WO1998021348A1 - Procede de production facteurs de croissance humain a partir de plantes entieres ou de cultures de cellules vegetales - Google Patents

Procede de production facteurs de croissance humain a partir de plantes entieres ou de cultures de cellules vegetales Download PDF

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WO1998021348A1
WO1998021348A1 PCT/US1997/020603 US9720603W WO9821348A1 WO 1998021348 A1 WO1998021348 A1 WO 1998021348A1 US 9720603 W US9720603 W US 9720603W WO 9821348 A1 WO9821348 A1 WO 9821348A1
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recited
plant
cdna
growth factor
hegf
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PCT/US1997/020603
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English (en)
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Brian S. Hooker
Ziyu Dai
Jianwei Gao
Mark T. Kingsley
Richard E. Weller
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Battelle Memorial Institute
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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
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates generally to a method for producing human growth factors from whole plants or plant cell culture. More specifically, the invention relates to producing a human growth factor from a plant cell encoded to produce the human growth factor with a length of at least 200 amino acids from transgenic plant cells.
  • hEGF human epidermal growth factor
  • hEGF-bearing fusion constructs include mitotoxins for treatment of restenosis (Frost and Sullivan, 1994) and radioconjugates for a variety of anti-neoplastic therapies (Grieg et al. , 1988).
  • Multimers of from 2 to 7 EGF units each having 53 amino acid residues have been produced from bacterial hosts, eg E. coli, Streptomyces and Bacillus, fungal hosts, eg Saccharomyces, Pichia and Aspergillus, insect cell host, and mammalian cell hosts, eg CHO cells and COS cells.
  • bacterial hosts eg E. coli, Streptomyces and Bacillus
  • fungal hosts eg Saccharomyces, Pichia and Aspergillus
  • insect cell host eg CHO cells and COS cells.
  • mammalian cell hosts eg CHO cells and COS cells.
  • hEGF production in Staphylococcus aureus (U.S. Patent No. 5004686, 1991) is by a fusion construct encoding hEGF linked to a protein.
  • Synthesis methods using transgenic bacterial strains have problems such as faulty antibody gene expression, protein folding difficulties, inability to glycosylate proteins
  • Transgenic plants can be used for the production of high value, medicinally important proteins, for example, production of Mabs (Hiatt et al. , 1989; During et al., 1990; Benvenuto et al. 1991 , Firek et al. 1993, Gao et al. 1993), human growth hormone (Kay et al. 1986) and human serum albumin (Sijmons et al. 1990).
  • Transformed cells synthesize, secrete, and accumulate functional antibodies including single (Benvenuto et al. 1991) and double (During et al. 1990, Hiatt et al. 1991) domain immunoglobulins.
  • none of these authors investigated production of any human growth factor from transgenic plants.
  • Plant cell culture media are well-defined and inexpensive compared to mammalian cell culture media. Further, plant cell products, unlike mammalian- derived protein formulations, are generally assumed as neither pathogenic nor oncogenic to humans (Crawford, 1995). Also, when compared to similar production in transgenic bacterial strains (Attaai and Shuler 1987), plant tissue culture methods showed greater stability of foreign gene expression, even without use of selection pressure (Gao et al. 1991).
  • Higo et al. (1993) produced a human growth factor, specifically hEGF in transgenic tobacco with cDNA fragment size of 180 bp. Unsatisfactory foreign peptide levels of 20 to 60 pg/mg (ppb) total soluble leaf protein were obtained.
  • Higo K Saito Y, Higo H. 1993. Expression of a chemically synthesized gene for human epidermal growth factor under the control of cauliflower mosaic virus 35S promoter in transgenic tobacco. Biosci Biotechnol Biochem
  • an object of the present invention to provide whole plant and plant cell culture derived human growth factors at higher overall concentrations and production rates, comparable to mammalian host cell systems.
  • the production of human growth factors is achieved in whole plants or plant cell culture wherein the human growth factor is produced with a length of at least 200 amino acids.
  • the human growth factor for epidermal growth factor this would comprise at least a tetramer of EGF units.
  • Modifying chimeric cDNA and subcloning into a plant expression vector are done using standard molecular cloning procedures (Ausubel et al. 1992) and splicing PCR techniques (Marks et al. 1992).
  • Effectiveness or production of the translation process has been increased according to the present invention by (1) cloning of pre-pro-EGF cDNA of approximately 4.5 kb into both whole plants and cell culture to increase overall titers of active hEGF, (2) synthesizing cDNA and transforming plants and cell culture for production of an oligomeric polypeptide consisting of repeated hEGF domains, and (3) increasing the overall size of the gene to be expressed with a fusion construct encoding hEGF linked to a protein that is efficiently produced in plant systems.
  • synthetic cDNA includes plant-specific proteolytic cleavage sites between EGF repeats to facilitate correct processing in planta.
  • FIG. 1 provides the size of EGF precursor (pre-pro-EGF) relative to correctly processed EGF.
  • FIG. 2 depicts schematically the construction of pZD203, a vector used to modify the restriction sites on pre-pro-EGF to develop cDNA suitable for cloning into the plant expression vector pGA643.
  • FIG. 3 depicts schematically the construction of pZD204, the plant expression vector carrying pre-pro-EGF.
  • FIG. 4 shows EGF levels seen in individual calli resulting from positive transformation and antibiotic selection. EGF concentrations were determined using enzyme-linked immunosorbent assay and are based on a 30 KD protein size.
  • the present invention is a method for production of human growth factors using whole plants as well as plant cell suspensions transformed with appropriately constructed vector plasmids, wherein the human growth factor is produced with a length of at least 200 amino acids. More specifically, the method of the present invention is stable expression of human growth factors of interest as direct therapeutics, targeted delivery systems and research reagents.
  • Human growth factors produced include human epidermal growth factor (hEGF), transforming growth factor (TGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), tumor necrosis factor (TNF), heparin-binding epidermal growth factor (HBEGF), insulin-like growth factor (ILGF), platelet-derived endothelial cell growth factor (PDECGF), platelet- derived angiogenesis factor (PDAF), and bone-and-cartilage inducing growth factor (BCIF).
  • hEGF human epidermal growth factor
  • TGF transforming growth factor
  • VEGF vascular endothelial growth factor
  • PDGF platelet-derived growth factor
  • FGF fibroblast growth factor
  • TNF tumor necrosis factor
  • HEGF heparin-binding epidermal growth factor
  • ILGF insulin-like growth factor
  • PDECGF platelet-derived endothelial cell growth factor
  • PDAF platelet
  • Preferred species include but are not limited to Nicotiana tabacum (tobacco), Solanum tuberosum (potato), Glycine max (soybean), and Zea mays (corn).
  • the method of the present invention a method of producing human growth factors from plant cells, has the steps of:
  • step of obtaining may be as simple as purchasing or more complex actual making by well known methods, for example direct particle bombardment as described in Gene Transfer by Particle Bombardment, Klein TM, Knowlton S, Arentzen R, Plant Tissue Culture Manual, DI, pp 1-12, 1991, Kluwer Academic Publishers, or by Agrobaterium mediated transformation as described in Hoekema et al.
  • the step of obtaining is by well known separation purification steps, for example ultrafiltration, affinity chromatography, and/or electrophoresis.
  • An Agrobacterium mediated transformation method of the present invention has the steps of:
  • modifying chimeric cDNA and subcloning into a plant expression vector are done using standard molecular cloning procedures (Ausubel et al. 1992) and splicing PCR techniques (Marks et al. 1992). More specifically, modifying chimeric cDNA, has the steps of: (a) adding a transcription promoter to the upstream or 5 ' end of the chimeric cDNA; and
  • the transcription promoter and the transcription terminator are regulatory elements.
  • an additional regulatory element encoding a signal peptide may be added between the transcription promoter and the 5' end of the chimeric cDNA in order to relegate the product human growth factor to a specific cellular organelle.
  • other regulatory elements may be added either between the promoter and the additional regulatory element encoding the signal peptide or at the 3' end of the chimeric cDNA to obtain greater mRNA stability between transcription and translation events.
  • the present invention further includes manipulation of a 35S promoter by duplication of the upstream region (-343 to -90 bp) of the CaMV 35S promoter to increase transcription activity, as well as use of TSC29 and TSC40 promoters. These promoters and their transcription activity have been reported by Gao et al. 1994, and Dai et al. 1995.
  • transcription promoters may include the upstream enhancer (nucleotides -343 to -90 relative to the transcription start site) of the CaMV 35S promoter (Benfey et al. 1989) or the chlorophyll a/b binding protein (cabl) promoter (Ha and An 1988).
  • upstream enhancer nucleotides -343 to -90 relative to the transcription start site
  • cabl chlorophyll a/b binding protein
  • Use of these types of regulatory elements confers human growth factor production characteristics into traditionally non-salable portions of crop plants, such as the leafy tops of potatoes.
  • Use of potato tops for example, under post-harvest conditions, results in overexpression and production of human growth factor in non-salable plant portions towards the end of the harvesting season, without affecting crop quality.
  • Transferring the plant expression vector into the agrobacterium is completed using the freeze-thaw method (An 1987).
  • super- binary vectors such as pTOK233 and pSB131, are used to achieve high transformation frequency (Ishida et al. 1996). Remaining cocultivation, selection, growth, and extraction steps (d through g) have been described by Magnusen et al. (1996), and are well known in the art of plant molecular biology.
  • hEGF has a length of 53 amino acids. Accordingly, obtaining a larger construct of at least 200 amino acids requires either (1) cloning the larger precursor cDNA, (2) synthesizing a concatemer consisting of multiple gene copies encoding the growth factor, or (3) increasing the overall size of a gene to be expressed using a fusion construct encoding a growth factor linked to a protein that is efficiently produced in plant systems.
  • cDNA encoding pre-pro-EGF is the cDNA encoding pre-pro-EGF.
  • This particular gene at approximately 4.5 kb, encodes a 1207 amino acid protein that, in vivo, is proteolytically cleaved to yield 53 amino acid EGF. In plant systems, this larger protein will provide additional stability against proteolytic degradation.
  • Synthesizing the cDNA concatemer is preferably done by ligating multiple gene copies using peptide linkers to obtain a processed protein length of at least 200 amino acids.
  • the multiple gene copies are preferably an oligomeric polypeptide having of repeated growth factor cDNA domains.
  • Peptide linkers may be used that are (1) proteolytically cleaved in planta, (2) proteolytically cleaved in a separate enzymatic treatment step, or (3) resistant to proteolytic cleavage.
  • Peptide linkers that are proteolytically cleaved by serine proteases in planta preferably possess the amino acid sequence Arg-Asn.
  • the processed protein is targeted either to the cell cytosol (no signal peptide) or vacuole (phytohemagglutinin signal peptide [Chrispeels et al.1991]).
  • the same amino acid sequence is preferably used (Arg- Asn) and the growth factor concatemer is either targeted to the chloroplast (pea photosystem II signal peptide) or secreted (PR-II signal peptide) to limit proteolytic degradation.
  • linkers would preferably possess the amino acid sequence Arg-Pro. This sequence is resistant to serine proteases. Specifically for EGF, linkage would preferably be achieved by synthesizing cDNA encoding a single proline unit between growth factor monomers cDNA.
  • Increasing the overall size of a gene may be done by ligating EGF with cDNA encoding a protective protein to protect from proteolytic cleavage, thereby forming a fusion construct.
  • Protective proteins include but are not limited to streptococcal protein G or -galactosidase, that have both been shown to inhibit proteolysis when attached to the C-terminus of other foreign proteins (Hellebust et al. 1989).
  • Gene size could also be increased by ligating EGF with cDNA encoding another protective protein of commercial interest that processes well in plant-based systems.
  • Protective proteins further include human serum albumin (Sijmons et al. 1990) and phytase (Verwoerd et al. 1995).
  • At least one genetic regulatory element may be included in the cDNA encoding the transcription of specific growth factors.
  • Regulatory elements include transcription promoters or enhancers that increase the frequency of transcription events, leader sequences that increase the stability of mRNA prior to translation, and signal peptides that target proteins to specific organelles for posttranslational modifications and accumulation.
  • transcription enhancers include but are not limited to the octapine synthase enhancer, a 16 bp palindrome (ACGTAAGCGCTTACGT) (Ellis et al. 1987) and the B-domain of the cauliflower mosaic virus 35S promoter (Kay et al. 1987).
  • leader sequence includes but is not limited to alfalfa mosaic virus RNA4 leader sequence (Jobling and Gehrke 1987).
  • signal peptides include but are not limited to the tobacco PR-S signal peptide (Cornelissen et al. 1986) and the phytohemagglutinin signal peptide (Hunt and Chrispeels 1991).
  • Example 1 The bacteriophage ⁇ EGF116 (ATCC No. 59956) containing the gene encoding the full length polypeptide of human kidney pre-pro-EGF was obtained from ATCC.
  • Pro-EGF (FIG. 1) is the 1207 amino acid precursor in which hEGF is flanked by polypeptide segments of 907 and 184 residues at its NH 2 - and COOH-termini, respectively (Bell et al., 1986).
  • the remainder of the 4.8 kb pre- pro-EGF gene encodes native signal peptides at both the NH 2 - and COOH- termini of pro-EGF.
  • the polypeptide contains a transmembrane (TM) binding region that facilitates proper cleavage in the endoplasmic reticulum.
  • TM transmembrane
  • cDNA was excised with Sma I, Hind III, and Eco RI restriction enzymes, as shown on FIG. 2, producing two separate fragments. These were sequentially ligated into compatible Sma I and Eco RI sites in pBluescript- creating the 7.5 kb plasmid pZD203. After proper orientation was confirmed, pre-pro-EGF cDNA was further excised with Xba I and Cla I restriction enzymes and ligated into compatible sites located between the CaMV 35S promoter and T 7 transcription terminator of binary vector pGA643, forming the 16 kb plasmid pZD204 (FIG. 3).
  • This plasmid was directly transferred into Agrobacterium tumefaciens LBA4404 using the freeze-thaw method (An 1987).
  • the transferred plasmid was introduced into tobacco whole plants (by leaf disks) and calli (by suspension culture) by co-cultivation with the Agrobacterium thereby producing transformants.
  • Over 200 specific samples of transformants were taken from the co-cultivation and separately placed on kanamycin selective media.
  • the co-cultivated transformants that grew were positive transformants.
  • the positive transformants were screened under kanamycin selection pressure and preliminary ELISA results indicated the presence of hEGF in tobacco calli. Accumulation levels of hEGF in select transgenic calli are shown on a ng/g fresh weight basis in FIG. 4. The bars in FIG.
  • the highest level of accumulation at approximately 400 ng/(g fresh weight cells) (ppb) corresponds to a concentration of 4.1 ng/(mg total soluble protein) (ppm) (based on a measured total soluble protein level of approximately 98 mg/(g fresh weight cells)).
  • the 4.1 ng/(mg total soluble protein) (ppm) corresponds to 4100 pg/(mg total soluble protein) (ppb) which is almost two orders-of-magnitude greater than the result of 60 pg/(mg total soluble protein) (ppb) reported by Higo et al. (1993).
  • the CaMV 35S enhancer contains at least two domains which can confer different developmental and tissue-specific expression patterns. EMBO J 8:2195-2202.
  • NAME/KEY human epithelial growth factor cDNA
  • ANTI-SENSE 5 ' -GTC CAG AGC ... CAG TGA TAA-3 end
  • FRAGMENT TYPE 5-copies of 159bp concatemer mature EGF linked with linkers
  • OORRIIGGIINNAALL S SOOUURRCCEE ::
  • GAATTCATCA ACAAATTACT CCTCAATCAC ACTCCTATAG AAAACGGTTT AAGCTATCAT 60 TACATGTCTA GTTGGTTTTA CTCAGCCCTA GAAGTGTTGT TTATTGCATC ACTTTCCACG 120 AAGCACAATT TTTCTTTTTT ACAATCACTA GACCTCACAG GCTCACACAT ATGCTTTAGA 180 GCACATTCTA AACTTTGAAC TATAAAAGCT GTTAACACTA ATACACTATG CGTTCTTTTT 240 TGCTCCAAAC ACTTTTGATC CATTATTAGG AGACACTCCA CTTAGAAAGA TTTTCTAATC 300 CTTTGGTCAA CTAGGAAGTT CAAGGTTTTT CTAAACAGAA ATTCATTTCA CAAGTAATTT 360 AATTTATAAG GAAATGAATA GAGAAATCAA ATCATTGAAG AACTACAAAA TATAGATTCA 420 AGGTCAGGTC TAAGAAAATA TTCCTGAAGC TCAAAAAAAAGA GTTTTCCTCT
  • MOLECULE TYPE (A) DESCRIPTION 5 ' -untranscription region of 35S gene from CaMV with 2 copies of B domains
  • CTTCGCAAGA CCCTTCCTCT ATATAAGGAA GTTCATTTCA TTTGGAGAGA ACACGGGGGA 1133

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Abstract

L'invention concerne la production de facteurs de croissance épidermique humaine (hEGF), réalisée à la fois dans des plantes entières et dans une culture de cellules végétales, lesdits hEGF ayant une longueur d'au moins 200 acides aminés. Pour un facteur de croissance épidermique, cela comprendrait au moins un tétramère d'unités EGF. L'efficacité ou la production du procédé de traduction a été améliorée conformément à la présente invention par: 1) clonage de l'ADN complémentaire pre-pro-EGF (précurseur EGF) d'environ 4,5 kb à la fois dans des plantes entières et dans des cultures de cellules végétales, afin d'augmenter tous les titrages de l'hEGF actif, 2) par synthèse de l'ADN complémentaire et par transformation des plantes et culture de cellules végétales pour produire un polypeptide oligomérique, qui consiste en des domaines de répétition hEGF, et 3) par augmentation de la taille globale du gène destiné à être exprimé avec un produit de recombinaison de fusion codant l'hEGF lié à une protéine qui est efficacement produite dans des systèmes végétaux. L'ADN complémentaire synthétique comporte des sites de coupure protéolytiques spécifiques d'un végétal entre des séquences répétées d'EGF, pour faciliter une maturation correcte in planta. Au besoin, des sites de coupure protéolytiques appropriés en amont et en aval des hEGF sont ajoutés pour obtenir le produit final. Dans les plantes entières, l'utilisation d'un élément régulateur permet de transformer les caractéristiques de la production d'hEGF en des parties traditionnellement non vendables de plantes cultivées, telles que la partie feuillue des pommes de terre. L'utilisation de cette partie feuillue après la récolte résulte en une surexpression d'hEGF, ce qui produit des parties de plantes non vendables à la fin de la période de récolte, sans pour autant affecter la qualité de ladite récolte.
PCT/US1997/020603 1996-11-12 1997-11-12 Procede de production facteurs de croissance humain a partir de plantes entieres ou de cultures de cellules vegetales WO1998021348A1 (fr)

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WO2001025456A2 (fr) * 1999-10-01 2001-04-12 Greenovation Biotech Gmbh Procede de production de substances proteiques
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US6783978B1 (en) 1999-11-19 2004-08-31 Battelle Memorial Institute Isolation and characterization of the genomic DNA clones of ribosomal protein gene L25 in tobacco
WO2005035768A1 (fr) * 2003-09-30 2005-04-21 Biolex, Inc. Expression de polypeptides biologiquement actifs dans du pourpier
US6884878B2 (en) 1999-04-21 2005-04-26 University Of Florida Research Foundation, Inc. Neuropeptides and their use for pest control
US7091401B2 (en) 1991-02-22 2006-08-15 Sembiosys Genetics Inc. Expression of epidermal growth factor in plant seeds
US7339092B2 (en) 2003-12-23 2008-03-04 Pioneer Hi-Bred International, Inc. Plant activation of Cry8Bb1 by insertion of a plant protease-sensitive site
US7700837B2 (en) * 2003-03-21 2010-04-20 Korea Research Institute Of Bioscience And Biotechnology Transformed plant cell expressing tandem repeats of beta-amyloid gene and plant produced by the same
US7709697B2 (en) 2002-01-08 2010-05-04 Agrivida, Inc. Transgenic plants expressing CIVPS or intein modified proteins and related method
US7947876B2 (en) * 2004-09-14 2011-05-24 Nippon Paper Industries Co., Ltd. Plant and plant storage organ having GLP-1 derivative accumulated therein and method of producing the same
US8022270B2 (en) 2000-07-31 2011-09-20 Biolex Therapeutics, Inc. Expression of biologically active polypeptides in duckweed
WO2012060666A2 (fr) * 2010-11-04 2012-05-10 한국생명공학연구원 Procédé de production à grande échelle de facteur de croissance épidermique humain à partir de levures
US8420387B2 (en) 2009-11-06 2013-04-16 Agrivida, Inc. Intein-modified enzymes, their production and industrial applications
US9249474B2 (en) 2009-11-06 2016-02-02 Agrivida, Inc. Consolidated pretreatment and hydrolysis of plant biomass expressing cell wall degrading enzymes
US9464333B2 (en) 2009-11-06 2016-10-11 Agrivida, Inc. Intein-modified enzymes, their production and industrial applications
US9598700B2 (en) 2010-06-25 2017-03-21 Agrivida, Inc. Methods and compositions for processing biomass with elevated levels of starch
US10407742B2 (en) 2009-11-06 2019-09-10 Agrivida, Inc. Intein-modified enzymes, their production and industrial applications
US10443068B2 (en) 2010-06-25 2019-10-15 Agrivida, Inc. Plants with engineered endogenous genes
US10889826B2 (en) 2017-06-16 2021-01-12 Arizona Board Of Regents On Behalf Of The University Of Arizona Methods and compositions for producing epidermal growth factor (EGF) in soybeans
US11098320B2 (en) 2017-02-03 2021-08-24 Arizona Board Of Regents On Behalf Of The University Of Arizona Engineering high-protein-content soybeans

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WO2001036649A3 (fr) * 1999-11-19 2002-01-03 Battelle Memorial Institute Isolation et caracterisation des clones d'adn genomique du gene l25 de la proteinique ribosomale chez le tabac
WO2001036649A2 (fr) * 1999-11-19 2001-05-25 Battelle Memorial Institute Isolation et caracterisation des clones d'adn genomique du gene l25 de la proteinique ribosomale chez le tabac
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