EP1272649A2 - Verfahren zur herstellung authentischer cytokine - Google Patents

Verfahren zur herstellung authentischer cytokine

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Publication number
EP1272649A2
EP1272649A2 EP01926581A EP01926581A EP1272649A2 EP 1272649 A2 EP1272649 A2 EP 1272649A2 EP 01926581 A EP01926581 A EP 01926581A EP 01926581 A EP01926581 A EP 01926581A EP 1272649 A2 EP1272649 A2 EP 1272649A2
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EP
European Patent Office
Prior art keywords
nucleic acid
acid sequence
host system
plant host
cytokine
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP01926581A
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English (en)
French (fr)
Inventor
Douglas Russell
Michael Schlittler
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Monsanto Technology LLC
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Monsanto Technology LLC
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Publication of EP1272649A2 publication Critical patent/EP1272649A2/de
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8257—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/61—Growth hormone [GH], i.e. somatotropin

Definitions

  • FIELD OF INVENTION This invention describes a novel method of producing and recovering bioactive recombinant proteins from plants. General methods of designing and engineering plants for expression of such proteins, and methods of purification, are also disclosed. Methods for the expression of proteins, such as growth hormone (GH) and granulocyte colony stimulating factor (G-CSF), in plants, and methods of isolating authentic heterologous proteins from plants are specifically disclosed.
  • the new method may be more cost-effective than other large-scale expression systems, by eliminating the need for refolding and other extensive manipulations that generate an active protein with a desired amino terminus.
  • Recombinant proteins that mimic or have the same structure as native proteins are highly desired for use in therapeutic applications, as components in vaccines and diagnostic test kits, and as reagents for structure/function studies. Mammalian, bacterial, and insect cells are commonly used to express recombinant proteins for such applications. Systems capable of accurately producing the desired protein within the host cell are preferred to systems that generate modified proteins or that require extensive procedures to remove the undesired forms.
  • baculovirus insect cell system can produce high levels of glycosylated proteins, these proteins are not secreted, however, thus making purification complex and expensive.
  • Transgenic animals are subject to lengthy lead times to develop herds with stable genetics, high operating costs, and contamination by prions or viruses.
  • Prokaryotic hosts may also suffer disadvantages in expressing heterologous proteins.
  • the post-translational modifications required for bioactivity may not be carried out in the prokaryote host.
  • Some of these post-translational modifications include signal peptide processing, pro-peptide processing, protein folding, disulfide bond formation, glycosylation, gamma carboxylation, and beta-hydroxylation.
  • complex proteins derived from prokaryote hosts are not always properly folded or processed to provide the desired degree of biological activity. Consequently, prokaryote hosts have generally been utilized for the expression of relatively simple foreign polypeptides that do not require folding or post-translational processing to achieve a biologically active protein.
  • the amino acid composition of protein termini are biased in many different manners. Berezovsky et al., 12(1) PROTEIN ⁇ NG'G 23-30 (1999). Systematic examination of N- exopeptidase activities led to the discovery of the 'N-terminar- or 'N-end rule': the N- terrninal (f)Met is cleaved if the next amino acid is Ala, Cys, Gly, Pro, Ser, Thr, or Val. If this next amino acid is Arg, Asp, Asn, Glu, Gin, He, Leu, Lys or Met, the initial (f)Met remains as the first amino acid of the mature protein. The radii of hydration of the amino acid side chains was proposed as physical basis for these observations.
  • Recombinant proteins that retain the N-terminal methionine in some cases, have biological characteristics that differ from the native species lacking the N-terminal methionine.
  • Human growth hormone that retains its N-terminal methionine (Met-hHG), for example, may be antigenic compared to hGH purified from natural sources or recombinant hGH that is prepared in such a way that has the same primary sequence as native hGH
  • One method of preparing native proteins in bacteria is to express the desired protein as part of a larger fusion protein containing a recognition site for an endoprotease that specifically cleaves upstream from the start of the native amino acid sequences.
  • the recognition and cleavage sites can be those recognized by native signal peptidases, which specifically cleave the signal peptide of the N-terminal end of a protein targeted for delivery to a membrane or for secretion from the cell.
  • recognition and cleavage sites can be engineered into the gene encoding a fusion protein so that recombinant protein is susceptible to other non-native endoproteases in vitro or in vivo.
  • the blood clotting factor Xa, collagenase, and the enzyme enterokinase can be used to release different fusion tags from a variety of proteins. Economic considerations, however, generally preclude use of endoproteases on a large scale for pharmaceutical use.
  • Preparation of hGH from bacterial systems, that encode genes having additional amino acids at the N-terminus are known in the art.
  • Derivatives of hGH containing amino acid substitutions are also known.
  • MAP methionine aminopeptidase
  • U.S. Pat. Nos. 4,870,017 and 5,013,662 describe the cloning, expression, and use of E. coli methionine aminopeptidase to remove Met from a variety of peptides and Met- IL-2.
  • WO 84/02351 discloses a process for preparing ripe (native) proteins, such as hGH or human proinsulin, from fusion proteins using leucine aminopeptidase.
  • a method of removing the N-terminal methionine from derivatives of human interleukin-2 and hGH using aminopeptidase M, leucine aminopeptidase, aminopeptidase PO, or aminopeptidase P has been described.
  • More complicated methods can also be used to generate recombinant proteins with a native amino terminus.
  • U.S. Pat. Nos. 5,565,330 and 5,573,923 refers to methods of removing dipeptides from the amino-terminus of precursor polypeptides involving treatment of the precursor with dipetidylaminopeptidase (dDAP) from the slime mold Dictostelium descoideum, which has a mass of about 225 kDa and a pH optimum of about 3.5.
  • dDAP dipetidylaminopeptidase
  • Precursors of human insulin, analogues of human insulin, and human growth hormone containing dipeptide extensions were processed by dDAP when the dDAP was in free solution and when it was immobilized on a suitable solid support surface.
  • Plant-based expression systems may be more cost-effective than other large-scale expression systems for the production of therapeutic proteins, by eliminating the need for refolding, and other extensive manipulations that generate a protein with a native amino terminus.
  • a wide variety of therapeutic proteins, for example, have already been expressed in many different plant hosts.
  • a nonexclusive list of the yield and quality of proteins recovered from transgenic plants is shown in Table 1.
  • the present invention contemplates producing bioactive cytokines from a plant host systems.
  • the cytokines of the present invention may be any mammalian soluble protein or peptide which acts as a humoral regulator at the nano- to pico-molar concentration, and which either under normal or pathological conditions, modulate the functional activities of individual cells and tissues.
  • the cytokines may also mediate interactions between cells directly and regulate processes talcing place in the extracellular environment.
  • the cytokines of the present invention belong to the cytokine superfamalies, which include, but are not limited to: the Tumor Growth Factor-beta (TGF-beta) superfamily (comprising various TGF-beta isoforms, Activin A, Inhibins, Bone Morphogenetic Proteins (BMP), Decapentaplegic Protein (DPP), granulocyte colony stimulating factor (G-CSF), Growth Hormone (GH) (including human growth hormone (hGH)), Interferons (IFN), and Interleukins (LL)); the Platelet Derived Growth Factor (PDGF) superfamily (comprising VEGF); the Epidermal Growth Factor (EGF) superfamily (comprising EGF, TGF-alpha, Amphiregulin (AR), Betacellulin, and HB-EGF); the Vascular Epithelial Growth Factor (VEGF) family; Chemokines; and Fibroblast Growth factors (FGF).
  • the methods of the present invention are applicable to any cytokine, whether or not yet discovered, and are not limited to any particular cytokine exemplified herein. See, e.g., Hill et al, 90 P.N.A.S. 5167-71 (1993).
  • More efficient strategies to process amino acids from the amino terminus of recombinant proteins are desirable to reduce the cost of generating therapeutic proteins that mimic the structure of native proteins.
  • Methods that increase the levels of expression or facilitate the downstream processing of recombinant proteins will also accelerate the selection and development of small chemical molecules and other protein-based molecules destined for large scale clinical trials. Therefore, the method and compositions provided by the present invention may yield more efficient and cost effective means for producing therapeutic proteins that mimic the structure of authentic proteins.
  • the present invention provides methods for producing a cytokine in a plant host system in which the plant host system had been transformed with a chimeric nucleic acid that encodes the cytokine, the method including cultivating the transformed plant under conditions that result in the expression of the cytokine in the plant host system.
  • a further aspect of this method includes the purification of the cytokine from the plant host system.
  • the cytokine produced in the plant host system is free from amino acid modifications such as hydoxyproline, and free from novel glycosylations.
  • the method of the present invention employs a chimeric nucleic acid sequence that includes a first nucleic acid that regulates the transcription in the plant host system of a
  • the chimeric nucleic acid sequence also contains a fourth nucleic acid sequence.
  • the fourth nucleic acid is a KDEL amino acid sequence.
  • the first nucleic acid is a plant-active transcription promoter.
  • the second nucleic acid sequence targets the cytokine to a sub-cellular location within the plant host system. Such sub-cellular locations are preferably the cytosol, plastid, or endoplasmic reticulum.
  • the second nucleic acid encodes a portion of ubiquitin, more preferably a monomer of yeast ubiquitin gene or a monomer of potato ubiquitin gene 3.
  • the second nucleic acid encodes a portion of the oleosin sufficient to provide sub-cellular targeting.
  • the oleosin portion is specifically cleavable by enzymatic or chemical means included between the oleosin portion and the cytokine.
  • the nucleic acid sequence encoding oleosin is derived from soy. i 0
  • the method of the present invention provides for the production in a plant host system of cytokines such as those of the cytokine superfamilies TGF-beta, PDGF, EGF, VEGF, chemokines, and FGF.
  • the cytokine is either GH, hGH, or G-CSF.
  • the invention described herein also provides a plant host system that has been transformed with a chimeric nucleic acid sequence that includes a first nucleic acid that regulates the transcription in the plant host system of a second nucleic acid sequence that encodes a signal sequence that is linked in reading frame to a third nucleic acid sequence that
  • the chimeric nucleic acid sequence also contains a fourth nucleic acid sequence.
  • the fourth nucleic acid is a KDEL amino acid sequence.
  • the first nucleic acid is a plant-active transcription promoter.
  • the second nucleic acid is a plant-active transcription promoter.
  • L 0 sequence targets the cytokine to a sub-cellular location within the plant host system.
  • Such sub-cellular locations are preferably the cytosol, plastid, or endoplasmic reticulum.
  • the second nucleic acid encodes a portion of ubiquitin, more preferably a monomer of yeast ubiquitin or a monomer of potato ubiquitin gene 3.
  • the second nucleic acid encodes a portion of the
  • the oleosin portion is specifically cleavable by enzymatic or chemical means included between the oleosin portion and the cytokine.
  • the nucleic acid sequence encoding oleosin is derived from soy.
  • cytokines such as those of the cytokine superfamilies TGF-beta, PDGF, EGF, VEGF, chemokines, and FGF.
  • the cytokme is either GH, hGH, or G-CSF.
  • the cytokine may be purified from the plant host system, and the cytokine produced in the plant host system is free from amino acid modifications such as hydoxyproline, and free from novel glycosylations.
  • the present invention also relates to a chimeric nucleic acid sequence expressed in a plant host system, that includes a first nucleic acid that regulates the transcription in the plant host system of a second nucleic acid sequence that encodes a signal sequence that is linked in reading frame to a third nucleic acid sequence that encodes a cytokine.
  • the chimeric nucleic acid sequence also contains a fourth
  • the fourth nucleic acid is a KDEL amino acid sequence.
  • the first nucleic acid is a plant- active transcription promoter.
  • the second nucleic acid sequence targets the cytokine to a sub-cellular location within the plant host system. Such sub-cellular locations are preferably the cytosol, plastid, or endoplasmic reticulum.
  • the second nucleic acid encodes a portion of ubiquitin, more preferably a monomer of yeast ubiquitin or a monomer of potato ubiquitin gene 3.
  • the second nucleic acid encodes a portion of the oleosin gene sufficient to provide sub-cellular targeting.
  • the oleosin portion is specifically cleavable by enzymatic or chemical means included between the oleosin portion and the cytokine.
  • the nucleic acid sequence that encodes oleosin is derived from soy.
  • the chimeric nucleic acid sequence provides for the production in a plant host system of cytokines such as those of the cytokine superfamilies TGF-beta, PDGF, EGF, VEGF, chemokines, and FGF. More preferably, the cytokine is either GH, hGH, or G-CSF. In another preferred embodiment of the invention, the hGH encoded by a portion of the chimeric nucleic acid sequence has an authentic N- terminus. In another preferred embodiment, the G-CSF encoded by a portion of the chimeric nucleic acid sequence has a authentic N-terminus.
  • the cytokines encoded by the chimeric nucleic acid sequences are free of novel glycosylations and modified amino acids such as hydroxyproline.
  • the chimeric nucleic acid sequence is included in an expression cassette.
  • the invention embodied herein also contemplates a plant, plant cell culture, or plant seed transformed with this chimeric nucleic acid sequence.
  • the invention herein also contemplates a cytokine produced in a plant that has been transformed by the chimeric nucleic acid sequence described herein.
  • the invention herein provides a method for preparing a bioactive, authentic mammalian growth hormone in com plants, by inserting a gene for said growth hormone into a com plant expression vector; transforming com plant cells with an expression vector; generating whole com plants from the transformed com cells; harvesting com seed from whole com plants; and purifying the growth hormone from powdered corn seed.
  • com plants and corn seed have been prepared by this method.
  • the mammalian growth hormone is human growth hormone.
  • the growth hormone accumulates to a level greater than 1% of the total soluble protein in a plant sample. More particularly, the growth hormone accumulates to level greater than 5% of the total soluble protein in a plant sample, hi another preferred aspect of the method, the growth hormone is not glycosylated.
  • the com plant expression vector is pwrg4825.
  • authentic human growth hormone from com seed is further purified by extracting com seed (that has been crashed or powdered) with buffered saline, wherein said extraction is carried out at a pH ranging from about pH 8 to about pH 10; adding urea to a concentration of about 2M to 3.5 M urea; adjusting the pH of the extract to about pH 5; clarifying the solution; purifying by cation exchange chromatography, wherein said cation exchange chromatography is carried out in the presence of urea at a pH from about 4.5 to about 5.5; and purifying by anion exchange chromatography, wherein said anion exchange chromatography is carried out in the absence of urea at apH from about 7.0 to about 8.0.
  • Figure 1 depicts the amino acid sequence of hGH, a single-chain polypeptide (22 kDa) (SEQ ID NO: 12), containing four cysteine residues involved in two disulfide bond linkages.
  • Figure 2 is a diagram of the com transformation vector pwrg4825. Restriction sites used for the construction are shown. Plant expression elements are defined as boxes, and bacterial vector sequences as a thin line.
  • Figure 3 is a chart summarizing different vectors constructed for the expression of hGH in plants.
  • Figure 4 is a Western blot of hGH transient expression (using CaMV 35S, or eFMV for CTP2) with different targeting signals: extensin, targeting secretion (EXT); '5 UTR, targeting cytosol (DSSU); chloroplast transit peptide, targeting plastids (CTP2); and hGH control (Stnd).
  • Figure 5 shows a Western blot of hGH fexpressed transiently in soy hypocotyl tissues from vectors with the CaMV 35S promoter and different targeting signals: standard (3 ng); null (--); cytosol (DSSU); extensin (EXT); potato ubiquitin (potato ubi); and yeast ubiquitin (yeast ubi).
  • Figure 6 shows a Western blot of an hGH oleosin fusion expressed transiently in soy hypocotyl tissues: null (--); standard (1 ng); oleosin fusion (OLE); and extensin (EXT).
  • Figure 7 is a chart summarizing the expression of hGH in transgenic soy seeds.
  • Figure 8 depicts a Western blot of hGH expression in transgenic soy seeds (A, B, C, and D, two seeds each from 2 different pods) compared to standards (1 ng and 0.2 ng).
  • Figure 9 charts a summary for transgenic tobacco cell and suspension media expression of hGH with different targeting designs.
  • Figure 10 is a Western blot showing hGH expression with different targeting signal sequences in tobacco cells: cytosol; endoplasmic reticulum (ER); plastid; null (N); and standard (32 ng) .
  • Figure 11 summarizes tobacco plant expression of hGH with different targeting designs.
  • Figure 12 depicts the bioactivity of hGH secreted and partially purified from transformed tobacco cells compared to an E. coli standard.
  • Figure 13 plots the mass spectrometry results for Phe-hGH expressed in tobacco cells.
  • Figure 14 tabulates the com seed expression and inheritance of different hGH transformation events.
  • Figure 15 is a Western blot comparing hGH expression found in seed extracts from independent first-generation transformation events, compared to a 0.5 ng hGH standard spiked into a non-expressing seed extract.
  • Figure 16 depicts graphically the bioactivity of com seed-derived hGH (Com sample) compared with that of refolded E. coli-de ⁇ ved hGH in null com extract (spiked control). Samples were diluted, and tested via a cell proliferation-based assay, to show bioactivity at a level expected from the ELISA-based quantitation.
  • Figure 17A-B presents mass spectrophotometry data of com-derived hGH. Com seed hGH was purified, and analyzed by mass spectrophotometry to show recovery of significant levels of authentic-sized hGH at 21 ,225 Da, consistent with proper disulfide linkages and no deleterious amino acid modifications.
  • Figure 18 shows a scheme for isolating human growth hormone from com seed.
  • Figure 19A-B illustrates anion exchange HPLC of hGH isolated from com seed and
  • Figure 19A shows an anion exchange HPLC profile of hGH isolated from com seed.
  • Figure 19B shows the profile of hGH isolated from E. coli.
  • Figure 20 shows the reverse-phase HPLC profile of hGH isolated from com seed and E. coli.
  • Panel A shows a reverse-phase HPLC profile of hGH isolated from com seed.
  • Panel B shows the profile of hGH isolated fromE. coli.
  • Figure 21 A-B depicts the tryptic peptide reverse phase HPLC chromatograms of hGH isolated from com seed (A) and E. coli(B).
  • Figure 22 compares graphically the weight gain in rats treated with either corn- derived or E. e ⁇ /t-derived hGH.
  • Figure 23 charts the vectors designed for the expression of G-CSF.
  • Figure 24 is a Western blot showing the transient expression (via the CaMV 35S promoter or eFMV promoter for CTP)of MetAla-GCSF targeted to different subcellular organelles of soy and com tissues.
  • Figure 25 is a Western blot reflecting transient expression of G-CSF in corn leaves, comparing different codon designs and non-transformed leaves against a 10 ng standard.
  • Figure 26 is a Western depicting transient expression of G-CSF in com, with (+ KD ⁇ L) and without the KD ⁇ L (- KD ⁇ L) fusion, comparing total com extract (total) to extracellular wash (wash), and a 5 ng standard.
  • Figure 27 presents a summary of G-CSF expression in tobacco cells and suspension media.
  • Figure 28 shows a Western blot of G-CSF expressed in transgenic tobacco cells and resultant suspension media, from different constructs. All constracts contained a secretion signal, but differ in codon design and use of KC ⁇ L fusion.
  • Figure 29 illustrates the results of electron spray mass spectrometry of purified MetAla G-CSF.
  • Figure 30 charts the results for liquid chromatography-electron spray mass spectrometry analysis of partially digested purified MetAla G-CSF.
  • Figure 31 illustrates the results of a bioassay of plant-derived (tobacco cell) MetAla G-CSF compared to an E coli derived refolded standard.
  • HSA human serum albumin
  • the present invention provides methods and compositions for producing and recovering bioactive recombinant proteins from plants.
  • recombinant proteins include cytokines.
  • the cytokines of the present invention may be any mammalian soluble protein or peptide which acts as a humoral regulator at the nano- to pico-molar concentration, and which either under normal or pathological conditions, modulate the functional activities of individual cells and tissues.
  • the cytokines may also mediate interactions between cells directly and regulate processes talcing place in the extracellular environment.
  • the cytokines of the present invention are belong to the cytokine superfamalies, which include, but are not limited to: the Tumor Growth Factor-beta (TGF-beta) superfamily (comprising various TGF-beta isoforms, Activin A, Inhibins, Bone Morphogenetic Proteins (BMP), Decapentaplegic Protein (DPP), G-CSF, Growth Hormone (GH, more particularly human growth hormoner (hGH)), Interferons (IFN), and Interleu ins (LL)); the Platelet Derived Growth Factor (PDGF) superfamily (comprising VEGF); the Epidermal Growth Factor (EGF) superfamily (comprising EGF, TGF-alpha, Amphiregulin (AR), Betacellulin, and HB-EGF); the Vascular Epithelial Growth Factor
  • a preferred aspect of the present invention relates to the production of bioactive, authentic growth hormone (GH) from a plant host system.
  • a preferred GH is human growth hormone (hGH).
  • This hormone depicted in Figure 1, is a single chain polypeptide hormone of 191 amino acids (SEQ ID NO: 12) produced mainly by the adenohypophysis (anterior pituitary), but is also expressed in mature lymphocytes.
  • Growth hormone also called somatotropin
  • the physiological effect of hGH is the promotion of bone growth, cartilage, and soft tissues.
  • hGH Overproduction of hGH leads to acromegaly, while a deficiency in hGH may result in dwarf ⁇ sm.
  • hGH also functions in the maintenance of lean body mass, and the regulation of the synthesis of other hormones, such as Insulin-like Growth Factor- 1 (IGF-1). Growth Hormone, Cytokines Online Pathfinder Encyclopedia ( ⁇ http : //www. cop ewithcytokines . de/>) .
  • the ubiquitin fusion showed activity, and some material of the correct mass, indicating no glycosylation and correct N-terminus.
  • Nuclear transformation showed expression lower than 0.03% for either secreted or chloroplast- targeted proteins, with no other data presented. Additionally, recovery of active somatotropin prepared from com plants has been reported, but the type of somatotropin, transformation details, expression levels, and protein quality were not discussed.
  • G-CSF biologically active, authentic granulocyte colony stimulating factor
  • G-CSF Cytokines Online Pathfinder Encyclopedia ( ⁇ http://www.copewithcytokines.de/>).
  • a particular therapeutic product is produced from mammalian cells, with 174 amino acids, the native N-terminus and mammalian-type O- glycosylation.
  • a product is also produced from bacterial cells, with 175 amino acids, a non-native methionine at the N- terminus, and no glycosylation.
  • PHYSICIAN'S DESK REFERENCE (2000) PHYSICIAN'S DESK REFERENCE (2000).
  • G-CSF is used in the treatment of transient phases of leukopenia that may follow chemotherapy and/or radiotherapy. It is also used to enhance immune system deficiency caused by diseases such as AIDS. G-CSF has been shown to expand the myleoid cell lineage. Thus, pretreatment with recombinant human G-CSF prior to bone marrow harvest can improve the graft by increasing the total number of myeloid lineage restricted progenitor cells. This may result in a stable, but not accelerated, myeloid engraftment of autologous marrow. Id.
  • modifying expression vector design to increase yield and improve quality of cytokines expressed in a plant host system.
  • the present invention contemplates optimizing expression vector design by modifying promoters, 5'UTRs, signal sequences, structural genes, and 3 'UTRs.
  • the design parameters of the present invention may include, but are not limited to codon usage, primary transcript structure, translational enhancing sequences, appropriate use of intron splice sites, RNA stabilizing, RNA destabilizing/processing sequences.
  • N- or C-terminal fusions may also be established to facilitate optimal yield, quality, and protein processing.
  • the present invention contemplates the recombinant cytokine fused to signal peptides, such as ubiquitin, soy oleosin oil binding protein, and extensin, to (1) target the expressed cytokine to specific sub-cellular locations within the plant host system, (2) enhance product accumulation and quality, and (3) provide a means for simple recovery of the recombinant cytokine from the plant host system.
  • signal peptides such as ubiquitin, soy oleosin oil binding protein, and extensin
  • the present invention envisions the C-terminus of the recombinant cytokine fused to a stabilizing element, such as the KDEL sequence, to enhance recombinant cytokine accumulation.
  • a protease site or self-processing site may be included to facilitate the release of the signal peptide or stabilizing element from the recombinant cytokine.
  • RNA transcription and translation protein expression
  • protein targeting e.g., nucleus, plastid, cytosol, endoplasmic reticulum
  • the recombinant nucleic acid encoding the cytokine may be part of all of a naturally occurring DNA sequence from any source, it may be a synthetic DNA sequence or it may be a combination of naturally occurring and synthetic sequences.
  • the present invention includes the steps, singly or in sequence, of preparing an expression vector that includes a first nucleic acid sequence that regulates the transcription of
  • a second nucleic acid sequence encoding a significant portion of a peptide that targets a protein to a sub-cellular location, and, fused to this second nucleic acid, a third nucleic acid encoding the cytokine of interest; generating a transformed plant host system in which the cytokine of interest is expressed; and purifying the cytokine of interest from the transgenic plant host system.
  • the first nucleic acid sequence may comprise a plant active promoter, such as the CaMV 35S promoter
  • the second nucleic acid sequence may comprise additional 5' regulatory sequences
  • the third nucleic acid sequence may comprise the cytokine of interest .
  • the 5' regulatory sequences may contain signal sequences which target the cytokine to a specific sub-cellular location within the plant host system.
  • a nucleic acid sequence encoding a cytokine of interest may be fused with a 5' regulatory sequence allowing significant accumulation of the mature cytokine in the cytosol.
  • the nucleic acid sequence encoding the cytokine of interest may be fused to a 5' regulatory sequence containing a signal peptide that targets the cytokine of interest to the endoplasmic reticulum.
  • the nucleic acid sequence encoding the cytokine of interest may be fused with a 5' regulatory sequence that targets the cytokine of interest to the plastid. Targeting the mature cytokine to a specific sub-cellular location may result in increased accumulation of the cytokine and easier purification of the cytokine from the plant host system.
  • a plant host system that has already been transformed with an expression vector comprising a first nucleic acid sequence that regulates the transcription of a second nucleic acid sequence encoding a significant portion of a peptide that targets a protein to a sub-cellular location and fused to this second nucleic acid, a third nucleic acid encoding the cytokine of interest.
  • Another aspect of this embodiment of the present invention comprises cultivating the plant host system under the appropriate conditions to facilitate the expression of the recombinant cytokine, and purifying the recombinant cytokine from the plant host system.
  • the present invention contemplates generating a recombinant cytokine that has a methionine-free N-terminus that is identical to the natural N-terminus of the mature cytokine. Furthermore, the present invention envisions producing a recombinant cytokine in a plant host system that is free from novel glycosylations and amino acid modifications (such as hydroxyproline).
  • a fusion protein is generated consisting of the N-terminus of the recombinant cytokine and ubiquitin.
  • the ubiquitin- cytokine fusion causes the expression of the fusion protein containing the ubiquitin gene at the 5' end, and subsequent in vivo processing cleaves the ubiquitin region from the recombinant cytokine, resulting in a cytokine free of both ubiquitin and methionine at the N-terminus.
  • a fusion protein is generated comprising a region of the soy oleosin oil binding protein, a protease site, and the cytolcine of interest. This fusion protein ultimately results in a mature cytolcine that is free of the oleosin/ protease fusion and a methionine N-terminus.
  • the transformed plant host system of the present invention may be any monocotyledonous or dicotyledonous plant or plant cell.
  • the monocotyledonous plants include, but are not limited to, com, cereals, grains, grasses, and rice.
  • the dicotyledonous plants may include, but are not limited to, tobacco, tomatoes, potatoes, and legumes including soybean and alfalfa.
  • Amino acid sequences includes an oligopeptide, peptide, polypeptide, or protein sequence, and fragment thereof, and to naturally occurring or synthetic molecules.
  • Asexual propagation producing progeny by regenerating an entire plant from leaf cuttings, stem cuttings, root cuttings, single plant cells (protoplasts) and callus.
  • Authentic means of the desired or natural form, being properly folded, having the proper disulfide bonds or other post-translational improvements, with no undesired post-translational modifications.
  • Bioactive as used herein, means displaying a measurable response by a cell, tissue, organ or organism.
  • Chemical derivative as used herein, a molecule is said to be a "chemical derivative" of another molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties can improve the molecule's solubility, absorption, biological half- life, and the like. The moieties can alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like.
  • Dicotyledon (dicot) a flowering plant whose embryos have two seed halves or cotyledons. Examples of dicots include: tobacco; tomatoes; potatoes, the legumes including alfalfa and soybeans; oaks; maples; roses; mints; squashes; daisies; walnuts; cacti; violets; and buttercups.
  • Enhancer sites which are standard and known to those in the art, may be included in the expression vectors to increase and/or maximize transcription of the cytolcine of interest in a plant host system. These include, but are not limited to, peptide export signal sequences,
  • regulatory sequences such as positively or negatively acting sequences, enhancers and silencers, as well as, chromatin structure can affect the rate of transcription in plants.
  • the present invention provides that at least one of these factors may be utilized in engineering plants to express a cytolcine of interest.
  • Fragments include any portion of an amino acid sequence which retains at least one structural or functional characteristic of the subject post-translational enzyme or heterologous polypeptide.
  • Functional equivalent a protein or nucleic acid molecule that possesses functional or ,5 structural characteristics that are substantially similar to a heterologous protein, polypeptide, enzyme, or nucleic acid.
  • a functional equivalent of a protein may contain modifications depending on the necessity of such modifications for the performance of a specific function.
  • Fusion protein a protein in which peptide sequences from different proteins are covalently linked together.
  • Introduction insertion of a nucleic acid sequence into a cell, by methods including infection, transfection, transformation or transduction.
  • Isolated refers to any element or compound separated not only from other elements or compounds that are present in the natural source of the element or compound, but also from other elements or compounds and, as used herein, preferably refers to an element or compound found in the presence of (if anything) only a solvent, buffer, ion, or other component normally present in a solution of the same.
  • Monocotyledon a flowering plant whose embryos have one cotyledon or seed leaf.
  • Examples of monocots include: lilies; grasses; com; rice, grains including oats, wheat and barley; orchids; irises; onions and palms.
  • Operably linked refers to the state of any compound, including but not limited to deoxyribonucleic acid, when such compound is functionally linked to any promoter.
  • Plant culture medium any combination of amino acids, salts, sugars, plant growth regulators, vitamins, and/or elements and compounds that will maintain and/or support the growth of any plant, plant cell, or plant tissue.
  • a typical plant culture medium has been described by Murashige & Skoog, 15 PHYSIOL. PLANT. 473-97 (1962).
  • Plant host system includes plants, including, but not limited to, monocots, dicots, and specifically maize, soybean, and tobacco. Plant host system also encompasses plant cells. Plant cells includes suspension cultures, embryos, merstematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, seeds and microspores. Plant host systems may be at various stages of maturity and may be grown in liquid or solid culture, or in soil or suitable medium in pots, greenhouses or fields. Expression in plant host systems may be transient or permanent.
  • Plant host system also refers to any clone of such a plant, seed, selfed or hybrid progeny, propagule whether generated sexually or asexually, and descendents of any of these, such as cuttings or seed.
  • Plant sample a tissue, organ, or subset of the plant, selected to have the preferred accumulation level, quality, or storability for production of the desired protein.
  • Plant transformation and cell culture broadly refers to the process by which plant cells are genetically altered and transferred to an appropriate plant culture medium for maintenance, further growth, and/or further development.
  • the expression of the heterologous protein may be under the direction of a plant promoter.
  • Promoters suitable for use in accordance with the present invention are described in the art. See e.g., WO 91/198696.
  • Examples of promoters that may be used in accordance with the present invention include non-constitutive promoters or constitutive promoters, such as, the nopaline synthetase and octopine synthetase promoters, cauliflower mosaic virus (CaMV) 19S and 35S promoters, and the figwort mosaic virus (FMV) 35 promoter. See U.S. Pat. No. 6,051,753.
  • the cytokine of interest may be expressed in a specific tissue, cell type, or under more precise environmental conditions or developmental control. Promoters directing expression in these instances are known as inducible promoters. In the case where a tissue-specific promoter is used, protein expression is particularly high in the tissue from which extraction of the protein is desired. Depending on the desired tissue, expression may be targeted to the endosperm, aleurone layer, embryo (or its parts as scutellum and cotyledons), pericarp, stem, leaves, tubers, roots, etc.
  • tissue-specific promoters include the tuber-directed class I patatin promoter, the promoters associated with potato tuber ADPGPP genes, the soybean promoter of beta-conglycinin (7S protein) which drives seed-directed transcription, and seed-directed promoters such as those from the zein genes of maize endosperm and rice glutelin-1 promoter. See, e.g., Bevan et al, 14 NUCLEIC ACIDS RES. 4625-38 (1986); MuUer et al., 224 MOL. GEN. GENET.
  • the cytokine of interest is produced from seed by way of seed-based production techniques using, for example, canola, com, soybeans, rice and barley seed. See, e.g., Russell, 240 CURRENT TECHNOLOGIES ⁇ N MICROBIOL. & IMMUNOL. 119-38 (1999). In such a process, the desired protein is recovered during or after seed maturation, or during the germination phase.
  • Protein purification broadly defined, any process by which proteins are separated from other elements or compounds on the basis of charge, molecular size, or binding affinity. More specifically, the expressed recombinant cytokines of the invention may be purified to homogeneity by cliromatography. In one embodiment, the cytokine produced in com seed is purified by extraction/precipitation, followed by cation exchange column chromatography, followed by purification by anion exchange column chromatography. However, other purification techniques known in the art can also be used, including ion exchange chromatography, and reverse-phase chromatography and selective phase separation. See, e.g. , MANIATIS ET AL., MOL. CLONING: A LAB.
  • Reading frame refers to the preferred way (of three possible) of reading a nucleotide sequence as a series of triplets. Reading "in frame” means that the nucleotide triplets (codons) are translated into a nascent amino acid sequence of the desired recombinant cytokine. Specifically, the present invention contemplates a first nucleic acid linked in reading frame to a second nucleic acid.
  • Recombinant as used herein, broadly describes various technologies whereby genes can be cloned, DNA can be sequenced, and protein products can be produced. As used herein, the term also describes proteins that have been produced following the transfer of genes into the cells of plant host systems.
  • Structural gene a gene coding for a polypeptide that may be equipped with a suitable promoter, termination sequence and optionally other regulatory DNA sequences, and having a correct reading frame.
  • Total soluble protein relative portion of desired measured protein compared to total extracted protein.
  • Transgene an engineered gene comprising a promoter to start gene expression, a 5' untranslated region to initiate translation, a protein coding region, and a polyadenylation/termination region to stop gene expression.
  • An intervening sequence may be included after the promoter, to potentially enhance expression.
  • the protein coding region may include the desired protein to be produced, and possibly a signal peptide or fusion to an additional region(s) that allows protein targeting, stabilization, and/or purification.
  • Transgenic a plant host system engineered to contain a novel, laboratory designed transgene.
  • Transgenic plants plant host systems that have been subjected to one or more methods of genetic transformation; plants that have been produced following the transfer of genes into the cells of plant host systems.
  • Variant an amino acid sequence that is altered by one or more amino acids.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan.
  • Analogous minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted may be found using computer programs well known in the art, for example, DNASTAR ⁇ software. Plant Expression Vectors
  • Expression vectors useful in the present invention comprise a nucleic acid sequence encoding a cytokine expression cassette, designed for operation in plants, with companion sequences upstream and downstream from the expression cassette.
  • the companion sequences may be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to be generated in bacteria and then introduced to the desired plant host system.
  • a cloning vector of this invention is designed so that a coding nucleic acid sequence inserted at a particular site will be transcribed and translated.
  • a typical expression vector may contain a promoter, selection marker, nucleic acids encoding signal sequences, and regulatory sequences, e.g., polyadenylation sites, 5'-untranslated regions, and 3'- untranslated regions, termination sites, and enhancers.
  • "Vectors” include viral derived vectors, bacterial derived vectors, plant derived vectors and insect derived vectors.
  • the basic bacterial/plant vector construct may preferably comprise a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T-DNA sequences for Agrobacterium-mediated transfer to plant chromosomes.
  • the construct will preferably also have a selectable marker gene suitable for determining if a plant cell has been transformed.
  • suitable markers for the members of the grass family is found in Wilrnink & Dons, 11(2) PLANT MOL. BIOL. REPTR. 165-85 (1993). Sequences suitable for permitting integration of the heterologous sequences into the plant genome may be used as well.
  • transposon sequences and the like, Cre/lox sequences and host genome fragments for homologous recombination, as well as Ti sequences which permit random insertion of a cytokine expression cassette into a plant genome.
  • Suitable prokaryote selectable markers useful for preparation of plant expression cassettes, include resistance toward antibiotics such as ampicillin, tetracycline, or kanamycin.
  • Other DNA sequences encoding additional functions may also be present in the vector, as is known in the art.
  • the plant selectable marker gene will encode antibiotic resistance, with suitable genes including at least one set of genes coding for resistance to the antibiotic spectinomycin, the streptomycin phosphotransferase (spt) gene coding for streptomycin resistance, the neomycin phosphotransferase (nptll) gene encoding kanamycin or geneticin resistance, the hygromycin phosphotransferase (hpt or aphiv) gene encoding resistance to hygromycin, acetolactate synthase (als) genes and modifications encoding resistance to, in particular, the sulfonylurea-type herbicides, genes coding for resistance to herbicides which act to inhibit the action of glutamine synthase such as phosphinothricin or basta (e.g., the bar gene), or other similar genes known in the art.
  • suitable genes including at least one set of genes coding for resistance to the antibiotic spectinomycin, the streptomycin phospho
  • the constructs of the subject invention will include the expression vector for expression of the cytokine of interest.
  • the recombinant expression vector contains, in addition to the nucleic acid sequence encoding the cytokine of interest, at least one of the following elements: a promoter region, signal sequence, 5' untranslated sequences, initiation codon depending upon whether or not the cytokine structural gene comes equipped with one, and transcription and translation termination sequences.
  • a gene encoding the cytokine of interest is inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation.
  • Methods for providing transgenic plants of the present invention include constructing expression vectors containing a protein coding sequence, and/or an appropriate signal peptide coding sequence, and appropriate franscriptional/translational control signals. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. See, e g., TRANSGENIC PLANTS: PROD. SYS. FOR INDUS.
  • chimeric genes used in the practice of the methods of the present invention are signal sequences.
  • the chimeric gene also encodes a signal peptide that allows processing and translocation of the protein, as appropriate.
  • the signal sequences may be derived from mammals, or from plants such as wheat, barley, cotton, rice, soy, and potato. These signal sequences will direct the cytokine of interest to a sub-cellular location (e.g., cytosol, endoplasmic reticulum, plastid, and chloroplast) within the plant host system. This may result in increased accumulation and easier purification of the cytokine of interest.
  • the signal peptides contemplated by the present invention include the tobacco extensin signal, the ubiquitin derived from yeast and potato, and the soy oleosin oil body binding protein.
  • Those of skill can routinely identify new signal peptides.
  • plant secretory signal peptides typically have a tripartite structure, with positively-charged amino acids at the N-terminal end, followed by a hydrophobic region and then the cleavage site within a region of reduced hydrophobicity.
  • sequence homology is not always present in the signal peptides, hydrophilicity plots demonstrate that the signal peptides of these genes are relatively hydrophobic.
  • the expression vectors of the present invention typically have a transcriptional termination region at the opposite end from the transcription initiation regulatory region.
  • the transcriptional termination region may normally be associated with the transcriptional initiation region or from a different gene.
  • the transcriptional termination region may be selected, particularly for stability of the mRNA to enhance expression.
  • Illustrative transcriptional termination regions include the NOS terminator from Agrobacterium Ti plasmid and the rice ⁇ -amylase terminator.
  • the transcription termination process also signals for the addition of polyadenylation tails added to the gene transcription product.
  • Polyadenylation sequences include but are not limited to those defined in the Agrobacteriiim octopine synthetase signal, (Gielen, et al., 3 EMBO J. 835-46 (1984)), or the nopaline synthase of the same species (Depicker, et al, 1 MOL. APPL. GENETICS 561-73 (1982)).
  • polynucleotide sequences which encode the cytolcine of interest may be used to generate recombinant nucleic acid sequences that direct the expression of such proteins, or functional equivalents thereof, in plant cells.
  • the present invention contemplates the production in plants of cytokines that have not yet been discovered.
  • New cytokines for which nucleic acid sequences are not available may be obtained from cDNA libraries prepared from tissues believed to possess a "novel" type of cytolcine at a detectable level.
  • a cDNA library could be constructed by obtaining polyadenylated mRNA from a cell line known to express the novel cytolcine, or a cDNA library previously made to the tissue/cell type could be used.
  • the cDNA library is screened with appropriate nucleic acid probes, and/or the library is screened with suitable polyclonal or monoclonal antibodies that specifically recognize other heterologous polypeptides.
  • Appropriate nucleic acid probes include oligonucleotide probes that encode known portions of the novel cytokine from the same or different species.
  • Other suitable probes include, without limitation, oligonucleotides, cDNAs, or fragments thereof that encode the same or similar gene, and/or homologous genomic DNAs or fragments thereof. Screening the cDNA or genomic library with the selected probe may be accomplished using standard procedures known to those in the art. See, e.g., Ch. 10-12, SAMBROOKET AL., MOL. CLONING: A LAB. MANUAL (Cold Spring Harbor Lab. Press, N.Y., 1989).
  • telomere shortening may involve known techniques of recombinant DNA technology, such as by direct expression cloning or using the polymerase chain reaction (PCR). See U.S. Pat. No. 4,683,195; Ch. 14 of SAMBROOK, supra; Ch. 15, CURRENT PROTOCOLS ⁇ N MOL. BIO. (Ausubel et al., eds., Greene Pub. Assocs. & Wiley-Intersci. 1991).
  • Altered DNA sequences which may be used in accordance with the invention include deletions, additions or substitutions of different nucleotide residues resulting in a sequence that encodes the same or a functionally equivalent gene product.
  • the gene product itself may contain deletions, additions or substitutions of amino acid residues within a cytokine sequence, which result in a functionally equivalent cytokine.
  • Altered nucleic acid sequences include nucleic acid sequences encoding a cytolcine, or functional equivalent thereof, including those sequences with deletions, insertions, or substitutions of different nucleotides resulting in a polynucleotide that encodes the same or a functionally equivalent cytokine.
  • polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding a cytolcine and improper or unexpected hybridization to alleles, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding a cytokine.
  • the encoded protein may also be "altered” and contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent cytokine.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues as long as the biological or immunological activity of the cytokine is retained.
  • negatively charged amino acids include aspartic acid and glutamic acid
  • positively charged amino acids include lysine and arginine
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, and valine; glycine and alanine; asparagine and glutamine; serine and threonine; and phenylalanine and tyrosine.
  • the nucleic acid sequences of the invention may be engineered in order to alter the coding sequence for a variety of ends including, but not limited to, alterations that modify expression and processing of the gene product.
  • alternative secretory signals may be substituted for or used in addition to the native secretory signal.
  • the KDEL sequence has been shown to increase the expression of single-chain antibody in tobacco. Schouten et al., 30(4) PLANT MOL. BlOL. 781-93 (1996). Additional mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis, to insert new restriction sites, or alter glycosylation or phosphorylation patterns.
  • the polynucleotides encoding the cytokine may be modified in the silent position of any triplet amino acid codon so as to better conform to the codon preference of the particular host organism. More specifically, translational efficiency of a protein in a given host organism can be regulated through codon bias, meaning that the available 61 codons for a total of 20 amino acids are not evenly used in translation, an observation that has been made for prokaryotes (Kane, 6 CURRENT OP. BIOTECH. 494-500 (1995)), and eukaryotes (ERNST, CODON USAGE & GENE EXPRESSION 196- 99 (Elsevier Pub., Cambridge 1988).
  • Codon usage tables have been established not only for organisms, but also for organelles and specific tissues (Kazusa DNA Research Inst., ⁇ www.lcazusa.or.jp>), and their general availability enables researchers to adopt the codon usage of a given gene to the host organism.
  • Other factors like the context of the initiator methionine start codon (Kozalc, 234 GENE 187-208 (1999)), may influence the translation rate of a given protein in a host organism, and can therefore be taken into consideration. See also Taylor et al., 210 MOL. GENETICS 572-77 (1987). Translation may also be optimized by reference to codon sequences that may generate potential signals of intron splice sites. PLANT MOL. BIO. LABFAX (Croy, ed. 1993), mRNA instability and polyadenylation signals (Perlak et al., supra).
  • the nucleic acid sequences of the invention are further directed to sequences that encode variants of the described cytokine.
  • These amino acid sequence variants of a cytolcine may be prepared by methods known in the art by introducing appropriate nucleotide changes into an authentic or variant cytokine encoding polynucleotide.
  • the amino acid sequence variants are preferably constructed by mutating the polynucleotide to give an amino acid sequence that does not occur in nature. These amino acid alterations can be made at sites that differ in cytokines, from different species (variable positions) or in highly conserved regions (constant regions).
  • Sites at such locations will typically be modified in series, e.g., by substituting first with conservative choices (e.g., hydrophobic amino acid to a different hydrophobic amino acid) and then with more distant choices (e.g., hydrophobic amino acid to a charged amino acid), and then deletions or insertions may be made at the target site.
  • conservative choices e.g., hydrophobic amino acid to a different hydrophobic amino acid
  • more distant choices e.g., hydrophobic amino acid to a charged amino acid
  • hydrophobic leu, met, ala, ile
  • neutral hydrophobic cys, ser, thr
  • acidic asp, glu
  • weakly basic asn, gin, his
  • strongly basic lys, arg
  • residues that influence chain orientation gly, pro
  • aromatic trp, tyr, phe.
  • Conservative changes encompass variants of an amino acid position that are within the same group as the native amino acid.
  • Moderately conservative changes encompass variants of an amino acid position that are in a group that is closely related to the native amino acid (e.g., neutral hydrophobic to weakly basic).
  • Non-conservative changes encompass variants of an amino acid position that are in a group that is distantly related to the "native" amino acid (e.g., hydrophobic to strongly basic or acidic).
  • Amino acid sequence deletions generally may range from about 1 to 30 residues, preferably about 1 to 10 residues, and are typically contiguous.
  • Amino acid insertions include amino- and/or carboxyl-terminal fusions ranging in length from one to one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Intrasequence insertions may range generally from about 1 to 10 amino residues, preferably from 1 to 5 residues.
  • terminal insertions include the heterologous signal sequences necessary for secretion or for intracellular targeting in different host cells.
  • polynucleotides encoding a cytokine are changed via site-directed mutagenesis.
  • This method uses oligonucleotide sequences that encode the polynucleotide sequence of the desired amino acid variant, as well as a sufficient adjacent nucleotide on both sides of the changed amino acid to form a stable duplex on either side of the site of being changed.
  • site-directed mutagenesis is well known to those of skill in the art and this technique is exemplified by publications such as, Adelman et al., 2 DNA 183-93 (1983).
  • PCR may also be used to create amino acid sequence variants of a recombinant cytolcine.
  • primer(s) that differs slightly in sequence from the co ⁇ esponding region in the template DNA can generate the desired amino acid variant.
  • PCR amplification results in a population of product DNA fragments that differ from the polynucleotide template encoding the cytolcine at the position specified by the primer. The product DNA fragments replace the co ⁇ esponding region in the plasmid and this gives the desired amino acid variant.
  • a further technique for generating amino acid variants is the cassette mutagenesis technique described in Wells et al., 34 GENE 315 (1985); and other mutagenesis techniques well l ⁇ iown in the art, such as, for example, the techniques in Sambrook et al., supra; Ausubel et al., CURRENT PROTOCOLS IN MOL. BIOL. supra.
  • DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence or polypeptide, specifically, comprising a consistent (Gly-X-Y), amino acid structure, that are natural, synthetic, semi-synthetic, or -recombinant, may be used in the practice of the claimed invention.
  • Such DNA sequences may be include those which are capable of hybridizing to the appropriate cytokine sequence under stringent conditions.
  • the invention further relates to nucleic acid sequences that hybridize to the above-described sequences.
  • the invention relates to nucleic acid sequences that hybridize under stringent conditions to the above-described nucleic acids.
  • stringent conditions and “stringent hybridization conditions” mean that hybridization will generally occur if there is at least 95% and preferably at least 97% identity between the sequences.
  • An example of stringent hybridization conditions is overnight incubation at 42°C in a solution comprising 50% 0 formamide, 5x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, followed by washing the hybridization support in O.lx SSC at approximately 65°C.
  • Other hybridization and wash conditions are well known and are exemplified in SAMBROOK, ET AL., MOLECULAR CLONING: A LABORATORY MANUAL (2d ed., Cold Spring Harbor, NY (1989)), particularly Chapter 11.
  • Transformation of Plant Cells Transfonnation is a process by which exogenous DNA enters and changes a recipient cell. It may occur under natural or artificial conditions using various methods well known in the art. Transformation may rely on any l ⁇ iown method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, A. tumefaciens-mediated transfection, and particle bombardment.
  • Suitable methods for corn transformation are provided by Fromm et al., 8 BIO/TECHNOLOGY 833-39 (1990); Gordon-Kamm et al, 2 PLANT CELL 603-18 (1990); Russell et al., 6 TRANSGENIC RES., 157-58 (1997); U.S. Pat. No. 5,780,708.
  • Vectors useful in the practice of the present invention may be microinjected directly into plant cells by use of micropipettes to mechanically transfer the recombinant DNA.
  • the genetic material may also be transferred into the plant cell by using polyethylene glycol, Krens et al., 96 NATURE 72-74 (1982).
  • nucleic acid segments Another method of introduction of nucleic acid segments is high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles, or on the surface. Klein et al., 327 NATURE 70-73 (1987); Knudsen & MuUer, 185 PLANTA 330-36 (1991).
  • Another method of introduction would be fusion of protoplasts with other entities, either minicells, cells, lysosomes or other fusible lipid-surfaced bodies, Fraley et al., 79 P.N.A.S. 1859-63 (1982).
  • the vector may also be introduced into the plant cells by electroporation.
  • electroporation fromm et al, 82 P.N.A.S. 5824-28 (1985).
  • plant protoplasts are electroporated in the presence of plasmids containing the gene construct. Electrical impulses of high field strength reversibly permeabilize biomembranes allowing the introduction of the plasmids. Electroporated plant protoplasts reform the cell wall, divide, and form plant callus. See U.S. Pat. No. 5,584,807.
  • Progeny containing the desired cytolcine can be identified by assaying for the presence of the biologically active heterologous protein using assay methods well known in the art. Such methods include Western blotting, immunoassays, binding assays, and any assay designed to detect a biologically functional heterologous protein. See, ' for example, the assays described in KLEIN, IMMUNOLOGY: SCI OF SELF-NONSELF DISCRIMINATION (John Wiley & Sons eds., New York, N.Y. 1982).
  • Prefe ⁇ ed screening assays detect the biological activity of the cytokine. These assays identify, for example, the production of a complex, fo ⁇ nation of a catalytic reaction product, the release or uptake of energy, cell growth, identification as authentic by the appropriate antibody, and the like.
  • a progeny containing a cytokine molecule produced by this method may be recognized by an antibody to binds to an authentic antigenic site on the cytokine in a standard immunoassay such as an ELISA or other immunoassays known in the art. See ANTIBODIES: A LAB. MANUAL (Harlow & Lane, eds., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 1988). Plant Regeneration
  • Plant regeneration from cultured protoplasts is described in EVANS, ET AL., HANDBOOK OF PLANT CELL CULTURES, Vol. 1 : (MacMillan Publishing Co. New York 1983); CELL CULTURE & SOMATIC CELL GENETICS OF PLANTS, (Vasil I.R., ed., Acad. Press, Orlando, Vol. 1 1984, and Vol. Ill 1986).
  • All plants from which protoplasts can be isolated and cultured to give whole regenerated plants can be transformed by the present invention so that whole plants are recovered which contain the transferred gene. It is l ⁇ iown that practically all plants can be regenerated from cultured cells or tissues, including but not limited to all major species of sugarcane, sugar beet, cotton, fruit and other trees, legumes and vegetables, dicots, and monocots.
  • Methods for regeneration vary from species to species of plants, but generally a cell capable of being cultured either alone or as part of a tissue and containing copies of the cytokine gene is first provided.
  • Callus tissue may be formed and shoots may be induced from callus and subsequently rooted, or shoots may be induced directly from a cell within a meristem.
  • embryo formation can be induced from the cell suspension. These embryos germinate as natural embryos to fonn plants.
  • the culture media will generally contain various amino acids and hormones, such as auxin and cytokinins. It is also advantageous to add glutamic acid and proline to the medium, especially for such species as com and alfalfa. Shoots and roots normally develop simultaneously. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these three variables are controlled, then regeneration is fully reproducible and repeatable.
  • a plant of the present invention containing the expression vector comprised of a first nucleic acid sequence that is capable of regulating the transcription of a second nucleic acid sequence encoding a significant portion of a peptide that is capable of targeting a protein to a sub-cellular location and fused to this second nucleic acid, a third nucleic acid encoding the cytokine of interest, is cultivated using methods well known to one slcilled in the art. Any of the transgenic plants of the present invention may be cultivated to isolate the desired cytolcine they contain. After cultivation, the transgenic plant is harvested to recover the produced cytokme. This harvesting step may consist of harvesting the entire plant, or only the leaves, or roots of the plant.
  • This step may either kill the plant or if only the portion of the transgenic plant is harvested may allow the remainder of the plant to continue to grow.
  • the transgenic plants according to this invention can be also be used to develop hybrids or novel varieties embodying the desired traits. Such plants would be developed using traditional selection type breeding.
  • the mature plants, grown from the transformed plant cells, are selfed and non- segregating, and the resulting homozygous transgenic plants is identified.
  • an outcross can be performed, to move the gene into another plant.
  • the transgenic plants produces seed containing the proteins of the present invention.
  • the transgenic plants according to this invention can be used to develop hybrids or novel varieties embodying the desired traits. Such plants would be developed using traditional selection type breeding.
  • Example 1 Construction of a vector for expression of hGH in corn seeds
  • the initial plant expression vector (accepting vector) used contained the CaMV 35S promoter (P-35S), a plant-active 5'utr and signal peptide with an Nc ⁇ l site for fusion to the start methionine of the hGH sequence, and a 3'utr/polyA addition site (nos). This combination has been used to express a single chain antibody in plant cells (Francisco et al., 1997).
  • the signal peptide for directing the protein through the secretory path is a 26 amino acid version from Nicotiana plumbaginifolia. De Loose et al., 99 GENE 95-100 (1991).
  • the plant cell expression cassette containing the hGH gene (GenBank accession number AF205361) was derived from an expression cassette originally designed for direct expression in E. coli. Staub et al., 18 NAT. BIOTECH. 333-38 (2000).
  • TheE. coli cassette contains methionine and alanine codons, in the context of an Ncol site immediately upstream from the codons encoding the authentic mature amino terminus (beginning Phe-Pro-Thr) of native hGH.
  • the downstream end of the coding sequence used a Hind ⁇ ll restriction site after the stop codon.
  • This hGH cassette was put into the Ncol-Pstl site of the above accepting vector, by using a linker: darlOO: (agcttgca) to allow joining of the HmdIII and Pstl sites, and to regenerate the HmdIII site.
  • the resulting plasmid was called pwrg4738.
  • darl39 ttagctagcgaaagctccgccttccgactatcccactgagccgcctgttcgacaacgctatgctgcgagct (S ⁇ Q ID NO: 01)
  • darl40 cgcagcatagcgttgtcgaacaggcggctcagtgggatagtcgggaaggcggagctttcgctagc (S ⁇ Q ID NO:02)
  • the com transformation vector was designed to include a com seed endosperm expression cassette, and a com selectable marker cassette.
  • the com seed endosperm expression cassette includes an endosperm-specific promoter from rice (P-OsGTl) that has been used in com seed previously (Russell & Fromm, 6 TRANSGENIC RES. 157-68 (1997); WO 98/10062), a com ⁇ SP70 intron (TVS) (WO 93/19189), a polyadenylation region previously used in com (nos) (WO 98/10062).
  • the com selectable marker cassette includes the 35S promoter, neomycin phosphotransferase II coding region (NPT2), and a polyadenylation region (nos).
  • the construction of the com transformation vector used the HmdIII to Blpl fragment of pwrg4768, encompassing the 5'utr, IVS, and amino terminus of the signal peptide.
  • a second fragment came from pwrg4776, extending from Blpl to Xbal, encompassing the carboxy-te ⁇ ninus of the signal peptide, the entire hG ⁇ coding region, and nos polyadenylation region.
  • These fragments were ligated into the com transformation vector pwrg4789, having a HmdIII site directly after the seed promoter, and an Xbal site directly before the selection cassette.
  • the resulting plasmid, pwrg4825 is illustrated in Figure 2.
  • General methods for constructing plant expression vectors have been described. See, e.g., Staub et al., 2000).
  • Transient expression allows examination of gene expression and protein accumulation in multiple plant tissues and species. Gene construct can be tested quickly for gross quality and quantity performance, although details of protein quality (N-terminus, glycosylation) may require transgenic plants.
  • the list of vectors encoding hG ⁇ for transient expression in several plant cells types is illustrated in Figure 3.
  • the 35S, extensin, nos, and kanamycin selection elements have been described. Russell et al, U.S. Pat. No. 6,140,075; Francisco et al. 8 BIOCONJUGATE C ⁇ EM. 708-13 (1997).
  • the Zm ⁇ SP70 intron is described in Brown et al., U.S. Pat. No. 5,859,347.
  • the Petunia HSP70 5 ' UTR is described in Austin et al., U.S. Pat. No. 5,659,122.
  • the rice glutellin promoter (OsGTl) for monocot seed expression is described in Brar et al. WO/9810062.
  • the bean 7S promoter for dicot seed expression is described in Chen et al., 83 P.N.A.S 8560-64.
  • the FMV promoter is described in Rogers, U.S. Pat. No. 6,018,100.
  • the DSSU 5' UTR and GUS selection cassette used for soy transformation is described in Kridl, WO/0009721.
  • the CTP2 and glyphosate selection cassette is described in Barry et al., U.S. Pat. No. 5,633,435.
  • the potato ubiquitin 3 used for fusion to hGH is described in Garbarino et al. 24 PLANT MOL. BlOL. 119-27 (1994).
  • Three different expression vectors were constructed for transiently expressing and targeting hGH to different locations within the plant cell. These expression vectors included an hGH expression cassette employing the CaMV 35S promoter, a plant active 3 'UTR/nos poly A, and different plant-active 5' regulatory regions. The differing 5 'regulatory regions that targeted the expressed hGH to different locations within the plant cell as follows: (1) a 5 'regulatory region that targeted hGH to the cytosol ("cytosolic form”) ;
  • the hGH gene cassette used in the three expression vectors was designed originally for the direct translation and expression of the hGH protein in E. coli.
  • the hGH cassette contained a Nco I restriction site at the N-te ⁇ ninal region, and yielded a methionine then an alanine codon immediately preceding the natural PheProThr N-terminus of mature hGH.
  • the first expression vector, targeting the cytosol included the hGH stractural gene, the CaMV 35S promoter, a plant-active 5 'UTR, and a 3'UTR Nos poly A signal. This generated a methionine-alanine N-terminus on the expressed hGH, which is not identical to the natural hGH N-te ⁇ ninus (PheProThr).
  • the second expression vector targeting the secretory pathway, included the hGH stractural gene, a 5 'regulatory region encoding a signal peptide to facilitate secretion of the nascent protein through the endoplasmic reticulum, and a 3 'UTR/nos poly A signal.
  • This expression vector also comprised the AlaSerAla/MetAlaPhe (S ⁇ Q ID NO:03) fusion point between the signal peptide and N-terminus of hGH and generated the methionine N-terminus on the expressed hGH protein.
  • This expression vector was further modified by introducing an intron from the com heat shock 70 gene between the promoter and the signal peptide.
  • the third expression vector, targeting the plastid comprised the hGH structural gene fused to the CaMV 35S promoter, a 5' regulatory region that encoding a plastid targeting sequence, and a 3 'UTR/nos poly A addition signal.
  • This expression vector was further modified by introducing an intron from the com heat shock 70 gene between the promoter and the signal peptide.
  • This expression vector also contained an CysMetLeuAla/MetAlaPhe (S ⁇ Q ID NO:04) fusion point, that also generated a methionine N-terminus on the expressed hGH.
  • the blot is analyzed via Western blot by reaction with rabbit-anti-hGH (Biodesign International D710071R), followed by detection with horse radish peroxidase- conjugated goat-anti-rabbit antibody (Sigma A0545) and substrate (ECL; Amersham).
  • Figure 4 shows the result for soy hypocotyls.
  • a comparison of the constracts indicated very low hGH expression with the plastid targeting signal (CTP2), higher hGH expression levels with the construct containing the secretion signal (EXT), and the highest hGH expression levels with the cytosolic construct (DSSU). Additionally, there was also a 14 kD truncation product associated with the secreted form. There was also a truncation product associated with the cytosolic form, but this was less prevalent in comparison to the secreted fonn.
  • the high level of hGH expression with the cytosolic construct was an unexpected, but otherwise desired result.
  • the advantages of the having high hGH expression levels with the cytosolic form include a reduced cost in production and easier purification.
  • a yeast ubiquitin monomer was placed between the end of the DSSU 5' UTR and the translational start of hGH.
  • This constract was named pwrg4834.
  • the second construct was generated by replacing the 5 'UTR, signal sequence, and fragment of hGH from pwrg4776 with a splicing PCR product that included the 5' UTR and ubiquitin monomer of potato ubiquitin gene 3, and a replacement fragment of hGH.
  • This constract was named pwrg4857.
  • Plant oil body-binding protein fusion expression constructs Plant oil body-binding protein fusion expression constructs
  • Oil body-binding protein has been shown to result in co ⁇ ect protein folding of some fused proteins normally destined for secretion, and ease protein purification from other host cell components. See, e.g., U.S. Pat. No. 5,650,554. This fusion protects the hGH from the apparent proteases in the secretory path that cleave hGH, thus yielding more, folded, intact hGH.
  • the design entailed a synthetic gene that encoded soy oleosin, an enterokinase protease recognition site, and a fragment of the hGH amino terminus. This was inserted between a plant 5' UTR, and the remaining fragment of hGH, to create pmon41324. While the oleosin fusion may aid in conect folding and potential purification of hGH, the enterokinase site allows later specific protease cleavage at AspAspAspAspLys/PheProThr (SEQ ID NO:06), to yield the mature natural amino terminus of hGH.
  • Example 3 Expression of hGH in soy plant with secretory targeting
  • Expression cassettes comprising the hGH structural gene operably linked to the plant extensin signal peptide, either the CAMV 35S or 7S seed storage protein promoter, and the nos poly A termination site, were used to generate transgenic soy plants.
  • the expression cassettes were transformed into soy by particle bombardment. All designs used the hGH gene cassette as in pwrg4776, having the desired PheProThr N-terminus. It was incorporated with a ⁇ -glucuronidase expression cassette, used for selecting transformed plants. Biolistic- based plant transformation was perfonned essentially as described by McCabe et al., 6 Bio TECH. 923-26 (1988). An alternative gene design used a promoter from the soy 7S seed storage protein. Chen et al.
  • Example 4 hGH stable cell expression with secretory targeting in stable tobacco cell lines
  • the expression constructs described in Example 2 were also used to generate stable transgenic tobacco cell lines. These expression constracts included the cytosolic targeting expression vector, the secreted targeting expression vector, and the plastid targeting expression vector.
  • TSM fresh tobacco suspension media
  • the medium is adjusted to pH 5.8 prior to autoclaving.
  • Early log phase cells were plated onto 15 mm target disks on tobacco culture medium (TCM) containing 0.3M osmoticum and held for one hour prior to bombardment.
  • TCM tobacco culture medium
  • the solid medium TCM consists of TSM plus 1.6 g/1 Gelrite (Scott Labs., West Warwick, R.I.).
  • the DNA constract was delivered into the plated NTl cells using a spark discharge particle acceleration device as described in U.S. Pat. No. 5,120,657. Delivery voltages ranged from 12-14 kV.
  • the disks containing the cells were held in the dark for one day, during which the disks were transfe ⁇ ed twice, at regular intervals, to solid media containing progressively lower concentrations of osmoticum.
  • the cells were then transferred to TCM containing 350 mg kanamycin sulphate/liter and grown for 3-12 weeks, with weekly transfers to fresh media.
  • kanamycin resistant calli of transgenic NTl cells may be used to start a suspension culture in TSM containing 350 mg kanamycin sulphate/liter.
  • Plasmid pWRG4738 was co-bombarded with a vector containing the kanaycin selection cassette, while the others had both gene cassettes on a single plasmid. Plasmid pWRG4803 was designed to have the desired PheProThr N-terminus.
  • transgenic calli and suspension cells were analyzed for the expression of the various forms of hGH by Western blotting with a rabbit-anti-hGH specific antibody.
  • the results showed higher levels of the 14 kD truncation band in the secreted version than in the cytosolic and plastid expression versions.
  • Figure 10 The absence of the 14 kD truncation product, with the cytosolic expression cassette, is a preferred result.
  • Example 5 hGH expression with secretory targeting in tobacco plants
  • the expression constracts as described in Example 2 were also used to generate stable transgenic tobacco plants. These expression constructs included the cytosolic targeting expression vector, the secreted targeting expression vector, and the plastid targeting expression vector. These expression constructs were mixed with a glyphosate selection cassette, and transformed into tobacco cells by accelerated particle delivery, as set forth previously.
  • Example 6 Plant cell hGH purification and quality tests MetAla-hGH purification and quality test
  • MetAla-hGH was purified from the media of tobacco cell lines expressing the secreted version of the protein, designed to have a MetAla N-terminus. Media was collected
  • the pooled major fractions were applied to a reducing 4-20% gradient SDS-PAGE,
  • MetAla-hGH yielded not only the expected MetAlaPhePro sequence, but also the nature-identical N-terminus of PheProThr as a minor product.
  • Activity tests of the partially purified MetAla-hGH were performed by the method of Dattani et al, 270 J. BlOL. CHEM. 9222-26 (1995), as shown in Figure 12.
  • Mammalian rat lymphoma Nb2 cells which respond to hGH, were incubated with different levels of purified MetAla-hGH. Following incubation, the mammalian cells were assayed for mitotic activity 5 and cell proliferation by the proportional conversion of tefrazolium dye to colored formazan product.
  • Phe-hGH was purified from the media of the cell line expressing the secreted version of hGH, with the desired N-terminus. Media was collected at 4-5 days post innoculation and loaded onto a Pharmacia DEAE Streamline column (Pharmacia, Peapack, NJ). The column 5 was washed with 25 mM Tris pH 8.3, followed by a step elution. Coomassie staining, as described above, revealed that the pooled major fractions contained an average of 272-293 ⁇ g/ml total protein. ELISA using an anti-hGH antibody revealed that the pooled major fractions contained an average of 5.4-10.1 ng/ ⁇ l Phe-hGH.
  • the pooled major fractions were then diluted, adjusted to pH 9.5 with Tris base, and 10 loaded onto to a SOURCE 30 Q column.
  • the SOURCE 30 Q column was developed with a linear gradient of 0-1 M NaCl.
  • the pooled major fractions were next applied to a reducing 4-20% gradient SDS- PAGE, and the SDS/PAGE-separated proteins were then transferred onto a polyvinylidene difluoride (PVFD) membrane (Schleicher & Schuell, Inc., Keene, NH).
  • PVFD polyvinylidene difluoride
  • the blots were 15 stained with 0.1% Ponceau S (Sigma, St. Louis, MO) in 1% acetic acid, then destained in water.
  • the band at the position corresponding to the appropriate size for hGH was marked and then sequenced on an Applied Biosystems sequencer (Applied Biosystems, Foster City, CA).
  • the sequencing results revealed the prefe ⁇ ed result of only the nature-identical N- terminus, PheProTMlePro, being present without the presence of any hydroxyproline. ⁇ 0
  • Mass Spectrophotometry of Phe-hGH The pooled major fractions of Phe-hGH were also analyzed by mass spectrometry.
  • the mass spectrometry results in Figure 13 show significant levels of authentic-sized hGH at 21,255 mass units, having the proper disulfide linkages, free of novel glycosylation and amino acid modifications.
  • Example 7 hGH expression in corn with secretory targeting
  • the com transformation vector included an endosperm-specific expression cassette, and a com selectable marker cassette as described in Example 1.
  • the endosperm-specific promoter obtained originally from rice (P-OsGTl) has been used previously in co seed. Russell & Fromm, 6 TRANSGENIC RESEARCH 157-68 (1997); WO 98/10062).
  • the constract also included a corn HSP70 intron (IVS) (WO 93/19189) and a nos polyadenylation region used previously in com (WO 98/10062).
  • the com selectable marker cassette included the 35S promoter, neomycin phosphotransferase II coding region (NPT2), and a polyadenylation region (nos).
  • the construction of the com transformation vector used the HmdIII to Blpl fragment of pwrg4768, encompassing the 5'UTR, IVS, and amino terminus of the signal peptide.
  • a second fragment came from pwrg4776, extending from Blpl to Xbal, encompassing the carboxy-terminus of the signal peptide, the entire hG ⁇ coding region, and nos polyadenylation region.
  • These fragments were ligated into the com transformation vector pwrg4789, having a HmdIII site directly after the seed promoter, and an Xbal site directly before the selection cassette.
  • the resulting plasmid, pwrg4825 is illustrated in Figure 2. General methods for constructing plasmid vectors have been described. AUSABEL ET AL., 1999.
  • Com transformation was performed by the biolistic method, using a kanamycin selection gene. Prior to use, the plasmid vector was cut with restriction enzyme Notl, cutting at sites on either side of the plant transgene cassettes. The transgene fragment was purified, eliminating the bacterial vector sequences. The transgene D ⁇ A can be precipitated onto microscopic metal particles, and delivered to com cell material that is competent to be regenerated into a fertile com plant. Gordon-Kamm et al., 2 PLANT CELL 603-18 (1990). The com material is then exposed to kanamycin, killing any cells that do not express the
  • NPT2 transgene The surviving cells are put into a series of media conditions of varied salts and plant growth regulators, stimulating the organized production of plant roots and shoots. The plantlets are then put to soil, and plants grown in the greenhouse to maturity, pollinated, and the resulting seed harvested. This seed can be either processed to purify the hGH, or replanted. Replanted mature plants can be either "selfed,” generating a pure-breeding transgenic strain, or out-crossed, placing the transgene in a novel genetic background, or used to create more transgenic material by transferring the transgenic pollen to multiple non- transgenic ears.
  • the ratio of positive seed to negative seed expression was generally as expected for each event: for selfed ears, a 3:1 ratio is expected, and for outcrossed, a 1:1 ratio is expected.
  • second generation seed was tested, even higher expression was noted, presumably due to higher gene dose.
  • Reduced SDS-PAGE Western blot indicated significant material of the co ⁇ ect mobility was seen in seed of multiple first generation events, though a truncation product was also observed.
  • Seeds from multiple first generation transgenic events were pooled, ground to a fine powder, and the hGH purified.
  • the powder was mixed with ten volumes of 100 mM Tris buffer, and shaken for one hr at room temperature. The material was centrifuged, the top fatty layer removed, and the remainder poured through cheesecloth to recover 163 ml of fluid.
  • the material was loaded at 2 ml/min. onto a Gibco Q HB2 column (10 x 75 mm) (Life Technologies, Rockville, MD), equilibrated in 25 mM Tris, 10 mM NaCl, pH 8.3, washed with ten volumes of equilibration buffer, and developed with 1 M NaCl. Fractions of 1.5 ml were collected. The flow through was reloaded on the column, rewashed, and developed with a step change to IM NaCl at 0.8 ml/min flow rate, with 1.6 ml fractions collected.
  • the fractions with the highest hGH levels from the two runs were pooled, and concentrated with buffer exchange to 20 mM Tris pH 9 using an A icon YM30 membrane (Millipore, Bedford, Massachusetts). This was loaded to a 5 ml BioRad High Q column (Bio-Rad Labs.), equilibrated in 25 mM Tris, 10 mM NaCl, pH 9. It was developed with a linear gradient to IM NaCl, with 5 ml fractions taken. Comparision of hGH levels by ELISA to total protein levels indicated a purity of 1.1 % at 225 mg/L.
  • the major fractions were subjected to amino terminal sequencing as follows.
  • the major fractions were applied to a reducing 4-20% gradient SDS-PAGE, and then the SDS PAGE-separated proteins were transferced onto a polyvmylidene difluoride (PVDF) membrane (Schleicher & Schuell, Inc., Keene, NH).
  • PVDF polyvmylidene difluoride
  • the blots were stained with 0.1% Ponceau S (Sigma, St. Louis, MO) in 1% acetic acid, then destained in water.
  • the upper band co ⁇ esponding to the appropriate size for hGH as seen in the Western blot above was marked and then sequenced on an Applied Biosystems sequencer (Applied Biosystems, Foster City, CA).
  • the diafiltration buffer was 3 M urea, 0.05 M acetic acid, pH 5.0.
  • the hGH recovered from the anion exchange pool was compared to hGH molecule purified from recombinant E. coli by anion exchange HPLC (Figure 19A-B), RP-HPLC ( Figure 20A-B), mass spectrometry (Figure 17A-B) and tryptic peptide mapping ( Figure 21A-B). All three assays showed similar HPLC profiles for the hGH purified from corn compared to hGH purified from E. coli. Amino te ⁇ ninal sequencing and electrospray mass spectrometry of hGH isolated from com seed showed that an intact hGH molecule with the co ⁇ ect amino terminus had been produced in com without hydroxyproliiie or sugar additions. The purification steps in this Example also removed the cleaved form of hGH. Sequencing of an earlier fraction from this purification scheme had showed cleavage near amino acid residue Serl 50.
  • transgenic proteins can greatly facilitate the initial purification of transgenic proteins from plants that have an acidic pi. Most transgenic proteins will bind to the cation resin, but most com proteins will not.
  • Example 8 Transient expression of G-CSF with different targeting signals
  • the G-CSF gene had been preceded immediately by methionine and alanine codons for the direct expression of the protein, in the context of aNcol restriction enzyme site, directly before the nature-identical G-CSF ThrProLeu N-terminus.
  • This G-CSF coding sequence had been further modified by performing a cysl7ser change (Kuga et al., 159 BIOCHEM. BIOPHYS. RES COMM. 103-111 (1989)), to minimize the potential of inconect disulfide linkages during E. coli expression and refolding.
  • the entire set of G-CSF vectors is in Figure 23. Three expression vectors were constructed that resulted in three different forms of
  • G-CSF G-CSF.
  • the first expression vector for the cytosolic form included the G-CSF gene, the CaMV 35S promoter, a plant active 5 'UTR, and a 3'UTR/Nos poly A signal.
  • the cytosolic expression vector yielded MetAlaThr as a translation start site.
  • the expression vector for the secreted form contained the G-CSF structural gene, a 5 'UTR that also contained a signal peptide to facilitate secretion of the nascent protein through the endoplasmic reticulum, and a 3'UTR/Nos poly A signal.
  • This expression cassettes comprised a AlaSerAla/MetAlaThr (SEQ LD NO: 13) fusion point between the signal peptide and the N-terminus, winch will lead to a methionine N-terminus during secretion.
  • the third expression vector which is the plastid form, comprised the G-CSF expression cassette fused to the CaMV 35S promoter, a 5' UTR that also contained a plastid targeting sequence, and a 3'UTR/Nos poly A addition signal. Also, an intron from the com heat shock 70 gene was placed in between the promoter and signal peptide.
  • This expression vector was designed to yield a CysMetLeuAla/MetAlaThr (SEQ ID NO: 14) fusion point, that is expected to generate a methionine N-terminus on the expressed G-CSF protein after import to the plastid.
  • Expression vectors without the intron were the same, except that the plastid version used an FMV promoter.
  • the expression vectors were delivered into soy hypocotyls and com leaves by particle bombardment as described above. Following delivery, transgenic plants were analyzed for the expression of the three forms of G-CSF via Western blotting with a rabbit-anti-G-CSF specific antibody.
  • Total soluble protein was extracted from about 250 mg of tissue of transgenic tisue in 0.5 ml of extraction buffer (25 mM Tris-acetate (pH 8.5), 0.5 M NaCl, 5 mM PMSF). The homogenate was centrifuged at 12,000 x g for 10 minutes. Protein concentration in the supernatant was measured by a Bradford assay. Proteins were separated by reducing SDS/PAGE (4-20%).
  • the SDS/PAGE-separated proteins were transferred onto a nitrocellulose membrane (Amersham).
  • the blots were probed with a rabbit-anti-G-CSF antibody, and detected with goat-anti-rabbit Ig-conjugated horse radish peroxidase, followed by ECL reagent (Amersham).
  • the vector was modified to alter the G-CSF N-terminus fusion to the signal peptide, incorporate the natural ser 17, and alter codon usage to improve expression levels as described previously.
  • the modified vectors yielded a fusion point between the signal peptide and the G-CSF N-terminus of AlaSerAla MetThrProLeu (SEQ ID NO:07) met-G-CSF), expected to yield a G-CSF amino acid sequence with a methionine terminus and cysl7, identical to commercial NEUPOGEN® (A gen).
  • Figure 25 shows accumulation from several different vectors with modified codons (mat, gmt, gpp, nsi), similar to that seen with the earlier secreted codon design in terms of relative presence of full sized compared to truncated product.
  • a carboxy-terminal "KDEL" fusion was added to the secreted G-CSF expression vector, yielding a carboxy-terminal fusion point of AlaGlnPro/AspAspLysGluAspLeu (SEQ ID NO:08).
  • This design has been used to increase expression of other proteins, presumably by stopping the secretion of the protein before traversing the golgi and later secretory compartments.
  • the newly modified expression vector was named pwrg4810.
  • the pwrg4810 expression vector was delivered into com leaves, extracted for total proteins, separated by reducing SDS-PAGE, and analyzed by Western blot for G-CSF as above.
  • Example 9 Stable tobacco cell expression of G-CSF with different targeting signals
  • G-CSF expression vectors described in Example 8 were used to generate stable transgenic tobacco cell culture. These expression vectors included the cytosolic form, the secreted form, the plastid form, and the KDEL fusion. The secreted forms included designs with different codon usage. These expression cassettes were mixed with a kanamycin resistance cassette, or the two cassettes were developed into a single vector, and then co-transformed by accelerated particle delivery as in Example 4.
  • the KDEL may then be valuable if the attached protein was purified from the whole cells. Designs which would allow later accurate removal of the KDEL, or allow retention in the secretory path without a fusion, may help minimize degradation, while still making the desired protein sequence. Plant cell MetAla-G-CSF purification and quality tests
  • MetAla-G-CSF was purified from the media of the tobacco cell line transformed with the secreted expression vector pwrg4743, having the AlaSerAla/MetAlaPhe (SEQ ID N0:03) fusion between the signal peptide and the N-terminus of G-CSF.
  • Media was collected four days post-inoculation, the pH adjusted to pH 3.6 with HC1, then loaded on a SBB cation exchange column (Amersham, Piscataway, NJ). The column was washed with 10 mM NaAc pH 4, and then the G-CSF was eluted with a linear salt gradient at pH 4, 250 mM NaCl.
  • the major fractions were pooled and applied to a POROS HS cation exchange (Amersham, Piscataway, NJ). Next, the column was washed in 50 mM NaCitrate pH 3.6, and then developed with a pH 3.6 to 7.5 gradient. G-CSF was eluted at pH6.3. This pool was applied to a Macroprep-Q column (Amersham, Piscataway, NJ), washed with 25 mM Tris-Cl pH 9.2, and developed with a 0 to 200 mM NaCl gradient. G-CSF eluted at 75 mM NaCl, pH 9.2.
  • the final material was 98% pure, determined by comparing G-CSF ELISA signal to total protein using a Coomassie Plus assay and bovine IgG as a standard(Pierce Chemicals, Rockford, IL). Comparing ELISA signal of the initial to final sample showed that the process yield was 43%.
  • the purified material was subjected to amino terminal sequencing as follows.
  • the final G-CSF material was applied to a reducing 4-20% gradient SDS-PAGE, and then the SDS/PAGE-separated proteins were transfe ⁇ ed onto a PVDF membrane (Schleicher & Schuell).
  • the blots were stained with 0.1% Ponceau S (Sigma) in 1% acetic acid and destained in water .
  • the band co ⁇ esponding to the appropriate size for G-CSF was marked and then sequenced on an Applied Biosystems sequencer.
  • the purified G-CSF material was also analyzed by electron spray mass spectrometry (ESMS).
  • ESMS electron spray mass spectrometry
  • the mass spectrometry results are shown in Figure 29.
  • the mass spectrometry results showed that roughly half of the purified material exhibited a molecular weight of 18,871 mass units, which is expected based upon the amino acid sequence of G-CSF.
  • the mass spectrometry results of the remaining half of the purified material was consistent with the hydroxylation also being the site of glycosylation, which added a molecular weight of 396 mass units. Other minor peaks were interpreted as methionine oxidations, occuring either during plant accumulation, or purification. Additional mass spectrometry indicated a ladder of masses consistent with a chain of three repeating units.
  • a cell-based proliferation assay was performed on the purified material derived from the cells expressing secreted MetAla G-CSF.
  • Final purified plant sample and E. coli refolded standard were each diluted to 30 ⁇ g/ml in 40 mM HEPES pH 6.3. They were used in an activity assay based on the ability of G-CSF to stimulate cell growth, as measured by 3 H-thymidine uptake for incorporation into cellular DNA.
  • the cell line used was a murine BAF3 line, transfected with the G-CSF receptor. Dong et al., 13 MOL. CELL BIO. 7774-81 (1993).
  • the results of the proliferation assay showed positive dose-dependent activity of plant-derived G-CSF, similar to that induced by of an E. coli-de ⁇ ved G-CSF.
  • Figure 31 It is also important to note that the E. co/z-derived G-CSF required ex vivo refolding, while the plant-derived G-CSF that was column purified had been properly folded in vivo.
  • G-CSF from cells transformed with Met G-CSF
  • G-CSF was purified from the media of the tobacco cell line transformed with the secreted expression vector pwrg4770, which contained the AlaSerAla/MetThrProLeu (SEQ LO NO:07) fusion between the signal peptide and the N-terminus of G-CSF.
  • the column purification was perfonned as described above. Following column purification, the purified material was subjected to amino terminal sequencing.
  • the column purified G-CSF material was applied to a reducing 4-20%> gradient SDS-PAGE, and then the SDS/PAGE-separated proteins were transferred onto a PVDF membrane (Schleicher & Schuell).
  • the blots were stained with 0.1% Ponceau S (Sigma) in 1% acetic acid and destained in water. The band co ⁇ esponding to the appropriate size for G- CSF was marked and then sequenced on an Applied Biosystems sequencer. The sequencing results showed the presence of the MetThrHypLeu N-terminus, rather than the desired MetThrProLeu (SEQ ID NO:l 1). Mass spectrophotometry indicated the sample was 18814 mass units, compared to the predicted 18815 mass for full length G-CSF, 2 disulfide bonds, and one hydroxyproline. This indicates that while the plant-modified amino acid hydroxyproline was present, sugars were not added. This is different than the results seen with the MetAla design of G-CSF.

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