for example, be part of an embryo, or a meristem, e.g. an apical meristem of a shoot, Preferably the cell is a cell containing chloroplasts, e.g. a leaf or stem cell, most 30 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 28 not.

Transformation techniques 5 Generation of the transplastome is brought about by the insertion of the polynucleotide fusion construct as defined above. The polynucleotide may be inserted by any method known in the art, such as recombinant techniques, random insertion, or site directed integration. Preferably the method of polynucleotide insertion is site directed integration, more preferably by the process of homologous recombination. The 10 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. Preferably, the plastid is within a cell, though it may be in isolated form.

15 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; Potrykus et al, 1985), injection by galinstan expansion ferntosyringe (Knoblauch et al, 1999) and 20 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 5,866,42 1). Particle bombardment is particularly preferred.

Selection of transformed cells and generation of homotransplastomic cells Homotransplastomic (see above) plastids, cells, plants, seeds, plant parts, plant tissues are preferred.

Cells generated by the transformation techniques discussed above will typically be 30 present in chimeric tissues, and thus will be surrounded by other non- transformed cells.

Furthermore, due to the multiple genome copies within each plastid, transplastomic 29 plastids will typically contain multiple copies of untransformed plastomes. In order to produce homotransplastomic cells, thaL 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.

5 Screening will be carried out via an expressed 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 10 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 hornotransplastomic nature of the thus generated cells are ascertained. Homotransplastomicity can be assayed by analysis of isolated plastornic DNA by Southern analysis or by performing polymerase chain 15 reaction amplification. These techniques are suitably sensitive such that the presence of a single untransformed plastome could be detected.

Generating stable transplastomic plants and seeds 20 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 transplastornic or homotransplastomic cell. Similarly, techniques such as somatic embryogenesis and meristern culture may be used. Regeneration techniques are well 25 known in the art and examples can be found in, e.g. US 4,459,355, US 4, 536,475, US 5,464,763, US 5, 177,010, US 5, 187,073, EP 267,159, EP 604, 662, EP 672, 752, US 4,945,050, US 5,036,006, US 5,100,792, US 5,371,014, US 5,478,744, US 5, 179,022, US 5,565,346, US 5,484,956, US 5,508,468, US 5,538,877, US 5,554,798, US 5,489,520, US 5,510,318, US 5,204,253, US 5,405,765, EP 442,174, EP 486, 233, EP 30 486,234, EP 539,563, EP 674,725, W091/02071, WO 95/06128 and WO 97/32977.

In many such techniques, one step is the formation of 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. Thus, for example, the invention provides transplastomic or homotransplastomic plant tissues and parts, 5 including embryos, meristems, seeds, shoots, roots, stems, leaves and flower parts.

These may be chimeric in the sense that some of their cells are transplastornic or homotransplastomic and some are not. Similarly they may be chimeric in the sense that all cells are transplastomic but only some are homotransplastomic.

10 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 transplastornic or homotransplastomic plant. The invention also provides methods of obtaining transplastomic or hornotransplastomic plants of further generations from this first generation plant. These 15 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.

Thus, the invention provides a method of obtaining a transplastornic or 20 homotransplastomic progeny plant comprising obtaining a second- generation transplastornic or homotransplastomic progeny plant from a firstgeneration transplastomic or homotransplastomic plant of the invention, an. 4optionally 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. For example, for the production of first generation transgenic plants, 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 30 or more subsequent generations of progeny plants before a transplastomic or homotransplastomic plant more suitable for cultivation is produced.

31 Progeny plants may be produced from their predecessors of earlier generations by any known technique. In particular, progeny plants may be produced by:

obtaining a transplastomic or homotransplastomic seed from a transplastomic or 5 homotransplastomic plant of the invention belonging to a previous generation, then obtaining a transplastornic or hornotransplastomic progeny plant of the invention belonging to a new generation by growing up the transplastornic or homotransplastomic seed; and/or 10 propagating clonally a 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; and/or 15 crossing a first-generation transplastornic 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 20 obtaining transplastomic or homotransplastomic progeny plants of one or more further generations from the progeny plant thus obtained.

These techniques may be used in any combination. For example, clonal propagation and sexual propagation may be used at different points in a process that gives rise to a 25 transplastornic or homotransplastomic plant suitable for cultivation. In particular, 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.

30 Also, 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 32 coding sequences other than the transforming construct of the invention. This may be carried out by conventional breeding techniques, e.g. fertilizing a transplastornic. or homotransplastomic plant of the invention with pollen from a plant with the desired additional characteristic. Alternatively, the characteristic can be added by further 5 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.

10 Preferably, 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.

15 Use of transplastomic plants 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. By crop product is meant any useful product obtainable from 20 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, 25 rye, rice, maize, sorghum, triticale, especially wheat; other seeds; shoots, especially tubers, such as potato tubers; fruit; and other plant parts, for example as defined herein. Alternatively, such a product may be obtainable indirectly, by harvesting and further processing. Examples of products obtainable by further processing are: flour; oil; rubber; beverages such as juices and fermented and/or distilled alcoholic beverages; food 30 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 33 cigars; fibres, e.g. cotton, linen, flax and hemp fibres and textile items made therefrom; paper or timber derived from woody plants.

Recovery of fusion proteins and proteins of interest 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.

10 Enrichinent In a preferred embodiment, a crude protein 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 15 protein sample. Typically following enrichment 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 20 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 25 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. In the most preferred embodiment, the fusion protein comprises the sequence of GUS, and the fraction (or fractions) containing the fusion protein can thus be detected by histochernical assays and 3 30 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 34 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 5 in the art.

Recove Fusion proteins can be recovered from a crude protein sample or in a preferred 10 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 15 99%, most preferably 100% of the fusion protein of the invention.

Typically the fusion protein recovered by affinity based methods suitable to the sequence of the fusion protein and any purification tag present therein. Typically the fusion protein comprises a His-tag and affinity purification is performed using a Ni- 20 NTA agarose column under suitable conditions. Thus, the fusion protein is retained in the affinity column whilst other proteins present in the fraction are washed out, and the conditions in the column are then altered leading to the rilease of the fusion protein, which can thus be collected in a purified form. The skilled person will understand that many other equivalent affinity based methods are equally suitable, 25 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).

Cleavage Fusion proteins can be processed in order to release the protein of interest, typically by 5 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. In a preferred embodiment the cleavage sequence is an Fa Xa site, IEGR, and is cleaved by incubation with factor Xa (Nagai, 1985; Quinlan, 1989; Wearne 1990).

Recovery qf the Protein qf Interes The resultant cleavage mixture comprises the protein of interest and polypeptide fragments representing the rest of the fusion protein, in particular the fusion protein 15 partner. The protein of interest can then be recovered by any method known in the art, for example either affinity based methods or fractionation. In a preferred embodiment following cleavage the fusion protein partner comprises a purification tag. Thus 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 20 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.

Use of Proteins of the Invention 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 30 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 36 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.

5 Thus 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. Thus 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.

Typically after the initial administration of the agent, the same or a different agent of 15 the invention can be given. In one embodiment the subject is given 1, 2, 3 or more separate administrations, each of which is separated by at least 12 hours, I day, 2, days, 7 days, 14 days, I month or more.

The agent may be in the form of a pharmaceutical composition which comprises the 20 agent and a pharmaceutically acceptable carrier or diluent. Suitable carriers and diluents include isotonic saline solutions, for example phosphate-buffered saline. Typically the composition is formulated for parenteral, intravenals, 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 30 patient. A suitable dose may however be from I OLg to I Og, for example from 100 Rg to I g of the agent. These values may represent the total amount administered in the 37 complete treatment regimen or may represent each separate administration in the regimen.

EXAMPLES 5

In the experimental details which follow, weights are given in grams (g), milligrams (mg) or micrograms ([tg), all temperatures are given in degrees centigrade ('C), concentrations are given as molar (M), millimolar (mM) or micromolar (mM), nanomolar (tM), picomolar (pM) and volumes are given in litres (L), millilitres (ml), 10 microlitres (tl), unless otherwise indicated.

Abbreviations: Base pairs (bp), Example 1: Expression vectors for ifnG in chloroplasts Polymerase chain reaction (PCR) was performed to amplify defined DNA fragments through appropriate primers (Sarkar and Sommer 1990). Convenient restriction sites were introduced into primers for easy cloning. Standard procedures were followed for cloning the PCR products (Sambrook et al, 1989).

The plastid transformation vector, pVSR326 (Fig. 2A), was constructed using the rrn and psbA promoters and 3' untranslated._regions of psbA and rbcL genes from rice plastome primary clones (Hiratsuka et al, 1988). The selectable aadA and reporter uidA genes were cloned from pUC-atpX-AAD (Goldschmidt-Clermont 1991) and 25 pGUSN358-S (Clontech) plasmids, respectively. The tobacco plastid genorne sequences spanning rbcL-accD genes (Shinozaki et al, 1986) were used for site specific integration of chimeric aadA and uid,4 genes into plastid DNA.

p3261FNG was a derivative of vector pVSR326. In the pVSR326, the uidA was 3 30 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 38 BglII and Sacl sites to create pVSRIFNGI. In the next step, (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 fnG coding region (Fig. 2C) was PCR amplified from an E. coli 5 expression vector pPLIFNG (Wang et al. 1992) using SR49 (SEQ ID NO: 49) and SR50 (SEQ ID NO: 50) primers and cloned at Apal site of pVSRIFNG2 to create p-')261FNG.

For the construction of pGUSIFNG, the uid,4 coding region, devoid of stop codon, was 10 PCR amplified from pGUSN358-S (Farrell and Beachy 1990) using SR51 (SEQ ID NO: 5 1) and SR52 (SEQ ID NO: 52) primers and cloned into vector pQE30 (Qiagen) at BamHI and KpnI sites to create pQEGUS. Parallely, 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 pQE3 I (Qiagen) at the Pstl site to create pQE3 I IFNG.

15 The uidA along with T7 promoter and (6x) His-tag was released from pQEGUS as BamHI and Smal fragment and cloned into pQE3 I IFNG digested with KpnI (end filled) and BamHI to create vector pQEGUSIFNG. Finally, the uidtifnG fusion gene was PCR amplified from pQEGUSIFNG using SR47 and SR55 (SEQ ID NO: 55), digested with Ncol and cloned into NcoI and SacI (end filled) digested pVSR326 to create vector 20 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.

To produce and purify recombinant human IFN-g, 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. IA). In the pBIIFNG, the coding region of ifnG was transcriptionally fused to a 30 reporter uidA gene (GUS) in pBI121 vector. Both uidA and ifnG have their own translation initiation (ATG) and termination (TGA) codons and both the genes were 39 under the transcription control of the same CaMV 35S promoter. Two vectors, p-')261FNG and pGUSIFNG, were used to express IFN-g in tobacco chloroplasts as an individual and as a fusion protein, respectively. Both the vectors were derived from pVSR326 that contained a selectable aadA gene that confers resistance to spectinomycin 5 and a reporter iddA gene under the regulation of rice psbA and rrn promoters, respectively (Fig. 2A). The rbcL-aecD gene sequences derived from tobacco plastid genome were provided in the vector flanking the transgenes for site-specific integration through two homologous recombinations. The p3261FNG was obtained from pVSR326 by replacing uidA with that of ifnG (Fig. 2B). The complete nucleotide and the deduced 10 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. In the vector, 15 pGUSIFNG, the ifnG was translationally fused at the C-terminal 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, The direction and size 20 of transcripts from uidA, ifnG, uidA:ifnG 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.

Example 2: Transformation and regeneration of stable transgenic plants The Agrobacterium mediated transformation method was followed for nuclear transformation of tobacco with pB1121 and pBlIFNG binary vectors under kanamycin selection. Particle bombardment of leaf tissue was used for chloroplast transformation under spectinomycin selection using DNA of vectors pVSR')26, p3261FNG and 3 30 pGUSIFNG. Tobacco (Nicotiana tabacum cv. Petit Havana) was transformed using particle delivery system PDS 1000 (BioRad) according to the method described by (Svab and Malica 1993)). In brief, vector DNA coated onto tungsten particles (M17 Bio-Rad) was bombarded on the abaxial side of a tobacco leaf placed on RMOP medium (Svab and Maliga 199')), a modified MS medium (Murashige and Skoog 1962) containing 0. 1 mg/I thiamine, 100 mg/I inositol, 3% sucrose, I mg/I BA and 0. 1 mg/l NAA, 0.6% agar, 5 pH 5.8). Transformed shoots were selected on RMOP medium containing 500 mg/I spectinomycin dihydrochloride. Three additional cycles of regeneration on spectinomycin (500 mg/1) containing RMOP medium was carried out to obtain homotransplastornic plastid containing plants (Svab and Maliga 1993).The Agrobacterium strain LBA 4404 containing vector pBIIFNG/pBI121 was used for 10 nuclear transformationfollowing a leaf disc method (Horsch 1985).

Although the vector DNA is randomly delivered into leaf cells in the particle bombardment method, the selectable aad,4 is expected to express and confer resistance to spectinomycin only when it entered the chloroplasts due to the specificity of the rrn 15 promoter. Substantially, 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. BI 12 1 - 1, Nt. BIIFNG 1/2, Nt. VSR326--)7, Nt. 3261FNG-1/2 and Nt. GUSIFNG-1 plants transformed with vectors pBI 12 1, pB I IFNG, pVSR326, p3261FNG and pGUSIFNG, respectively, were 20 subjected to molecular analysis to confirm the transgenic nature of regenerated plants and the expression of recombinant IFN-g protein. Total DNA isolated from transgenic and control plants (Mettler 1987) was digested with relevant restriction endonucleases, separated on 0.8% agarose gets and transferred on to nylon membrane. About 3 Ig of total RNA isolated from leaf tissue (Hughes and Galarn 1988) was separated in 25 denaturing formaldehyde agarose gel (1.5%) and blotted to nylon membranes. The membranes were UV crosslinked and then probed with 3 2p labeled psbA (SEQ ID NO:

39),16S rRNA (SEQ ID NO: 32), uid4 (SEQ ID NO: 33), aadA (SEQ ID NO: 34) and targeting sequence (SEQ ID NO: 3 5) amplified using SR 17 (SEQ ID NO: 17) - SR 18 (SEQ ID NO: 18), SR 19 (SEQ ID NO: 19) - SR20 (SEQ ID NO: 20), SR41 (SEQ ID 30 NO:41)-SR42(SEQIDNO:42),SR43(SEQIDNO:43)-SR44(SEQIDNO:44) AND SR12 (SEQ ID NO: 12) - SR13 (SEQ ID NO: 13) primer pairs respectively.

41 Standard procedures were followed for hybridization (Sambrook et al, 1989) and membranes were subjected to autoradiography, Example 3: Integration of uidA, aadA, ifnG into nuclear/plastid genomes 5 Southern hybridization analysis using uidA, aadA, ifnG and rbcL-accD probes confirmed the stable integration of vector DNA into tobacco nuclear and plastid genomes (Fig. IB, Fig. 3A-C). The genomic DNA isolated from Nt. BIIFNG-1, Nt. BIIFNG-2 and Nt. BI121-1 plants were subjected to Southern hybridization analysis.

10 The presence of 2.65 kb and 0.45 kb size fragments can be seen in the PstI-EcoRJ and Xbal digested DNA, respectively, when probed with ifnG coding regions (Fig. 1B).

Reprobing the blot containing Pstl-EcoRl digested DNA with uidA reconfirmed the presence of an expected 2.65 kb fragment (Fig. 113).

15 Integration of vector pVSR326 into the plastid genome was confirmed by digesting the total genomic DNA with Clal and probing with uidA and aadA gene probes. Presence of a 3.4 kb signal in the wild-type plant (Fig. 3A, lane 1) and a 7.3 kb signal in all six transformed plants tested (Fig. 3A, lanes 2-7) when probed with the rbcL-accD sequences confirmed the sitespecific integration of uidA and aadA. The complete 20 absence of a 3.4 kb signal in transplastomic lines is a clear evidence for the homoplasmic nature of the transplastome. Similar analysis was performed to confirm the transgenic nature of Nt. 3261FNG-1, Nt. 3261FNG- 2 (Fig. 313) and Nt. GUSIFNG-1 (Fig. 3Q. As shown in Fig. 313, the size of the fragments that hybridized to the aadA and ifnG in Nt. 3261FNG- I and Nt. 3261FNG-2 plants were in agreement with the predicted 25 size DNA fragments when transgenes are integrated into the plastid genome sitespecifically. Similarly, Southern hybridization confirmed the stable integration of uidA:ifnG into Nt. GUSIFNG-1 plastorne (Fig. 3C). As predicted by the restriction map, all the three probes (aadA, ifnG and uidA) hybridized to a common 11.2 kb and 9.18 kb band in Xhol and Clal digested DNA, respectively (Fig. 3C). Hybridization with the 30 targeting rbcL-accD probe further confirmed the stable site-specific integration of uidA:ifnG and aadA into the plastome (Fig. 3C). The complete absence of 3.74 kb and 42 8.72 kb signals in Clal and Xhol digested DNA in the rbcL-accD hybridized blot is a clear evidence for the complete honioplasmic nature of the transplastome in Nt. GUSIFNG-l plant.

5 Example 4: Transcription of chimeric uidA, ifnG and tddA.-/hG genes In the Nt. BIIFNG-1 plant, transcription of fnG gene was examined by Northern hybridization (data not shown) and RT-PCR (Fig. IC). A 2.3 kb size DNA fragment amplified in RT-PCR (Fig. IC, lane 2) when ifnG (SR56) and uidA (SR18) gene 10 specific primers were used, confirmed the presence of a fusion transcript in the RNA from Nt. BIIFNG-1. No amplification was observed when cDNA made from RNAse treated RNA was used as a template (Fig. I C, lane 1). The RT-PCR analysis using ifnG gene specific primers (SR56 and SR57) amplified a fragment of about 0.45 kb in size from cDNA template made from DNAse treated RNA. In the chloroplast transformed 15 plants, Northern blot analysis was performed to confirm the transcription of chimeric OdA, aadA, ifnG and uidA.-ifnG genes (Fig. -')D-F). As shown in Fig. 3D, a 0.45 kb transcript corresponding to the expected size of ifnG was observed in the RNA from Nt.

3261FNG-I and Nt. 3261FNG-2 plants. Hybridization using uidA probe confirmed transcription of uidA and uidA:ifnG in Nt. 326-37 and Nt. GUS:IFNG-I plants, 20 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- I (Fig. -3F, lanes I and 2). The RNA sample from Nt. VSR326-37 (Fig. 3E, lane 3) 25 included in the blot for direct comparison of uidA and uidA:ifnG transcript levels additionally confirmed the presence of expected size uidA.-ifnG fusion transcript in Nt.

GUSIFNG- I plant (Fig. 4E, lanes I and 2).

Example 5: Expression of GUS, IFN-g and GUS:IFN-g For Western blotting (Sambrook et al, 1989), proteins at various stages of purification 43 (see below) were subjected to SDS-PAGE (Laemmli 1970), electroblotted on to nitrocellulose membranes, blocked with bovine serum albumin (BSA), incubated by anti-GUS (Clontech), anti-IFN-g (Calbiochem), anti-His antibodies (Qiagen) and detected by goat anti-rabbit alkaline phosphatase (Sigma) as per the supplier 5 instructions.

For pulse labeling, total protein was labeled by incubating the leaf discs in 10 ml of MS medium containing 3.5 mCi labeled amino acid mix (S-3) 5 Express, NEN/DuPont) and incubated at 250C under 4,000 lux light. After one hour, leaf discs were thoroughly 10 washed with NIS medium and continued the 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 I D Image analysis software 15 (Kodak).

Western blotting was used to confirm the presence of GUS, IFN-g and GUS:IFN-g proteins in Nt.326-37, Nt., 3261FNG-1 and Nt. GUSIFNG-1 plants, respectively. In addition to anti-IFN-g, the anti-His antibodies were also expected to recognize the 20 recombinant IFN-g produced in the Nt. 3261FNG- I plant, as it contained a (6x) His-tag at the N-terminal region. As shown in Fig. 4A, both anti-His and anti-IFN- g antibodies recognized a 17 kDa protein, an expected size for IFN-g, in Nt. 3261FNG-1, confirming the expression of IFN-g. Similar analysis using anti-His-tag, anti-IFN-g (data not shown) and anti-GUS antibodies revealed the presence of a 85 kDa GUS:IFN- g fusion 25 protein in the leaf extracts of Nt. GUSIFNG- I (Fig. 4B).

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. 3261FNG-1 and Nt. 326-37 plants, respectively. To understand the reasons for 300 fold difference in the expression levels 30 between IFN-g and GUS, the role of protein degradation was investigated. The leaf discs from Nt. VSR326-37 and Nt. 3261FNG-1 plants were pulse labeled with S-35 44 (Methionine and Cysteine) to analyze the half life of recombinant IFN-g and GUS proteins in the chloroplasts. As shown Fig. 4C, the GUS protein had a half life of about 48 hours whereas IFN-g had comparatively very short half life (about 4-6 hours) in chloroplasts.

Example 6: Purification of IFN-g and assaying its biological activity Soluble leaf protein extract obtained from the greenhouse grown Nt. GUS:IFN-g- I plant in buffer A (50 mM Tris-HC1 pH 7.0,5 mM DTT, I mM Na2EDTA, 0.1% SDS, 0.1% 10 Triton X- 100, one protease inhibitor cocktail tablet per each 5 0 ml of buffer) was loaded onto a 200 ml DEAE cellulose column equilibrated with the same buffer. The column was washed with 5 vol buffer B (buffer A, 50 mM NaCI) 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 15 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 20 separate the IFN-g from the fusion protein as per the supplier instructions. After the cleavage, protein was passed once again through a fresh Ni-NTA column to remove the undigested GUS:IFN-g and GUS protein containing His-tag. The flow through was loaded directly on to a 10 ml S- sepharose column equilibrated with buffer E (20 mM Tris. Hcl pH 8). After washing the column with 50 ml buffer E, the bound protein was 25 eluted with 80 ml buffer E + 1.2 M NaCI gradient. The fractions containing recombinant IFN-g was dialyzed extensively, analyzed on SDS- PAGE for purity, quantified the protein and then subjected to bioassay (Lewis 1988). In brief, 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. One unit of antiviral activity was 30 defined as the amount of rh-IFN-g required to produce equivalent antiviral activity expressed by I U of the (NIH IFN-g reference standard (Gg 23-901-530)).

Only 10% of the total estimated GUS:IFN-g fusion protein with less than 85% purity, as judged on a comassie blue stained SDS-PAGE gel, was recovered when purified directly using Ni-NTA column. However, the fusion protein was purified to near 5 homogeneity using a two step column procedure (Table 1).

Table 1. Purification of GUS:IFNG fusion protein starting from 50 grams (fresh weight) of greenhouse grown plant leaves Purification step Total protein Total Specific activity Yield (MEO (U) (U/mg) (%) Crude extract 750 5,791,666 7,722 100 Cellulose DE52 353 4,866,000 13,784 84 Ni-NTA colum 18 4,402,000 244,555 76 (One unit is defined as the amount of activity required to release one tmole of MU from MUG in one minute at 37C.) In the first step, the crude extract was loaded on to DE-52 column and the bound 15 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, 20 The activity of GUS was detected by histochernical assay and measured by fluorometric assay (Gallagher 1992). The fractions containing GUS:IFN- g fusion protein were identified using a histochernical 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 37C. Protein 25 concentration was determined with the Bradford reagent (BioRad) using BSA as 46 standard. 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) virus. The expression levels of IFN-g and GUS were quantified by comparing with E. coli derived IFN-g (Boeringer Mannheim) and GUS 5 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 10 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. Finally, the IFN- g present 15 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). In a bioassay, a 24 h pretreatment of human lung carcinomas (A 549) with 25 pg of the purified IFN-g offered complete 20 protection against the infection of by EMC virus (Fig. 4F), whereas the untreated cells were completely infected (Fig. 4G). These results suggested that the purified recombinant IFN-g have a specific activity of >I 0' IU mg-' protein.

The expression of IFN-y was very low (0.001%) in the nuclear transformed plants 25 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 30 methods followed for protein extraction, the expression levels are generally low, especially, when compared to microbial expression systems (Goddijin and Pen 1995).

47 Considerable improvement in the expression level was achieved through the modification of codon usage (Perlok et al, 199 1), by the addition of ER retention signals (Fiedler et al, 1997), by subcellular targeting (Wong et al, 1992; Fiedler 1997; Schouten et al, 1996; 1997) and through the use of strong promoters in combination with enhancer 5 elements (Fiedler et al, 1997). Recently, very high level expression of maize phosphoenolpyruvate carboxylase was reported when transformed into rice plants accumulating up to 12% of the total leaf soluble protein (Ku et al, 1999). However, the same gene expressed at low level in tobacco (Hudspeth et al, 1991) and potato (Gehlen et al, 1996) suggesting that such high level expression is specific for few taxonomically 10 related species (Ku et al, 1999).

So far, the expression levels of foreign genes in transplastomic plants were found to be very high due to possible amplification of gene copy number. Amplification of Bacillus thuringiensis cryIA(c) gene in chloroplasts led to extraordinarily high levels, 3-5% of 15 the soluble protein, of insecticidal protein production (McBride et al, 1995). Recently, expression of aroA from petunia that confer low levels of tolerance to glyphosate, a herbicide, was shown to confer very high levels of tolerance due to overproduction of enzyme 5-enol-pyruvyl shikimate-3 -phosphate synthase (EPSPS) when expressed in chloroplasts (Daniell et al, 1998). Similarly, expression of uidA resulted into high level 20 accumulation of GUS in chloroplasts reaching up to 2.5% of the total cellular protein (Stuab and Maliga 1993). In the present study, similar level of GUS expression (3%) was observed in the chloroplast transformed Nt. 326-3)7 plant using vector pVSR326.

In order to improve the IFN-,y expression, the ifnG was cloned into plastid 25 transformation vector p3261FNG and transformed into tobacco chloroplasts. Although there was 100 fold increase in the IFN-y expression levels in the plastid transformed Nt. 3261FNG-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 30 reasons for such low levels of IFN-y expression could be due to the lack of efficient transcription/mRNA stability and/or fast degradation of the recombinant protein. The 48 Northern hybridization analysis revealed efficient transcription of ifnG in Nt. 3261FNGI 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. On the other hand, 5 the pulse labeling experiments have shown that the IFN-,y 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-,y was the reason for such a low accumulation.

To increase the expression levels of IFN-y further, the ifnG was translationally fused to 10 high expressing uidA in chloroplasts and integrated again into tobacco chloroplast genome. In the Nt. GUSIFNG-1 plants, about 5% of the total cellular protein were found to be of GUS:IFN-y fusion protein. When compared to 0.001% expression of IFN-T in the nuclear transformed plants and 0. 0 1 % in the chloroplast transformed plants, the 5. 0% expression levels achieved through GUS-fusion strategy are exceptionally significant.

15 Therefore, GUS fusion offers an attractive way to increase the low expressing proteins/peptides in transgenic plants. Recently, enhanced expression of antimicrobial peptide sarcotoxin IA was reported by GUS fusion in transgenic tobacco plants (Okamoto et al, 1998). 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 20 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-y fusion protein very rapidly using a simple and inexpensive GUS assay (Gallagher 1992). This feature is particularly useful for those proteins that are highly labile under certain 25 harsh conditions employed during purification steps and difficult to detect with simple analytical assays. Therefore, the 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 30 (Gilissen et al, 1998).

49 The number of steps involved in the downstream processing to recover the recombinant protein from relatively large volumes of plant biomass play critical role in the use of plants as bioreacters (Krebbers and Vandekerckhove 1990, Goddijin and Pen 1995,).

Affinity based purification greatly reduces the number of chromatography steps 5 involved in the purification of protein. Translational fusion of (6x) His-tag at the N or C-terminal end facilitates separation of recombinant protein using nikel nitrilotriacetic acid (Ni-NTA). While such a strategy has been often used for the purification of E coli expressed proteins (Hochuli et al, 1987), it has been rarely used in plant systems (Flachmann and Kuhlbrandt 1996; Sugiura 1999). The His- tag can be 10 removed, if necessary, by providing a specific protease site separating His-tag from the recombinant protein. Unlike E coli expressed proteins, our efforts to purify GUS:IFN-Y fusion protein from Nt. GUSIFNG- I 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 15 extract that might be competing directly for Ni-NTA binding. However, the GUS:IFN-7 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-,y from the GUS fusion partner and the cleaved-IFN-y was purified further by S-sepharose column. The purified recombinant 20 human IFN-y offered complete protection against the infection of human lung carcinomas (A 549) by EMC virus suggesting that IFN-y expressed in chloroplasts folded correctly and retained it's biological activity at a level that is comparable to E, coli derived r-IFN-y. In the present study, as a test case, we have used His-tag. However, there are number of ligands described in the literature for the purification of recombinant 25 proteins expressed in microbial, yeast, insect and mammalian systems which can replace His-tag depending on the need.

During the past one decade, a number of laboratories have experimented with the use of plants for 'biomanufacturing' of specialty products as plants can express, fold, 30 assemble and process foreign proteins (Krebbers and Vandekerckhove 1990; Goddijin and Pen 1995). The recent clinical trials in humans of plant expressed heat labile enterotoxin subunit B (LT-B) from enterotoxogenic E. coli for edible vaccine (Tacket el al, 1998) and a monoclonal secretary antibody that recognize surface adhesion protein of Streptococcus mutans as a preventive immunotherapy (Ma et al, 1998) have further strengthened this approach. Plant derived proteins with high level of purity may be more 5 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 10 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

15 of stringent quality control tests imposed by various national/international health agencies/organizations (Miele 1997). In 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 20 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 immensely while providing better healtS for mankind.

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Springer Verlag, Heidelberg: 3-38 Wang et al (1992) International Centre for Genetic Engineering and Biotechnology, Activity Report, Trieste, New Delhi: 58-60 Wearne (1990) FEBS Lett. 236(l): 23-26 Weening et al (1995) Eur. J Pediatr. 154: 295-298 54 SEQUENCE LISTING <110> INTERNATIONAL CENTRE FOR GENETIC ENGINEERING AND BIOTECHNOLOGY 5 <120> PLASTIDTRANSFORMATION <141> 10 <160> 60 <170> PatentIn Ver. 2.1 <210> 1 15 <211> 36 <212> DNA <213> Artificial Sequence <220> 20 <223> Description of Artificial Sequence: PRIMER <400> 1 aaaactgcag tcgactttca cagtttccat tctgaa 36 25 <210> 2 <211> 27 <212> DNA <213> Artificial Sequence 30 <220> <223> Description of Artificial Sequence: PRIMER <400> 2 catgccatgg taagatcttg gtttatt 27 <210> 3 <211> 33 <212> DNA <213> Artificial Sequence <220> 5 <223> Description of Artificial Sequence: PRIMER <400> 3 aattgagctc gaggtaccgc ggtctagaag ctt 33 10 <210> 4 <211> 33 <212> DNA <213> Artificial Sequence 15 <220> <223> Description of Artificial Sequence:: PRIMER <400> 4 aattaagctt ctagaccgcg gtacctcgag ctc 33 <210> 5 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 5 attcgagctc taattaatta aggcttttct gctaacatat ag 42 <210> 6 <211> 24 <212> DNA 3 <213> Artificial Sequence <220> 56 <223> Description of Artificial Sequence: PRIMER <400> 6 ggggtaccat catttattgg caaa 24 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 7 15 ctggggtacc tccccccgcc acgatcg 27 <210> 8 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER. 'T' at position 25 was converted to 'a' to eliminate an upstrem atg (start codon).

<400> 8 cgcggatcct ccctacaact tccaagcgct tcagattatt ag 42 <210> 9 30 <211> 30 <212> DNA <213> Artificial Sequence <220> 35 <223> Description of Artificial Sequence: PRIMER <400> 9 57 cgcggatcca ctaagtggat aaaattagat 30 <210> 10 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 10 gctctagatt gtatttattt attgtattat ac 32 <210> 11 15 <211> 42 <212> DNA <213> Artificial Sequence <220> 20 <223> Description of Artificial Sequence: PRIMER <220> <221> misc-feature <222> (19)..(24) 25 <223> Restriction site sensitive to Xho I and Bsu MI <400> 11 aaggtagttg gcaaataact cgagactaag tggataaaat ta 42 30 <210> 12 <211> 27 <212> DNA <213> Artificial Sequence 35 <220> <223> Description of Artificial Sequence: PRIMER <400> 12 58 cccaagcttg aaagagataa attgaac 27 <210> 13 <211> 23 5 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 13 ccggaattct atctgaacta ctc 23 <210> 14 15 <211> 20 <212> DNA <213> Artificial Sequence <220> 20 <223> Description of Artificial Sequence: PRIMER <400> 14 cggccagcaa cgtcggttcg 20 25 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence 30 <220> <223> Description of Artificial Sequence: PRIMER <400> 15 atgcccacag gccgtcgagt 20 <210> 16 <211> 26 59 <212> DNA <213> Artificial Sequence <220> 5 <223> Description of Artificial Sequence: PRIMER <400> 16 ttcatagttg cattacttat agcttc 26 10 <210> 17 <211> 25 <212> DNA <213> Artificial Sequence 15 <220> <223> Description of Artificial Sequence: PRIMER <400> 17 atgactgcaa ttttagagag acgcg 25 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 18 JO ttatccattt gtagatggag cttcg 25 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 19 cctggctcag gatgaacgct 20 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 20 15 ttcatgcagg cgagttgcag cc 22 <210> 21 <211> 34 <212> DNA 20 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER

25 <400> 21 aaaactgcag tcgactagaa aggagcaatc tgag 34 <210> 22 <211> 38 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 22 aaaactgcag tcgacataaa aaaatttcac tattctga 38 61 <210> 23 <211> 32 <212> DNA 5 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER

10 <400> 23 catgccatgg taaaatcttg gtttatttaa tc 32 <210> 24 <211> 36 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 24 aaaactgcag tcgacgcaac ccactagcat atcgaa 36 <210> 25 25 <211> 452 <212> DNA <213> Rice <400> 25 30 actttcacag tttccattct gaaatgttct ctgtactata ataaatagta agtgaatcaa 60 cttactacta aaaaaattag tagacttcct cttcggaata gaaatagcct atttctacat 120 agggaaagtc gtgtgcaatg aaaaatgcaa gcacgatttg gggagaggtt ttttctctat 180 tgtaacaagg aataattatc tactccatcc gactagttcc gggttcgagt cccgggcaac 240 ccatatggaa actagaaagg agcaatctga gttttgattt ttcactcact tcatttacaa 300 35 aattttttgg tttggtaaat tttgttgtat ggatatacaa ctgtcggggc tggcttggtt 360 gacattggta tatagtctat attatactgt taaataacaa gccttctatt atctttctag 420 ttaatacgtg tgcttgggag tccttgcaat ttaatttgaa taaaccaaga tcttaccatg 480 62 <210> 26 <211> 203 <212> DNA 5 <213> Rice <400> 26 actagaaagg agcaatctga gttttgattt ttcactcact tcatttacaa aattttttgg 60 tttggtaaat tttgttgtat ggatatacaa ctgtcggggc tggcttggtt gacattggta 120 10 tatagtctat attatactgt taaataacaa gccttctatt atctttctag ttaatacgtg 180 tgcttgggag tccttgcaat tt 203 <210> 27 <211> 374 15 <212> DNA <213> Rice <400> 27 ggcttttctg ctaacatata gcaatttttg aagaaaggaa agctagaaat acccaatatc 60 20 ttgctgaagc aagatattgg gtatttcttt tttttttatt ttgaatcttt ctattctgaa 120 ttcagttaac gacgagattt agtatccttt cttgcacttt cataactcgt gaaatgccga 180 gttggtacga attcccccaa tttgcgacct accataggat ttgttatgta aataggtata 240 tgttcctttc cattatgaat cgcgattgta tggccaacca ttgcgggtag aatgctagat 300 gcccgggacc acgttactat tgtttctttc tcctccttca tattgagctt ttctattttt 360 25 gccaataaat gatg 374 <210> 28 <211> 117 <212> DNA 30 <213> Rice <400> 28 ctccccccgc cacgatcgaa cgggaatgga taagaggctt gtgggattga cgtgataggg 60 tagggttggc tatactgctg gtggcgaact ccaggctaat aatctgaagc gcaagga 117 3 5 <210> 29 <211> 256 63 <212> DNA <213> Rice <400> 29 5 actaagtgga taaaattaga tagaaaaaag gtctaaataa aaaagaagag aaatagaaag 60 atcaaaaatc agttacgaaa atgcagtaat tcttcttttt tcttctaatt gattgcaatt 120 aaactcgtct caatctgaaa aaagattgag ccgagtttaa atagattttg atacgatcat 180 gagacttgac aaatcgggat tcctctattc tatatattta gaagatataa aggtataata 240 caataaataa atacaa 256 <210> 30 <211> 484 <212> DNA <213> Tobacco <400> 30 ataaaaaaat ttcactattc tgaaatgttg attgtaatag taattaaggg gtaaatcaac 60 tgagtattca actttttaaa gtctttctaa tttctataag aaaggaactg atgtatacat 120 agggaaagcc gtgtgcaatg aaaaatgcaa gcacggcttg gggaggggtc tttacttgtt 180 20 tatttaattt aagattaaca tttattttat ttaacaagga acttatctac tccatccgac 240 tagttccggg ttcgaatccc gggcaaccca ctagcatatc gaaattctaa ttttctgtag 300 agaagtccgt atttttccaa tcaacttcat taaaaatttg aatagatcta catacacctt 360 ggttgacacg agtatataag tcatgttata ctgttgaata aaaagccttc cattttctat 420 tttgatttgt agaaaactag tgtgcttggg agtccctgat gattaaataa accaagattt 480 25 tacc 484 <210> 31 <211> 222 <212> DNA 30 <213> Tobacco <400> 31 gcaacccact agcatatcga aattctaatt ttctgtagag aagtccgtat ttttccaatc 60 aacttcatta aaaatttgaa tagatctaca tacaccttgg ttgacacgag tatataagtc 120 35 atgttatact gttgaataaa aagccttcca ttttctattt tgatttgtag aaaactagtg 180 tgcttgggag tccctgatga ttaaataaac caagatttta cc 222 <210> 32 64 <211> 1261 <212> DNA <213> Rice 5 <400> 32 cctggctcag gatgaacgct ggcggcatgc ttaacacatg caagtcggac gggaagtggt 60 gtttccagtg gcggacgggt gagtaacgcg taagaacctg cccttgggag gggaacaaca 120 gctggaaacg gctgctaata ccccgtaggc tgaggagcaa aaggaggaat ccgcccgagg 180 aggggctcgc gtctgattag ctagttggtg aggcaatagc ttaccaaggc gatgatcagt 240 agctggtccg agaggatgat cagccacact gggactgaga cacggcccag actcctacgg 300 gaggcagcag tggggaattt tccgcaatgg gcgaaagctg acggagcaat gccgcgtgga 360 ggtagaaggc ccacgggtcg tgaacttctt ttcccggaga agaagcaatg acggtatctg 420 gggaataagc atcggctaac tctgtgccag cagccgcggt aatacagagg atgcaagcgt 480 tatccggaat gattgggcgt aaagcgtctg taggtggctt tttaagtccg ccgtcaaatc 540 ccagggctca accctggaca ggcggtggaa actaccaagc tggagtacgg taggggcaga 600 gggaatttcc ggtggagcgg tgaaatgcgt agagatcgga aagaacacca acggcgaaag 660 cactctgctg ggccgacact gacactgaga gacgaaagct aggggagcga atgggattag 720 ataccccagt agtcctagcc gtaaacgatg gatactaggc gctgtgcgta tcgacccgtg 780 cagtgctgta gctaacgcgt taagtatccc gcctggggag tacgttcgca agaatgaaac 840 tcaaaggaat tgacgggggc ccgcacaagc ggtggagcat gtggtttaat tcgatgcaaa 900 gcgaagaacc ttaccagggc ttgacatgcc gcgaatcctc ttgaaagaga ggggtgcctt 960 cgggaacgcg gacacaggtg gtgcatggct gtcgtcagct cgtgccgtaa ggtgttgggt 1020 taagtcccgc aacgagcgca accctcgtgt ttagttgcca tcgttgagtt tggaaccctg 1080 aacagactgc cggtgataag ccggaggaag gtgaggatga cgtcaagtca tcatgcccct 1140 tatgccctgg gcgacacacg tgctacaatg gccgggacaa agggtcgcga tcccgcgagg 1200 tgagctaacc ccaaaaaccc gtcctcagtt cggattgcag gctgcaactc gcctgcatga 1260 a 1261 <210> 33 <211> 1812 <212> DNA <213> Artificial Sequence <220> 3 35 <223> Description of Artificial Sequence: PCR generated uidA coding region <400> 33 atggtccgtc ctgtagaaac cccaacccgt gaaatcaaaa aactcgacgg cctgtgggca 60 ttcagtctgg atcgcgaaaa ctgtggaatt gatcagcgtt ggtgggaaag cgcgttacaa 120 gaaagccggg caattgctgt gccaggcagt tttaacgatc agttcgccga tgcagatatt 180 5 cgtaattatg cgggcaacgt ctggtatcag cgcgaagtct ttataccgaa aggttgggca 240 ggccagcgta tcgtgctgcg tttcgatgcg gtcactcatt acggcaaagt gtgggtcaat 300 aatcaggaag tgatggagca tcagggcggc tatacgccat ttgaagccga tgtcacgccg 360 tatgttattg ccgggaaaag tgtacgtatc accgtttgtg tgaacaacga actgaactgg 420 cagactatcc cgccgggaat ggtgattacc gacgaaaacg gcaagaaaaa gcagtcttac 480 10 ttccatgatt tctttaacta tgccggaatc catcgcagcg taatgctcta caccacgccg 540 aacacctggg tggacgatat caccgtggtg acgcatgtcg cgcaagactg taaccacgcg 600 tctgttgact ggcaggtggt ggccaatggt gatgtcagcg ttgaactgcg tgatgcggat 660 caacaggtgg ttgcaactgg acaaggcact agcgggactt tgcaagtggt gaatccgcac 720 ctctggcaac cgggtgaagg ttatctctat gaactgtgcg tcacagccaa aagccagaca 780 15 gagtgtgata tctacccgct tcgcgtcggc atccggtcag tggcagtgaa gggcgaacag 840 ttcctgatta accacaaacc gttctacttt actggctttg gtcgtcatga agatgcggac 900 ttgcgtggca aaggattcga taacgtgctg atggtgcacg accacgcatt aatggactgg 960 attggggcca actcctaccg tacctcgcat tacccttacg ctgaagagat gctcgactgg 1020 gcagatgaac atggcatcgt ggtgattgat gaaactgctg ctgtcggctt ttcgctctct 1080 20 ttaggcattg gtttcgaagc gggcaacaag ccgaaagaac tgtacagcga agaggcagtc 1140 aacggggaaa ctcagcaagc gcacttacag gcgattaaag agctgatagc gcgtgacaaa 1200 aaccacccaa gcgtggtgat gtggagtatt gccaacgaac cggatacccg tccgcaaggt 1260 gcacgggaat atttcgcgcc actggcggaa gcaacgcgta aactcgaccc gacgcgtccg 1320 atcacctgcg tcaatgtaat gttctgcgac gctcacaccg ataccatcag cgatctcttt 1380 25 gatgtgctgt gcctgaaccg ttattacgga tggtatgtcc aaagcggcga tttggaaacg 1440 gcagagaagg tactggaaaa agaacttctg

* gcctggcagg agaaactgca tcagccgatt 1500 atcatcaccg aatacggcgt ggatacgtta gccgggctgc actcaatgta caccgacatg 1560 tggagtgaag agtatcagtg tgcatggctg gatatgtatc accgcgtctt tgatcgcgtc 1620 agcgccgtcg tcggtgaaca ggtatggaat ttcgccgatt ttgcgacctc gcaaggcata 1680 30 ttgcgcgttg gcggtaacaa gaaagggatc ttcactcgcg accgcaaacc gaagtcggcg 1740 gcttttctgc tgcaaaaacg ctggactggc atgaacttcg gtgaaaaacc gcagcaggga 1800 ggcaaacaat ga 1812 <210> 34 35 <211> 813 <212> DNA <213> Artificial Sequence 66 <220> <223> Description of Artificial Sequence: PCR generated aadA coding region <400> 34 atggctcgtg aagcggttat cgccgaagta tcgactcaac tatcagaggt agttggcgtc 60 atcgagcgcc atctcgaacc gacgttgctg gccgtacatt tgtacggctc cgcagtggat 120 ggcggcctga agccacacag tgatattgat ttgctggtta cggtgaccgt aaggcttgat 180 10 gaaacaacgc ggcgagcttt gatcaacgac cttttggaaa cttcggcttc ccctggagag 240 agcgagattc tccgcgctgt agaagtcacc attgttgtgc acgacgacat cattccgtgg 300 cgttatccag ctaagcgcga actgcaattt ggagaatggc agcgcaatga cattcttgca 360 ggtatcttcg agccagccac gatcgacatt gatctggcta tcttgctgac aaaagcaaga 420 gaacatagcg ttgccttggt aggtccagcg gcggaggaac tctttgatcc ggttcctgaa 480 15 caggatctat ttgaggcgct aaatgaaacc ttaacgctat ggaactcgcc gcccgactgg 540 gctggcgatg agcgaaatgt agtgcttacg ttgtcccgca tttggtacag cgcagtaacc 600 ggcaaaatcg cgccgaagga tgtcgctgcc gactgggcaa tggagcgcct gccggcccag 660 tatcagcccg tcatacttga agctagacag gcttatcttg gacaagaaga agatcgcttg 720 gcctcgcgcg cagatcagtt ggaagaattt gtccactacg tgaaaggcga gatcactaag 780 20 gtagttggca aataa 795 <210> 35 <211> 2572 <212> DNA 25 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR generated plastid targeting sequence <400> 35 ttgaaagaga taaattgaac aagtatggtc gtcccctgtt gggatgtact attaaaccta 60 aattggggtt atctgctaaa aactacggta gagccgttta tgaatgtctt cgcggtggac 120 ttgattttac taaagatgat gagaacgtga actcacaacc atttatgcgt tggagagatc 180 35 gtttcttatt ttgtgccgaa gcactttata aagcacaggc tgaaacaggt gaaatcaaag 240 ggcattactt gaatgctact gcaggtacat gcgaagaaat gatcaaaaga gctgtatttg 300 ctagagaatt gggcgttccg atcgtaatgc atgactactt aacgggggga ttcaccgcaa 360 67 atactagctt ggctcattat tgccgagata atggtctact tcttcacatc caccgtgcaa 420 tgcatgcggt tattgataga cagaagaatc atggtatcca cttccgggta ttagcaaaag 480 cgttacgtat gtctggtgga gatcatattc actctggtac cgtagtaggt aaacttgaag 540 gtgaaagaga cataactttg ggctttgttg atttactgcg tgatgatttt gttgaacaag 600 5 atcgaagtcg cggtatttat ttcactcaag attgggtctc tttaccaggt gttctacccg 660 tggcttcagg aggtattcac gtttggcata tgcctgctct gaccgagatc tttggggatg 720 attccgtact acagttcggt ggaggaactt taggacatcc ttggggtaat gcgccaggtg 780 ccgtagctaa tcgagtagct ctagaagcat gtgtaaaagc tcgtaatgaa ggacgtgatc 840 ttgctcagga aggtaatgaa attattcgcg aggcttgcaa atggagcccg gaactagctg 900 10 ctgcttgtga agtatggaaa gagatcgtat ttaattttgc agcagtggac gttttggata 960 agtaaaaaca gtagacatta gcagataaat tagcaggaaa taaagaagga taaggagaaa 1020 gaactcaagt aattatcctt cgttctctta attgaattgc aattaaactc ggcccaatct 1080 tttactaaaa ggattgagcc gaatacaaca aagattctat tgcatatatt ttgactaagt 1140 atatacttac ctagatatac aagatttgaa atacaaaatc tagaaaacta aatcaaaatc 1200 15 taagactcaa atctttctat tgttgtcttg gatccacaat taatcctacg gatccttagg 1260 attggtatat tcttttctat cctgtagttt gtagtttccc tgaatcaagc caagtatcac 1320 acctctttct acccatcctg tatattgtcc cctttgttcc gtgttgaaat agaaccttaa 1380 tttattactt atttttttat taaattttag atttgttagt gattagatat tagtattaga 1440 cgagatttta cgaaacaatt atttttttat ttctttatag gagaggacaa atctcttttt 1500 20 tcgatgcgaa tttgacacga cataggagaa gccgcccttt attaaaaatt atattatttt 1560 aaataatata aagggggttc caacatatta atatatagtg aagtgttccc ccagattcag 1620 aacttttttt caatactcac aatccttatt agttaataat cctagtgatt ggatttctat 1680 gcttagtctg ataggaaata agatattcaa ataaataatt ttatagcgaa tgactattca 1740 tctattgtat tttcatgcaa atagggggca agaaaactct atggaaagat ggtggtttaa 1800 25 ttcgatgttg tttaagaagg agttcgaacg caggtgtggg ctaaataaat caatgggcag 1860 tcttggtcat attgaaaata ccaatgaaga tccaaataga aaagtgaaaa acattcatag 1920 ttggaggaat agtgacaatt ctagttgcag taatgttgat tatttattcg gcgttaaaga 1980 cattcggaat ttcatctctg atgacacttt tttagttagt gataggaatg gagacagtta 2040 ttccatatat tttgatattg aaaatcatat ttttgagatt gacaacgatc attcttttct 2100 30 gagtgaacta gaaagttctt tttatagtta tcgaaactcg aattatagga ataatggatt 2160 taggggcgaa gatccctact ataattctta catgtatgat actcaatata gttggaataa 2220 tcacattaat agttgcattg atagttatct tcagtctcaa atctgtatag atacttccat 2280 tataagtggt agtgagaatt acggtgacag ttacatttat agggccgttt gtggtggtga 2340 aagtcgaaat agtagtgaaa acgagggttc cagtagacga actcgcacga agggcagtga 2400 35 tttaactata agagaaagtt ctaatgatct cgaggtaact caaaaataca ggcatttgtg 2460 ggttcaatgc gaaaattgtt atggattaaa ttataagaaa tttttgaaat caaaaatgaa 2520 tatttgtgaa caatgtggat atcatttgaa aatgagtagt tcagatagaa tt 2572 68 <210> 36 <211> 28 <212> DNA 5 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER

10 <400> 36 attcgagctc ttatttcaat gatattat 28 <210> 37 <211> 28 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 37 ggggtaccga tcctggccta gtctatag 28 <210> 38 25 <211> 375 <212> DNA <213> Rice <400> 38 30 tcttatttca atgatattat tatttcaaag ataagagata ttcaaagata agagataaga 60 agaagtcaaa atttgatttt ttttttggaa aaaaaaaatc aaaaagatat agtaacatta 120 gcaagaagag aaacaagttc tatttcacaa tttaaacaaa tacaaaatca aaatagaata 180 ctcaatcatg aataaatgca agaaaataac ctctccttat ttttctataa tgtaaacaaa 240 aaagtatatg taagtaaaat actagtaaat aaataaaaag aaaaaaagaa aggagcaata 300 35 gcacactatt gatagaacaa gaaaatgatt attgatcatt tcttttcaaa acctcctata 360 gactaggcca ggatc 375 69 <210> 39 <211> 1062 <212> DNA <213> Rice <400> 39 ttatccattt gtagatggag cttcgatagc agataggtat agagggaagt tgtgagoatt 60 acgttcatgc ataacttcca taccaaggtt agcacggtta atgatatcag cccaagtatt 120 10 aattacacgg ccttgactgt caactacaga ttggttgaaa ttgaaaccat ttaggttgaa 180 agccatagtg ctgataccta aagcggtaaa ccagatacct actacaggcc aagcagctag 240 gaagaagtgt aacgaacgag agttgttgaa actagcatat tggaagatca atcggccaaa 300 ataaccatga gcggctacga tgttataagt ttcttcctct tgaccgaatc tgtaaccttc 360 attagcagat tcattttctg tggtttccct gatcaaacta gaagttacca aggaaccatg 420 15 catagcactg aatagggagc cgccgaatac accagctacg cctaacatgt gaaatgggtg 480 cataaggatg ttgtgctcag cctggaatac aatcatgaaa ttgaaagtac cagagattcc 540 tagaggcata ccatcagaaa aacttccttg accaattggg tagatcaaga aaactgcggt 600 agcagctgca acaggagctg aatatgcaac agcaatccaa ggtcgcatac ccagacggaa 660 actaagctcc cactcacgac ccatgtaaca agctacgcca agtaagaagt gtagaacaat 720 20 tagttcataa ggaccaccgt tgtataacca ttcatgaacg gatgccgctt cccagattgg 780 gtaaaaatgt aaacctatag ctgcagaagt aggaataatg gcaccggaaa taatattgtt 840 tccgtaaagt agagaccctg aaacaggttc acgaatacca tcaatgtcta ctggaggagc 900 agcaatgaag gcaataataa atacagaagt tgccgtcaat aaggtaggga tcatcaaaac 960 accaaaccat ccaatgtaaa gacggttttc agtgctagtt atccagttac agaagcgacc 1020 25 ccataggctt tcgctttcgc gtctctctaa aattgcagtc at 1062 <210> 40 <211> 10 <212> DNA 30 <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Cla I site linker <400> 40 gatcatcgat 10 <210> 41 <211> 32 5 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PRIMER <400> 41 cgcggatcca tggtccgtcc tgtagaaacc cc 32 <210> 42 15 <211> 36 <212> DNA <213> Artificial Sequence <220> 20 <223> Description of Artificial Sequence: PRIMER <400> 42 gctcgagctc ccgggtcatt gtttgcctcc ctgctg 36 25 <210> 43 <211> 31 <212> DNA <213> Artificial Sequence 30 <220> <223> Description of Artificial Sequence: PRIMER <400> 43 cgcggatcct atggctcgtg aagcggttat c 31 <210> 44 <211> 32 <212> DNA <213> Artificial Sequence <220> 5 <223> Description of Artificial Sequence: PRIMER <400> 44 ccgctcgagt tatttgccaa ctaccttagt ga 32 10 <210> 45 <211> 38 <212> DNA <213> Artificial Sequence 15 <220> <223> Description of Artificial Sequence: Primer <400> 45 gatcttacca tgggcccgcg gaggcctatc gatgagct 38 <210> 46 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer

30 <400> 46 catgcatagg cctccgcggg cccatggtaa 30 <210> 47 35 <211> 36 <212> DNA <213> Artificial Sequence 72 <220> <223> Description of Artificial Sequence: Primer

5 <400> 47 catgccatgg gacacgtgca tcaccatcac catcac 36 <210> 48 10 <211> 29 <212> DNA <213> Artificial Sequence <220> 15 <223> Description of Artificial Sequence: Primer <400> 48 ccgcgggccc cgtgatggtg atggtgatg 29 <210> 49 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 49 30 ggccggggcc catcgaaggt cgccaggacc cgtacgttaa agaa 44 <210> 50 <211> 33 35 <212> DNA <213> Artificial Sequence 73 <220> <223> Description of Artificial Sequence: Primer <400> 50 5 cgccgggccc ttactgagaa gcacgacgac cgc 33 <210> 51 <211> 32 10 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 51 cgcggatcca tgttacgtcc tgtagaaacc cc 32 <210> 52 20 <211> 35 <212> DNA <213> Artificial Sequence <220> 25 <223> Description of Artificial Sequence: Primer <400> 52 ggggtaccct tagattgttt gcctcccctg ctgcg 35 <210> 53 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer

74 <400> 53 aaactgcaga tcgaaggtcg ccaggacccg tacgttaaag aa 42 <210> 54 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 54 15 ctgcatgcat ctctagacta ttactgagaa g 31 <210> 55 <211> 30 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 55 tgctctagac tattactgag aagcacgacg 30 30 <210> 56 <211> 30 <212> DNA <213> Artificial Sequence 35 <220> <223> Description of Artificial Sequence: Primer <400> 56 tgctctagat gcaggacccg tacgttaaag 30 5 <210> 57 <211> 30 <212> DNA <213> Artificial Sequence 10 <220> <223> Description of Artificial Sequence: Primer <400> 57 tgctctagac tattactgag aagcacgacg 30 <210> 58 <211> 39 <212> DNA <213> Artificial Sequence 20 <220> <223> Description of Artificial Sequence: Primer <400> 58 ggatcctccc tacaacttcc aagcgcttca gattattag 39 <210> 59 <211> 22 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 59 ttcatgcagg cgagttgcag cc 22 76 5 <210> 60 <211> 10 <212> DNA <213> Artificial Sequence 10 <220> <223> Description of Artificial Sequence: ClaI site-containing linker <400> 60 gatcatcgat 10 77

Claims (1)

1. I A method of producing a protein of interest comprising allowing a polynucleotide fusion construct to be expressed in a plastid thereby to 5 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 10 of interest; (b) is resistant to internal cleavage in vivo; and (c) comprises a cleavage site.

   2. A method according to claim I wherein the plastid is a chloroplast.

   A method wherein the accumulation of the fusion protein as a percentage of the total cell protein in a cell comprising a plastid according claim I or 2 is from I O-fold to I 000-fold higher than the accumulation of the individually expressed protein of interest.

   4. A method according to any one of the preceding claims wherein the protein of interest comprises a human protein, or a biologically active variant or fragment thereof.

   25 5. A method according to any one of the preceding claims wherein the protein of interest comprises a pharmaceutically active protein.

   6. A method according to any one of the preceding claims wherein the protein of interest comprises an interferon, or a biologically active variant or fragment 30 thereof.

   78 7. A method according to any one of the preceding claims wherein the protein of interest comprises a human interferon-gamma (hIFN-,y), or a biologically active variant or fragment thereof, 5 8. A method according to any one of the preceding claims wherein the fusion protein partner has a scorable property.

   9. A method according to claim 8 wherein the fusion protein partner comprises GUS or GFP, or a variant or fragment of either that has GUS or GFP activity.

   10. A method according to any one of the preceding claims wherein the N terminal of the fusion protein partner is fused to the C-terminal of the polypeptide of interest.

   15 11. A method according to any one of the preceding claims wherein the cleavage site is IEGR.

   12. A method according to any one of the preceding claims wherein the fusion protein comprises a purification tag, which purification tag comprises a 20 sequence which facilitates affinity purification.

   13. A method according to claim 12 wherein the purification- Tag is a His- tag.

   14. A method according to claim 12 or 13 wherein the purification tag is retained 25 at a terminus of the fusion protein partner following in vitro cleavage of the cleavage site.

   15. A method according to any one of the preceding claims further comprising obtaining a sample comprising the thus expressed fusion protein.

   16. A method according to claim 15 further comprising enriching the thus 79 obtained sample in the fusion protein.

   17. A method according to claim 16 wherein the step of enriching comprises ftactionating the sample comprising the fusion protein and selecting the 5 ftaction or fractions enriched in the fusion protein by detecting the scorable property of the fusion protein partner, thereby to provide a sample enriched in the fusion protein.

   18. A method according to anyone of claims 15 to 17 further comprising 10 recovering the fusion protein from a sample by an affinity-based technique that uses a purification tag.

   19. A method wherein prior to recovery of the fusion protein by a method according to claim 18 the sample is enriched in the fusion protein by a 15 method according to claim 16 or 17.

   20. A method to any one of claims 15 to 19 which further comprises cleaving the fusion protein in vitro to release the protein of interest.

   A method according to claim 20 wherein cleavage has a fidelity of at least 95%.

   22. A method according to claim 20 or 21 wherein the sample is enriched by a method according to claim 16 or 17 prior to cleavage.

   23. A method according to any one of claims 20 or 22 wherein the fusion protein is recovered by a method according to claim 18 or 19 prior to cleavage.

   24. A method according to any one of claims 20 to 23 comprising recovering the 30 thus released protein of interest 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.

   25. A method of obtaining a transplastornic plastid, which method comprises transforming a plastome within a plastid by a fusion construct as defined in any one of the claims I or 3 to 14.

   26. A method of obtaining a transplastornic cell, which method comprises transforming a plastid within a cell by a method according to claim 25.

   10 27. A method of obtaining a homotransplastomic cell, which method comprises obtaining transplastornic cells by a method according to claim 26 and selecting for the presence of the transplastome.

   28, A method of obtaining a first-generation transplastomic or 15 homotransplastomic plant, wherein the method comprises regenerating a transplastornic or homotransplastomic plant cell obtainable by the method of claim 26 or 27 to give a transplastornic or homotransplastomic plant.

   29. A method of obtaining a transplastornic or homotransplastomic plant seed, 20 wherein the method comprises obtaining a transplastornic or homotransplastomic seed from a transplastornic or homotransplastomic plant obtainable by the method of claim 28.

   30. A method of obtaining a transplastornic or homotransplastomic progeny plant 25 comprising obtaining a second-generation transplastornic or homotransplastomic progeny plant from a first-generation transplastomic or hornotransplastomic plant obtainable by a method according to claim 28, and optionally obtaining transplastomic or homotransplastomic plants of one or more further generations from the second-generation progeny plant thus 30 obtained.

   81 31. A method according to claim 30 comprising:

   (1) obtaining a transplastomic or homotransplastomic seed from a first-generation transplastomic or homotransplastomic plant obtainable by the method according to claim 28, then obtaining a 5 second-generation transplastomic or homotransplastomic progeny plant from the seed; and/or (ii) propagating clonally a first-generation transplastomic or homotransplastomic plant obtainable by the method according to claim 28 to give a second-generation transplastomic or 10 homotransplastomic progeny plant; and/or (iii) crossing a first-generation transplastomic or homotransplastomic plant obtainable by a method according to claim 28 with another plant to give a second-generation transplastomic or homotransplastomic progeny plant; and optionally; 15 (iv) obtaining transplastomic or homotransplastomic progeny plants of one or more further generations from the second-generation transplastomic or homotransplastomic progeny plant thus obtained.

   2. Use of a polynucleotide encoding a fusion protein partner to increase the 20 stability of a recombinantly expressed protein of interest in a plastid.

   3.). Use of a polynucleotide fusion construct as defined in any one of the claims 1 or to 14 to increase the stability of a recombinantly expressed protein of interest in a plastid.

   34. A transplastome, a transplastomic or homotransplastomic plastid, transplastomic or homotransplastomic plant cell, transplastomic or homotransplastomic callus, a transplastomic or homotransplastomic first generation plant, transplastomic or homotransplastomic plant seed or progeny 30 plant obtainable by a method according to any one of claims 25 to 31 or comprising a polynucleotide fusion construct as defined in claim 32 or 33.

   82 3 5. A transplastomic or homotransplastornic plastid, transplastornic or homotransplastomic plant cell, transplastomic or hornotransplastomic callus, a transplastornic or homotransplastomic first-generation plant, transplastornic or homotransplastomic plant seed or progeny plant according to claim 34 wherein the polynucleotide fusion construct is integrated into the plastome.

   _3 6. A fusion protein or protein of interest obtained by a method according to any one of claims 15 to 24 or from a cell or plant as defined in any one of claims 26 to ") 1.

   3 7. A method of obtaining a crop product comprising harvesting a crop product from a cell or plant according to claim 34 or 35 and optionally further processing the harvested product.

   38. A crop product obtainable by a method according to claim 37.

   83