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WO1998036085A1 - Production de proteines matures dans des plantes - Google Patents

Production de proteines matures dans des plantes Download PDF

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Publication number
WO1998036085A1
WO1998036085A1 PCT/US1998/003068 US9803068W WO9836085A1 WO 1998036085 A1 WO1998036085 A1 WO 1998036085A1 US 9803068 W US9803068 W US 9803068W WO 9836085 A1 WO9836085 A1 WO 9836085A1
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Prior art keywords
mature
die
ala
seq
leu
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PCT/US1998/003068
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English (en)
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Thomas D. Sutliff
Raymond L. Rodriguez
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Applied Phytologics, Inc.
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Priority to CA002280894A priority Critical patent/CA2280894A1/fr
Priority to EP98906507A priority patent/EP0981635A1/fr
Priority to AU61716/98A priority patent/AU746826B2/en
Priority to JP53599798A priority patent/JP2001512318A/ja
Publication of WO1998036085A1 publication Critical patent/WO1998036085A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8128—Antithrombin III
    • 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
    • C07K14/76—Albumins
    • C07K14/765—Serum albumin, e.g. HSA
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8235—Fruit-specific
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline
    • 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
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

Definitions

  • the present invention relates to the production of mature proteins in plant cells, and in particular, to the production of proteins in mature secreted form.
  • Therapeutic proteins are commonly produced recombinantly by microbial expression systems, such as in E. coli and the yeast system S. cerevisiae. To date, the cost of recombinant proteins produced in a microbial host has limited the availability of a variety of therapeutically important proteins, such as human serum albumin (HSA) and ⁇ -antitrypsin (AAT), to the extent that the proteins are in short supply.
  • HSA human serum albumin
  • AAT ⁇ -antitrypsin
  • Some therapeutic proteins appear to rely on glycosylation for optimal activity or stability, and the general inability of microbial systems to glycosylate or properly glycosylate mammalian proteins has also limited the usefulness of these recombinant expression systems. In some cases, proper protein folding cannot take place, because of the need for mammalian-specific foldases or other folding conditions.
  • protein expression in cultured mammalian cells, or in transgenic animals may overcome the limitations of microbial expression systems.
  • the cost per weight ratio of the protein is still high in mammalian expression systems, and the risk of protein contamination by mammalian viruses may be a significant regulatory problem.
  • Protein production by transgenic animals also carries the risk of genetic variation from one generation to another. The attendant risk is variation in the recombinant protein produced, for example, variation in protein processing to yield a nature active protein with different N-terminal residue.
  • AAT Human ⁇ -antitrypsin
  • Normal AAT contains 394 residues, with three complex oligosaccharide units exposed to the surface of the molecule, linked to asparagines 46, 83, and 247 (Carrell, P., et al, Nature (1982) 298:329).
  • AAT is the major plasma proteinase inhibitor whose primary function is to control the proteolytic activity of trypsin, elastase, and chymotrypsin in plasma.
  • the protein is a potent inhibitor of neutrophil elastase, and a deficiency of AAT has been observed in a number of patients with chronic emphysema of the lungs.
  • a proportion of individuals with serum deficiency of AAT may progress to cirrhosis and liver failure ⁇ e.g., Wu, Y., et al, BioEssa s 13(4): 163 (1991).
  • Antithrombin III is the major inhibitor of thrombin and factor Xa, and to a lesser extent, other serine proteases generated during the coagulation process, e.g., factors IXa, XIa, and Xlla.
  • the inhibitory effect of A ⁇ II is accelerated dramatically by heparin. In patients with a history of deep vein thrombosis and pulmonary embolism, the prevalence of A ⁇ II deficiency is 2-
  • ATIII protein has been useful in treating hereditary A ⁇ II deficiency and has wide clinical applications for the prevention of thrombosis in high risk situations, such as surgery and delivery, and for treating acute thrombotic episodes, when used in combination with heparin.
  • ATIII is a glycoprotein with a molecular weight of 58,200, having 432 amino acids and containing three disulfide linkages and four asparag ⁇ ne-linked biantennary carbohydrate chains. Because of the key role of ATIII as an anti-thrombotic agent, and because of the broad clinical potential in anti-thrombosis therapy, there has been an active interest in recombinant synthesis of ATIII, for human therapeutic use. To date, this approach has not been satisfactory for ATEI produced by microbial or mammalian recombinant methods, for the reasons discussed above. Human Serum Albumin
  • Serum albumin is the main protein component of plasma. Its main function is regulation of colloidal osmotic pressure in the bloodstream. Serum albumin binds numerous ions and small molecules, including Ca2 + , Na + , K + , fatty acids, hormones, bilirubin and certain drugs.
  • Human serum albumin (HSA) is expressed as a 609 amino acid prepro-protein which is further processed by removal of an amino-terminal peptide and an additional six amino acid residues to form the mature protein.
  • the mature protein found in human serum is a monomeric, unglycosylated protein 585 amino acids in length (66 kDal), with a globular structure maintained by 17 disulfide bonds.
  • the pattern of disulfide links forms a structural unit of one small and two large disulfide-Iinked double loops (Geisow, M.J. et al. (1977) Biochem. J. 163:477-484) which forms a high-affinity bilirubin binding site.
  • HSA is used to expand blood volume and raise low blood protein levels in cases of shock, trauma, and post-surgical recovery. HSA is often administered in emergency situations to stabilize blood pressure.
  • HSA osmotic stabilizing agent
  • Subtilisin BPN' (BPN') is an important industrial enzyme, particularly for use as a detergent enzyme.
  • BPN' is an important industrial enzyme, particularly for use as a detergent enzyme.
  • Several groups have reported amino acid substitution modifications of the enzyme that are effective in enhancing the activity, pH optimum, stability and/or therapeutic use of the enzyme.
  • BPN' is expressed in as a 381 amino acid preproenzyme, including 35 amino acid sequence required for secretion and a 77 amino acid moiety which serves as a chaperon to facilitate folding. Studies indicate that the pro moiety acts in trans outside of cells.
  • the invention includes a method of producing, in monocot plant cells, a mature heterologous protein selected from the group consisting of (i) mature, glycosylated ⁇ - antitrypsin (AAT) having the same N-terminal amino acid sequence as mature AAT produced in humans and a glycosylation pattern which increases serum halflife substantially over that of non- glycosylated mature AAT; (ii) mature, glycosylated antithrombin III (A ⁇ II) having the same N- terminal amino acid sequence as mature ATIII produced in humans; (iii) mature human serum albumin (HSA) having the same N-terminal amino acid sequence as mature HSA produced in humans and having the folding pattern of native mature HSA as evidenced by its bilirubin-binding characteristics; and (iv) mature, active subtilisin BPN' (BPN'), glycosylated or non-glycosylated, having the same N-terminal amino acid sequence as BPN' produced in Bacillus.
  • AAT glycosylated
  • the method includes obtaining monocot cells transformed with a chimeric gene having (i) a monocot transcriptional regulatory region, inducible by addition or removal of a small molecule, or during seed maturation, (ii) a first DNA sequence encoding the heterologous protein, and (iii) a second DNA sequence encoding a signal peptide.
  • the second DNA sequence is operably linked to the transcriptional regulatory region and to the first DNA sequence.
  • the first DNA sequence is in translation-frame with the second DNA sequence, and the two sequences encode a fusion protein.
  • the transformed cells are cultivated under conditions effective to induce the transcriptional regulatory region, thereby promoting expression of the fusion protein and secretion of the mature heterologous protein from the transformed cells.
  • the mature heterologous protein produced by the transformed cells is then isolated.
  • the first DNA sequence encodes pro-subtilisin BPN' (proBPN')
  • the cultivating includes cultivating the transformed cells at a pH between 5 and 6
  • the isolating step includes incubating the proBPN' to under condition effective to allow its autoconversion to active mature BPN'.
  • the first DNA sequence encodes mature BPN'
  • the cells are transformed with a second chimeric gene containing (i) a transcriptional regulatory region inducible by addition or removal of a small molecule, (ii) a third DNA sequence encoding the pro-peptide moiety of BPN', and (iii) a fourth DNA sequence encoding a signal polypeptide.
  • the fourth DNA sequence is operably linked to d e transcriptional regulatory region and to the third DNA sequence, and the signal polypeptide is in translation-frame with the pro-peptide moiety and is effective to facilitate secretion of expressed pro-peptide moiety from the transformed cells.
  • the cultivating step includes cultivating the transformed cells at a pH between 5 and 6, and the isolating step includes incubating the mature BPN' and the pro-moiety under conditions effective to allow the conversion of BPN' by the pro- moiety to active mature BPN'.
  • the signal peptide is the RAmy3D signal peptide (SEQ ID NO:l) or the RAmylA signal peptide (SEQ ID NO:4).
  • the coding sequence of the signal peptide may be a codon-optimized sequence, such as the codon-optimized RAmy3D sequence identified as SEQ ID NO:3.
  • the first DNA sequence may also be codon-optimized.
  • Exemplary codon-optimized signal peptide-heterologous protein fusion protein coding sequences include 3D- AAT (SEQ ID NO:18), 3D-A ⁇ II (SEQ ID NO:19), and 3D-HSA (SEQ ID NO:20).
  • the first DNA sequence may further contain codon substitutions which eliminate one or more potential glycosylation sites present in the native amino acid sequence of the heterologous protein, such as the codon-optimized sequence encoding 3D-proBPN * (SEQ ID NO:21).
  • the transcriptional regulatory region may be a promoter derived from a rice or barley ⁇ -amylase gene, including RAmylA, RAmylB, RAmy2A, RAmy3A, RAmy3B, RAmy3C, RAmy3D, RAmy3E, pM/C, gKAmyl41, gKAmyl55, Amy32b, or HV18.
  • the chimeric gene may further include, between the transcriptional regulatory region and the fusion protein coding sequence, the 5' untranslated region (5' UTR) of an inducible monocot gene such as one of the rice or barley ⁇ -amylase genes described above.
  • an inducible monocot gene such as one of the rice or barley ⁇ -amylase genes described above.
  • One preferred 5' UTR is that from the RAmylA gene, which is effective to enhance the stability of the gene transcript.
  • the chimeric gene may further include, downstream of the coding sequence, the 3' untranslated region (3' UTR) from an inducible monocot gene, such as one of the rice or barley ⁇ -amylase genes mentioned above.
  • One preferred 3' UTR is from the RAmylA gene.
  • preferred promoters are the RAmy3D and RAmy3E gene promoters, which are upregulated by sugar depletion in cell culture.
  • a preferred promoter is the RAmylA gene promoter, which is upregulated by gibberellic acid during seed germination.
  • a preferred promoter is the barley endosperm-specific Bl-hordein promoter.
  • the invention also includes a mature heterologous protein produced by the above method.
  • the protein has a glycosylation pattern characteristic of the monocot plant in which the protein is produced.
  • the glycosyated protein is selected from the group consisting of (i) mature glycosylated ⁇ -antitrypsin (AAT) having the same N-terminal amino acid sequence as mature AAT produced in humans and having a glycosylation pattern which increases serum halflife substantially over that of non-glycosylated mature AAT; (ii) mature glycosylated antithrombin III (ATIII) having the same N- terminal amino acid sequence as mature A ⁇ II produced in humans; and (iii) mature glycosylated subtilisin BPN' (BPN') having the same N-terminal amino acid sequence as BPN' produced in Bacillus.
  • AAT mature glycosylated ⁇ -antitrypsin
  • ATIII mature glycosylated antithrombin III
  • BPN' mature glycosylated subtilisin BPN'
  • the invention also includes plant cells and seeds capable of producing the mature heterologous proteins according to the above method.
  • Fig. 1 shows, in the lower row, the amino acid sequence of a RAmy3D signal sequence portion employed in the invention, identified as SEQ ID NO:l; in the middle row, the corresponding native coding sequence, identified as SEQ ID NO:2; and in the upper row, a corresponding codon-optimized sequence, identified as SEQ ID NO:3;
  • Fig. 2 illustrates the components of a chimeric gene constructed in accordance with an embodiment of the invention
  • Figs. 3A and 3B illustrate the construction of an exemplary transformation vector for use in transforming a monocot plant, for production of a mature protein in cell culture in accordance with one embodiment of the invention (native mature AAT coding sequence under control of the RAmy3D promoter and signal sequence);
  • Fig. 4 illustrates factors in the metabolic regulation of AAT production in rice cell culture
  • Fig. 5 shows immunodetection of AAT using antibody raised against the C-terminal region of AAT
  • Fig. 6 shows Western blot analysis of AAT produced by transformed rice cell lines 18F,
  • Fig. 7 shows the time course of elastase:AAT complex formation in human and rice- produced forms of AAT;
  • Fig. 8 shows an N-terminal sequence for mature ⁇ -antitrypsin (AAT) produced in accordance with the invention, identified herein as SEQ ID NO:22;
  • Fig. 9 shows a Western blot of ATIII produced in accordance with the invention.
  • Fig. 10 shows a Western blot of plant-produced BPN', comparing expression from codon- optimized and native coding sequences
  • Fig. 11 compares the specific activity of BPN' codon-optimized (AP106) vs. BPN' native (AP101) expression in rice callus cell culture;
  • Fig. 12 shows a western blot of HSA produced in germinating seeds in accordance with the invention.
  • SEQ ID NO:l is the amino acid sequence of the RAmy3D signal peptide
  • SEQ ID NO:2 is the native sequence encoding the RAmy3D signal peptide
  • SEQ ID NO:3 is a codon-optimized sequence encoding the RAmy3D signal peptide
  • SEQ ID NO:4 is the amino acid sequence of the RAmylA signal peptide
  • SEQ ID NO:5 is the 5' UTR derived from the RAmylA gene
  • SEQ ID NO:6 is the 3' UTR derived from the RAmylA gene
  • SEQ ID NO:7 is the amino acid sequence of mature ⁇ r antitrypsin (AAT);
  • SEQ ID NO: 8 is the native DNA coding sequence of mature AAT
  • SEQ ID NO:9 is the amino acid sequence of mature antithrombin III (AT111);
  • SEQ ID NO: 10 is the native DNA coding sequence of mature AT111;
  • SEQ ID NO: 11 is the amino acid sequence of mature human serum albumin (HSA);
  • SEQ ID NO: 12 is the native DNA coding sequence of mature HSA;
  • SEQ ID NO: 13 is the amino acid sequence of native proBPN'
  • SEQ ID NO: 14 is the native DNA coding sequence of proBPN'
  • SEQ ID NO: 15 is the amino acid sequence of the "pro" moiety of BPN';
  • SEQ ID NO: 16 is the amino acid sequence of native mature BPN';
  • SEQ ID NO: 17 is the amino acid sequence of a mature BPN' variant in which all potential N-glycosylation sites are removed according to Table 2;
  • SEQ ID NO: 18 is a codon-optimized sequence encoding the RAmy3D signal sequence/mature ⁇ -antitrypsin fusion protein
  • SEQ ID NO: 19 is a sequence encoding the RAmy3D signal sequence/mature antithrombin
  • SEQ ID NO:20 is a sequence encoding the RAmy3D signal sequence/mature human serum albumin fusion protein, with a codon-optimized RAmy3D coding sequence fused to the native mature HSA coding sequence;
  • SEQ ID NO:21 is a codon-optimized sequence encoding the RAmy3D signal sequence/prosubtilisin BPN' fusion protein
  • SEQ ID NO: 22 is the N-terminal sequence of mature ⁇ -antitrypsin produced in accordance with the invention
  • SEQ ID NO:23 is an oligonucleotide used to prepare the intermediate p3DProSig construct of Example 1;
  • SEQ ID NO:24 is the complement of SEQ ID NO:23;
  • SEQ ID NO:25 is an oligonucleotide used to prepare the intermediate p3DProSigENDlink construct of Example 1;
  • SEQ ID NO:26 is the complement of SEQ ID NO:25;
  • SEQ ID NO:27 is one of six oligonucleotides used to prepare the intermediate plAProSig construct of Example 1;
  • SEQ ID NO:28 is one of six oligonucleotides used to prepare the intermediate plAProSig construct of Example 1
  • SEQ ID NO:29 is one of six oligonucleotides used to prepare the intermediate plAProSig construct of Example 1;
  • SEQ ID NO:30 is one of six oligonucleotides used to prepare the intermediate plAProSig construct of Example 1;
  • SEQ ID NO:31 is one of six oligonucleotides used to prepare the intermediate plAProSig construct of Example 1
  • SEQ ID NO:32 is one of six oligonucleotides used to prepare the intermediate plAProSig construct of Example 1;
  • SEQ ID NO:33 is the N-terminal primer used to PCR-amplify the AAT coding sequence according to Example 1; and SEQ ID NO: 34 is me C-terminal primer used to PCR-amplify the AAT coding sequence according to Example 1.
  • Cell culture refers to cells and cell clusters, typically callus cells, growing on or suspended in a suitable growth medium.
  • “Germination” refers to the breaking of dormancy in a seed and the resumption of metabolic activity in the seed, including the production of enzymes effective to break down starches in the seed endosperm.
  • “Inducible” means a promoter that is upregulated by the presence or absence of a small molecules. It includes both indirect and direct inducement.
  • “Inducible during germination” refers to promoters which are substantially silent but not totally silent prior to germination but are turned on substantially (greater than 25%) during germination and development in the seed. Examples of promoters that are inducible during germination are presented below.
  • Small molecules in the context of promoter induction, are typically small organic or bioorganic molecules less than about 1 kDal. Examples of such small molecules include sugars, sugar-derivatives (including phosphate derivatives), and plant hormones (such as, gibberellic or absissic acid).
  • Specifically regulatable refers to the ability of a small molecule to preferentially affect transcription from one promoter or group of promoters (e.g., the ⁇ -amylase gene family), as opposed to non-specific effects, such as, enhancement or reduction of global transcription within a cell by a small molecule.
  • promoter or group of promoters e.g., the ⁇ -amylase gene family
  • “Seed maturation” or “grain development” refers to the period starting with fertilization in which metabolizable reserves, e.g., sugars, oligosaccharides, starch, phenolics, amino acids, and proteins, are deposited, with and without vacuole targeting, to various tissues in the seed (grain), e.g., endosperm, testa, aleurone layer, and scutellar epithelium, leading to grain enlargement, grain filling, and ending with grain desiccation.
  • “Inducible during seed maturation” refers to promoters which are turned on substantially (greater than 25%) during seed maturation.
  • Heterologous DNA or “foreign DNA” refers to DNA which has been introduced into plant cells from ano er source, or which is from a plant source, including the same plant source, but which is under the control of a promoter or terminator that does not normally regulate expression of the heterologous DNA.
  • Heterologous protein is a protein, including a polypeptide, encoded by a heterologous DNA.
  • a "transcription regulatory region” or “promoter” refers to nucleic acid sequences that influence and/or promote initiation of transcription. Promoters are typically considered to include regulatory regions, such as enhancer or inducer elements.
  • a "chimeric gene,” in the context of the present invention, typically comprises a promoter sequence operably linked to DNA sequence that encodes a heterologous gene product, e.g., a selectable marker gene or a fusion protein gene.
  • a chimeric gene may also contain further transcription regulatory elements, such as transcription termination signals, as well as translation regulatory signals, such as, termination codons.
  • “Operably linked” refers to components of a chimeric gene or an expression cassette that function as a unit to express a heterologous protein.
  • a promoter operably linked to a heterologous DNA which encodes a protein, promotes the production of functional mRNA corresponding to the heterologous DNA.
  • a "product" encoded by a DNA molecule includes, for example, RNA molecules and polypeptides.
  • Removal in the context of a metabolite includes both physical removal as by washing and the depletion of the metabolite through the absorption and metabolizing of the metabolite by the cells.
  • Substantially isolated is used in several contexts and typically refers to the at least partial purification of a protein or polypeptide away from unrelated or contaminating components. Methods and procedures for the isolation or purification of proteins or polypeptides are known in the art.
  • “Stably transformed” as used herein refers to a cereal cell or plant that has foreign nucleic acid stably integrated into its genome which is transmitted through multiple generations.
  • ⁇ -antitrypsin or "AAT” refers to the protease inhibitor which has an amino acid sequence substantially identical or homologous to AAT protein identified by SEQ ID NO:7.
  • Antithrombin III or “ATIII” refers to the heparin-activated inhibitor of thrombin and factor Xa, and which has an amino acid sequence substantially identical or homologous to ATJJl protein identified by SEQ ID NO:9.
  • Human serum albumin or “HSA” refers to a protein which has an amino acid sequence " substantially identical or homologous to the mature HSA protein identified by SEQ ID NO: 11.
  • Subtilisin or “subtilisin BPN'” or “BPN”' refers to the protease enzyme produced naturally by B. amyloliquefaciens, and having the sequence of SEQ ID NO: 16, or a sequence homologous therewith.
  • proBPN refers to a form of BPN' having an approximately 78 amino-acid "pro” moiety that functions as a chaperon polypeptide to assist in folding and activation of the BPN', and having the sequence in SEQ ID NO: 13, or a sequence homologous therewith.
  • Codon optimization refers to changes in the coding sequence of a gene to replace native codons with those corresponding to optimal codons in the host plant.
  • a DNA sequence is "derived from" a gene, such as a rice or barley ⁇ -amylase gene, if it corresponds in sequence to a segment or region of that gene. Segments of genes which may be derived from a gene include me promoter region, the 5' untranslated region, and the 3' untranslated region of the gene.
  • the plants used in the process of the present invention are derived from monocots, particularly the members of the taxonomic family known as the Gramineae.
  • This family includes all members of the grass family of which the edible varieties are known as cereals.
  • the cereals include a wide variety of species such as wheat (Triticum sps.), rice (Oryza sps.) barley (Hordeum sps.) oats, (Avena sps.) rye (Secale sps.), corn (Zea sps.) and millet (Pennisettum sps.).
  • preferred family members are rice and barley.
  • Plant cells or tissues derived from the members of the family are transformed with expression constructs (i.e., plasmid DNA into which the gene of interest has been inserted) using a variety of standard techniques (e.g., electroporation, protoplast fusion or microparticle bombardment).
  • the expression construct includes a transcription regulatory region (promoter) whose transcription is specifically upregulated by the presence of absence of a small molecule, such as the reduction or depletion of sugar, e.g., sucrose, in culture medium, or in plant tissues, e.g., germinating seeds.
  • particle bombardment is the preferred transformation procedure.
  • the construct also includes a gene encoding a mature heterologous protein in a form suitable for secretion from plant cells.
  • the gene encoding the recombinant heterologous protein is placed under the control of a metabolically regulated promoter.
  • Metabolically regulated promoters are those in which mRNA synthesis or transcription, is repressed or upregulated by a small metabolite or hormone molecule, such as the rice RAmy3D and RAmy3E promoters, which are upregulated by sugar-depletion in cell culture.
  • a preferred promoter is the Ramy 1A promoter, which is up-regulated by gibberellic acid during seed germination.
  • the expression construct also utilizes additional regulatory DNA sequences e.g., preferred codons, termination sequences, to promote efficient translation of AAT, as will be described.
  • Expression vectors for use in the present invention comprise a chimeric gene (or expression cassette), designed for operation in plants, with companion sequences upstream and downstream from the expression cassette.
  • the companion sequences will be of plasmid or viral origin and provide necessary characteristics to the vector to permit the vectors to move DNA from bacteria to the desired plant host.
  • Suitable transformation vectors are described in related application PCT WO 95/14099, published May 25, 1995, which is incorporated by reference herein.
  • Suitable components of the expression vector including an inducible promoter, coding sequence for a signal peptide, coding sequence for a mature heterologous protein, and suitable termination sequences are discussed below.
  • One exemplary vector is the p3D(AAT)vl.O vector illustrated in Figs 3A and 3B.
  • the transcription regulatory or promoter region is chosen to be regulated in a manner allowing for induction under selected cultivation conditions, e.g., sugar depletion in culture or water uptake followed by gibberellic acid production in germinating seeds.
  • Suitable promoters, and their method of selection are detailed in above-cited PCT application WO 95/14099. Examples of such promoters include those that transcribe the cereal ⁇ -amylase genes and sucrose synthase genes, and are repressed or induced by small molecules, like sugars, sugar depletion or phytohormones such as gibberellic acid or absissic acid.
  • promoters include the promoters from the rice ⁇ -amylase RAmylA, RAmylB, RAmy2A, RAmy3A, RAmy3B, RAmy3C, RAmy3D, and RAmy3E genes, and from the pM/C, gKAmyl41, gKAmyl55, Amy32b, and HV18 barley ⁇ - amylase genes. These promoters are described, for example, in ADVANCES IN PLANT BIOTECHNOLOGY Ryu, D.D.Y., et al, Eds., Elsevier, Amsterdam, 1994, p.37, and references cited therein.
  • Other suitable promoters include the sucrose synthase and sucrose-6-phosphate-synthetase (SPS) promoters from rice and barley.
  • promoters which are regulated in a manner allowing for induction under seed-maturation conditions.
  • promoters include those associated with the following monocot storage proteins: rice glutelins, oryzins, and prolamines, barley hordeins, wheat gliadins and glutelins, maize zeins and glutelins, oat glutelins, and sorghum kafirins, millet pennisetins, and rye secalins.
  • a preferred promoter for expression in germinating seeds is the rice ⁇ -amylase RAmylA promoter, which is upregulated by gibberellic acid.
  • Preferred promoters for expression in cell culture are the rice ⁇ -amylase RAmy3D and RAmy3E promoters which are strongly upregulated by sugar depletion in the culture. These promoters are also active during seed germination.
  • a preferred promoter for expression in maturing seeds is the barley endosperm-specific Bl-hordein promoter (Brandt, A., et al, (1985) Carlsberg Res. Commun. 50:333-345).
  • the chimeric gene may further include, between the promoter and coding sequences, the 5' untranslated region (5' UTR) of an inducible monocot gene, such as the 5' UTR derived from one of the rice or barley ⁇ -amylase genes mentioned above.
  • 5' UTR 5' untranslated region
  • an inducible monocot gene such as the 5' UTR derived from one of the rice or barley ⁇ -amylase genes mentioned above.
  • One preferred 5' UTR is that derived from the RAmylA gene, which is effective to enhance the stability of the gene transcript. This 5' UTR has the sequence given by SEQ ID NO: 5 herein.
  • the chimeric gene encodes a signal sequence
  • signal peptide that allows processing and translocation of the protein, as appropriate.
  • Suitable signal sequences are described in above-referenced PCT application WO 95/14099.
  • One preferred signal sequence is identified as SEQ ID NO:l and is derived from the RAmy3D promoter.
  • Another preferred signal sequence is identified as SEQ ID NO:4 and is derived from the RAmylA promoter.
  • the plant signal sequence is placed in frame with a heterologous nucleic acid encoding a mature protein, forming a construct which encodes a fusion protein having an N-terminal region corresponding to the signal peptide and, immediately adjacent to the C-terminal amino acid of the signal peptide, the N-terminal amino acid of the mature heterologous protein.
  • the expressed fusion protein is subsequently secreted and processed by signal peptidase cleavage precisely at the junction of the signal peptide and the mature protein, to yield the mature heterologous protein.
  • the coding sequence in die fusion protein gene in at least the coding region for the signal sequence, may be codon-optimized for optimal expression in plant cells, e.g., rice cells, as described below.
  • the upper row in Fig. 1 shows one codon- optimized coding sequence for the RAmy3D signal sequence, identified herein as SEQ ID NO:3.
  • P1 -Antitrypsin Mature human AAT is composed of 394 amino acids, having the sequence identified herein as SEQ ID NO:7. The protein has N-glycosylation sites at asparagines 46, 83 and 247. The corresponding native DNA coding sequence is identified herein as SEQ ID NO:8.
  • Antithrombin III Mature human ATM is composed of 432 amino acids, having the sequence identified herein as SEQ ID NO:9. The protein has N-glycosylation sites at the four asparagine residues 96, 135, 155, and 192.
  • the corresponding native DNA coding sequence is identified herein as SEQ ID NO: 10.
  • Human serum albumin Mature HSA as found in human serum is composed of 585 amino acids, having the sequence identified herein as SEQ ID NO: 11. The protein has no N-linked glycosylation sites. The corresponding native DNA coding sequence is identified herein as SEQ ID NO:12.
  • Subtilisin BPN' Native proBPN' as produced in B. amyloliquefaciens is composed of 352 amino acids, having the sequence identified herein as SEQ ID NO: 13, The corresponding native DNA coding sequence is identified herein as SEQ ID NO: 14.
  • the proBPN' polypeptide contains a 77 amino acid "pro" moiety which is identified herein as SEQ ID NO: 15.
  • the remainder of the polypeptide, which forms the mature active BPN' is a 275 amino acid sequence identified herein by SEQ ID NO: 16.
  • Native BPN' as produced in Bacillus is not glycosylated.
  • the method will be illustrated for expression of a heterologous gene in rice plant cells, it being recognized that the method is generally applicable to any monocot.
  • a representative set of known coding gene sequence from rice is assembled.
  • the sequences are then analyzed for codon frequency for each amino acid, and the most frequent codon is selected for each amino acid.
  • This approach differs from earlier reported codon matching methods, in which more than one frequent codon is selected for at least some of the amino acids.
  • the optimal codons selected in this manner for rice and barley are shown in Table 1.
  • the fusion protein coding sequence in die chimeric gene is constructed such that the final (C-terminal) codon in the signal sequence is immediately followed by the codon for the N-terminal amino acid in the mature form of the heterologous protein.
  • Exemplary fusion protein genes, in accordance witii the present invention, are identified herein as follows:
  • SEQ ID NO: 18 corresponding to codon-optimized coding sequences of me fusion protein consisting of RAmy3D signal sequence/mature ⁇ -antitrypsin;
  • SEQ ID NO: 19 corresponding to the fusion protein coding sequence consisting of the codon-optimized RAmy3D signal sequence and me native mature antithrombin III sequence;
  • SEQ ID NO:20 corresponding to the fusion protein coding sequence consisting of the codon-optimized RAmy3D signal sequence and die native mature human serum albumin sequence;
  • prosubtilisin is considered the "mature" protein, in that secreted prosubtilisin can autocatalyze to active, mature subtilisin.
  • the BPN' coding sequence is further modified to eliminate potential N-glycosylation sites, as native BPN' is not glycosylated.
  • Table 2 illustrates preferred codon substitutions, which eliminate all potential N-glycosylation sites in subtilisin BPN'.
  • SEQ ID NO: 17 corresponds to a mature BPN' amino acid sequence containing the substitutions presented in Table 2.
  • thermostability 'improved thermostability; Bryan, et al., Proteins: Structure, Function, and Genetics 326 (1986).
  • the chimeric gene may also include, downstream of the coding sequence, die 3' untranslated region (3 ' UTR) from an inducible monocot gene, such as one of the rice or barley ⁇ - amylase genes mentioned above.
  • Die 3' UTR is that derived from me RAmylA gene, whose sequence is given by SEQ ID NO:6. This sequence includes non-coding sequence 5' to me polyadenylation site, die polyadenylation site, and die transcription termination sequence.
  • the transcriptional termination region may be selected, particularly for stability of the mRNA to enhance expression. Polyadenylation tails (Alber and Kawasaki, 1982, Mol. and Appl. Genet.
  • Polyadenylation sequences include but are not limited to the Agrobacterium octopine synthetase signal (Gielen, et al, EMBO J. 3:835- 846 (1984) or the nopaline synthase of the same species (Depicker, et al., Mol. Appl. Genet. 1:561- 573 (1982).
  • Fig. 2 shows the elements of one preferred chimeric gene constructed in accordance widi the invention, and intended particularly for use in protein expression in a rice cell suspension culture.
  • the gene includes, in a 5' to 3' direction, me promoter from the RAmy3D gene, which is inducible in cell culture with sugar depletion, the 5' UTR from the RAmylA gene, which confers enhanced stability on the gene transcript, the RAmy3D signal sequence coding region, as identified above, die coding region of a heterologous protein to be produced, and a 3' UTR region from the RAmylA gene.
  • the chimeric gene is placed in a suitable expression vector designed for operation in plants.
  • the vector includes suitable elements of plasmid or viral origin that provide necessary characteristics to the vector to permit the vectors to move DNA from bacteria to the desired plant host.
  • suitable transformation vectors are described in related application PCT WO 95/14099, published May 25, 1995, which is incorporated by reference herein.
  • Suitable components of the expression vector, including the chimeric gene described above, are discussed below.
  • One exemplary vector is the p3Dvl.O vector described in Example 1.
  • Vectors containing a chimeric gene of the present invention may also include selectable markers for use in plant cells (such as the nptll kanamycin resistance gene, for selection in kanamycin-containing or the phosphinothricin acetyltransferase gene, for selection in medium containing phosphinothricin (PPT).
  • selectable markers for use in plant cells such as the nptll kanamycin resistance gene, for selection in kanamycin-containing or the phosphinothricin acetyltransferase gene, for selection in medium containing phosphinothricin (PPT).
  • the vectors may also include sequences mat allow their selection and propagation in a secondary host, such as sequences containing an origin of replication and a selectable marker such as antibiotic or herbicide resistance genes, e.g., HPH (Hagio et al, Plant Cell Reports 14:329 (1995); van der Elzer, Plant Mol. Biol. 5:299-302 (1985).
  • Typical secondary hosts include bacteria and yeast.
  • the secondary host is Escherichia coli
  • the origin of replication is a colEl-type
  • die selectable marker is a gene encoding ampicillin resistance.
  • sequences are well known in the art and are commercially available as well (e.g., Clontech, Palo Alto, CA; Stratagene, La Jolla, CA).
  • the vectors of the present invention may also be modified to intermediate plant transformation plasmids that contain a region of homology to an Agrobacterium tumefaciens vector, a T-DNA border region from Agrobacterium tumefaciens, and chimeric genes or expression cassettes (described above). Further, the vectors of the invention may comprise a disarmed plant tumor inducing plasmid of Agrobacterium tumefaciens.
  • the vector described in Example 1, and having a promoter from me RAmy3D gene is suitable for use in a method of mature protein production in cell culture, where the RAmy3D promoter is induced by sugar depletion in cell culture medium. Other promoters may be selected for other applications, as indicated above. For example, for mature protein expression in germinating seeds, me coding sequence may be placed under me control of the rice ⁇ -amylase RAmylA promoter, which is inducible by gibberellic acid during seed germination.
  • promoters directing expression of selectable markers used for plant transformation should operate effectively in plant hosts.
  • selectable markers used for plant transformation e.g., nptll
  • promoters directing expression of selectable markers used for plant transformation should operate effectively in plant hosts.
  • One such promoter is the nos promoter from native Ti plasmids (Herrera-Estrella, et al, Nature 303:209-213 (1983).
  • Others include the 35S and 19S promoters of cauliflower mosaic virus (Odell, et al, Nature 313:810-812 (1985) and die 2' promoter (Velten, et al. , EMBO J. 3:2723-2730 (1984).
  • me embryo and endosperm of mature seeds are removed to exposed scutulum tissue cells.
  • the cells may be transformed by DNA bombardment or injection, or by vectored transformation, e.g., by Agrobacterium infection after bombarding me scuteller cells with microparticles to make them susceptible to Agrobacterium infection (Bidney et al. , Plant Mol. Biol. 18:301-313, 1992).
  • Callus mass is men detached from the seed, and placed on fresh NB media, and incubated again for about 14 days at 28°C. After the second incubation period, satellite calli developed around die original "mother” callus mass. These satellite calli were slightiy smaller, more compact and defined dian die original tissue. It was these calli were transferred to fresh media. The "mother " calli was not transferred. The goal was to select only the strongest, most vigorous growing tissue for further culture.
  • Calli to be bombarded are selected from 14-day-old subcultures.
  • the size, shape, color and density are all important in selecting calli in me optimal physiological condition for transformation.
  • the calli should be between .8 and 1.1 mm in diameter.
  • the calli should appear as spherical masses with a rough exterior. Transformation is by particle bombardment, as detailed in the references cited above.
  • the cells are typically grown under conditions that permit expression of the selectable marker gene.
  • die selectable marker gene is HPH. It is preferred to culture the transformed cells under multiple rounds of selection to produce a uniformly stable transformed cell line.
  • Transgenic cells are cultured under conditions that favor plant cell growth, until the cells reach a desired cell density, tiien under conditions diat favor expression of the mature protein under the control of the given promoter. Preferred culture conditions are described below and in Example 2. Purification of me mature protein secreted into me medium is by standard techniques known by those of skill in the art.
  • me culture medium contains a phosphate buffer, e.g., me 20 IDM phosphate buffer, pH 6.8 described in Example 2, to reduce AAT degradation catalyzed by metals.
  • a metal chelating agent such as EDTA, may be added to me medium.
  • Lane 4 contains human AAT, and its migration position corresponds to about 52 kdal.
  • me plant-produced AAT having an apparent molecular weight of about 49-50 kdal, indicating an extent of glycosylation of up to 60-80% of the glycosylation found in human AAT (non-glycosylated AAT has a molecular weight of 45 kdal). Similar results are shown in the Western blots in Fig. 6.
  • Lanes 1-3 in this figure correspond to decreasing amount (15, 10, and 5 ng) of human AAT; lane 4, to 10 ⁇ l supernatant from a non-expressing plant cell line; lanes 5 and 6, to 10 nl supernatant from AAT-expressing plant cell lines 11B and 27F, respectively, and lane 7, to 10 nl supernatant from cell line 27F plus
  • Fig. 7 shows the shift in molecular weight over a 30 minute binding interval for the 52 kdal human AAT (lanes 1-4) and the 49-50 kdal plant-produced AAT.
  • the culture medium contains a
  • the chaperon "pro" moiety of the enzyme facilitates enzyme folding and is cleaved from me enzyme, leaving the active mature form of BPN'.
  • me mature enzyme is co-expressed and co-secreted widi me “pro” chaperon moiety, with conversion of the enzyme to active form occurring in presence of the free chaperon (Eder et al, Biochem. (1993) 32:18-26; Eder et al, (1993) J. Mol. Biol 223:293-304).
  • the BPN' is secreted in inactive form at a pH that may be in the 6-8 range, with subsequent activation of the inactive form, e.g., after enzyme isolation, by exposure to the "pro" chaperon moiety, e.g., immobilized to a solid support.
  • me culture medium is maintained at a pH of between 5 and 6, preferably about 5.5 during the period of active expression and secretion of BPN', to keep the BPN', which is normally active at alkaline pH, at a pH below optimal activity.
  • Codon optimization to me host plant's most frequent codons yielded a severalfold enhancement in the level of expressed heterologous protein in cell culture as shown in Fig. 11.
  • the extent of enhancement is seen from the Western blot analysis shown in Fig. 10 for two cells lines and further substantiated in Fig. 11.
  • Lane 2 (second from left) in Fig. 10 shows a Western blot of BPN' obtained in culture from cells transformed widi a native proBPN' coding sequence. Two bands observed correspond to a lower molecular weight protein whose approximately 35 kdal molecular weight corresponds to tiiat of proBPN'. The upper band corresponds to a somewhat higher molecular weight species, possibly glycosylated.
  • the first lane in the figure shows BPN' polypeptides produced in culture by plant cells transformed wi i the codon-optimized proBPN' sequence identified by SEQ ID NO:21.
  • the amount of BPN' enzyme produced with a codon-optimized sequence was severalfold higher than for subtilisin BPN' produced widi die native coding sequence.
  • Fig. 11 compares the specific activity of BPN' codon-optimized (AP106) versus BPN' native (AP101) expression in rice callus cell culture, assayed using the chromogenic peptide substrate suc-Ala-Ala-Pro-Phe-pNA as described by DelMar, E.G. et al. (1979; Anal. Biochem. 99:316-320). As shown if Fig. 11, several of the cell lines transformed widi codon-optimized chimeric genes produced levels of BPN', as evidenced by measured specific activity in culture medium, mat were 2-5 times the highest levels observed for plant cells transformed with native proBPN' sequence.
  • Plant regeneration from cultured protoplasts or callus tissue is carried by standard methods, e.g., as described in Evans et al, HANDBOOK OF PLANT CELL CULTURES Vol. 1: (MacMillan Publishing Co. New York, 1983); and Vasil I.R. (ed.), CELL CULTURE AND SOMATIC CELL
  • the transgenic seeds obtained from the regenerated plants are harvested, and prepared for germination by an initial steeping step, in which me seeds immersed in or sprayed wi i water to increase the moisture content of die seed to between 35-45%. This initiates germination. Steeping typically takes place in a steep tank which is typically fitted widi a conical end to allow me seed to flow freely out. The addition of compressed air to oxygenate the steeping process is an option. The temperature is controlled at approximately 22°C depending on die seed. After steeping, the seeds are transferred to a germination compartment which contains air saturated widi water and is under controlled temperature and air flows. The typical temperatures are between 12-25°C and germination is permitted to continue for from 3 to 7 days.
  • heterologous protein coding gene is operably linked to a inducible promoter requiring a metabolite such as sugar or plant hormone, e.g., 2 to 100 nM gibberellic acid
  • mis metabolite is added, removed or depleted from the steeping water medium and/or is added to me water saturated air used during germination.
  • the seed absorbs die aqueous medium and begins to germinate, expressing the heterologous protein.
  • the medium may then be wididrawn and me malting begun, by maintaining the seeds in a moist temperature controlled aerated environment. In this way, the seeds may begin growth prior to expression, so that the expressed product is less likely to be partially degraded or denatured during the process.
  • the temperature during the imbibition or steeping phase will be maintained in d e range of about 15-25°C, while the temperature during the germination will usually be about 20°C.
  • the time for the imbibition will usually be from about 1 to 4 days, while me germination time will usually be an additional 1 to 10 days, more usually 3 to 7 days. Usually, die time for the malting does not exceed about ten days.
  • the period for the malting can be reduced by using plant hormones during the imbibition, particularly gibberellic acid.
  • die malting procedure may be modified to accommodate de-hulled and de-embryonated seeds, as described in above-cited PCT application WO 95/14099.
  • die absence of sugars from the endosperm there is expected to be a 5 to 10 fold increase in RAmy3D promoter activity and dius expression of heterologous protein.
  • embryoless half-seeds are incubated in 10 mM CaCl 2 and 5 MM gibberellic acid, there is a 50 fold increase in RAmylA promoter activity.
  • the seeds and tiieir heterologous proteins may be used directly, that is, widiout protein isolation, where for example, the heterologous protein is intended to confer a benefit on the seed as a whole, for example, to enrich the seed in the selected protein.
  • the seeds may be fractionated by standard methods to obtain die heterologous protein in enriched or purified form.
  • the seed is first milled, men suspended in a suitable extraction medium, e.g., an aqueous or an organic solvent, to extract the protein or metabolite of interest.
  • a suitable extraction medium e.g., an aqueous or an organic solvent
  • die heterologous protein can be further fractionated and purified, using standard purification methods.
  • the 3 kb BamHI fragment containing the 35S promoter-Hph-NOS was removed from e plasmid pMON410 (Monsanto, St. Louis, MO) and placed into an site-directed mutagenized BglR site in the pUC18 at 1463 to form the plasmid pUCH18+ .
  • pOSglABK5 is a 5 kb BamBl-Kpnl fragment from lambda clone ⁇ OSglA (Huang, N., et al, (1990) Nuc. Acids Res. 18:7007) cloned into pBluescript KS- (Stratagene, San Diego, CA). Plasmid pOSglABK5 was digested with Mspl and blunted widi T4 DNA polymerase followed by Spel digestion.
  • the 350 bp terminator fragment was subcloned into pUC19 (New England BioLabs, Beverly, MA), which had been digested widi BamHI, blunted widi T4 DNA polymerase and digested widi Xbal, to form pUC19/terminator.
  • pGEM5zf-(3D/2V -P.jtI) was then digested widi Pstl and Sacl, and two non-kinased 30mers having me complementary sequences 5' GCTTG ACCTG TAACT CGGGC CAGGC GAGCT 3' (SEQ ID NO:23) and 5' CGCCT AGCCC GAGTT ACAGG TCAAG CAGCT 3' (SEQ ID NO:24) were ligated in to form p3DProSig.
  • the promoter fragment prepared by digesting p3DProSig with Ncol, blunting widi T4 D ⁇ A polymerase, and digesting widi Sstl was subcloned into pUC19/terminator which had been digested widi EcoRI, blunted widi T4 D ⁇ A polymerase and digested widi Sstl, to form p3DProSig ⁇ D.
  • p3DProSigEND was digested with Sstl and Smal followed by die ligation of a new synthetic linker fragment constructed widi die non-kinased complementary oligonucleotides 5' AGCTC CATGG CCGTG GCTCG AGTCT AGACG CGTCC CC 3' (SEQ ID NO:25) and 5' GGGGA CGCGT CTAGA CTCGA GCCAC GGCCA TGG 3' (SEQ ID NO:26) to form p3DProSigENDlink.
  • p3DProSigENDlink was digested with Sail and blunted widi T4 DNA polymerase followed by EcoRV digestion. The blunt fragment was then inserted into pBluescript KS + (Stratagene) in the EcoRV site so that the Hindlll site is proximal to the promoter and the EcoRI is proximal to the terminator sequence. The /fi/idlll-EcoRI fragment was then moved into die polylinker of pUCH18+ to form the p3Dvl.O expression vector.
  • pGEM5zf-(lA/ ⁇ .I-PstI) was digested widi Pstl and Sacl and two non-kinased 35mers and four kinased 32mers were ligated in, with the complementary sequences as follows: 5' GCATG CAGGT GCTGA ACACC ATGGT GAACA AACAC 3' (SEQ ID NO:27); 5' TTCTT GTCCC TTTCG GTCCT CATCG TCCTC CT 3' (SEQ ID NO:28); 5' TGGCC TCTCC TCCAA CTTGA CAGCC GGGAG CT 3' (SEQ ID 0:29); 5' TTCAC CATGG TGTTC AGCAC CTGCA TGCTG CA 3' (SEQ ID NO:30); 5' CGATG AGGAC CGAAA GGGAC AAGAA GTGTT TG 3' (SEQ ID NO:31); 5' CCCGG CTGTC AAGTT GGAGG AGAGG CCAAG GAGGA 3' (SEQ ID NO
  • the Hin m-Sacl 0.8 kb promoter fragment was subcloned from plAProSig into the p3Dvl.0 vector digested witii Hi ⁇ zdIII--S' ⁇ cI to yield the plAvl .0 expression vector.
  • Two PCR primers were used to amplify a fragment encoding AAT according to me sequence disclosed as Genbank Accession No. K01396: N-terminal primer 5' GAGGA TCCCC AGGGA GATGC TGCCC AGAA 3' (SEQ ID NO:33) and C-terminal primer 5' CGCGC TCGAG
  • the N-terminal primer amplifies to a blunt site for in-frame insertion with the end of die p3D signal peptide and die C-terminal primer contains a Xhol site for cloning the fragment into the vector as shown in Figs. 3A and 3B.
  • sequence encoding mature AAT (SEQ ID NO: 8) or codon-optimized AAT may be chemically synthesized using techniques known in the art, incorporating a -XTioI restriction site 3' of the termination codon for insertion into the expression vector as described above.
  • Example 2 Production of mature p-antitrypsin in cell culture After selection of transgenic callus, callus cells were suspended in liquid culture containing
  • AA2 media Thimpson, J.A., et al, Plant Science 47:123 (1986), at 3% sucrose, pH 5.8. Thereafter, the cells were shifted to phosphate-buffered media (20 mM phosphate buffer, pH 6.8) using 10 mL multi-well tissue culture plates and shaken at 120 rpm in the dark for 48 hours. The supernatant was then removed and stored at -80°C prior to western blot analysis. Supernatants were concentrated using Centricon-10 filters (Amicon cat. #4207) and washed with induction media to remove substances interfering widi electrophoretic migration. Samples were concentrated approximately 10 fold, and mature AAT was purified by SDS PAGE electrophoresis.
  • the purified protein was extracted from the electrophoresis medium, and sequenced at its N-terminus, giving the sequence shown in Fig. 8, identified herein as SEQ ID NO:22.
  • Example 3 HSA Induction in Germinating Seeds After selection of transgenic plants which tested positive for the presence of a codon- optimized HSA gene driven by die GA 3 -responsive RAmylA promoter, seeds were harvested and imbibed for 24 hours with 100 rpm orbital shaking in the dark at 25°C. GA 3 was added to a final concentration of 5 ⁇ M and incubated for an additional 24-120 hours. Total soluble protein was isolated by double grinding each seed in 120 ⁇ l grinding buffer and centrifuging at 23,000 x g for 1 minute at 4°C. The clear supernatant was carefully removed from the pellet and transferred to a fresh tube.
  • Bilirubin binding to its high-affinity site on mature HSA is assayed using d e method described by Jacobsen, J. et al. (1974; Clin. Chem. 20:783) and Reed, R.G. et al. (1975; Biochemistry 14:4578-4583). Briefly, the concentration of free bilirubin in equilibrium with protein-bound bilirubin is determined by the rate of peroxide-peroxidase catalyzed oxidation of free bilirubin. Stock solutions of bilirubin (Nutritional Biochemicals Corp.) are prepared fresh daily in
  • AATTTCAACC TCACGGAGAT TCCGGAGGCT CAGATCCATG AAGGCTTCCA GGAACTCCTC 300
  • ACTTTCTATC AGCACCTGGC AGATTCCAAG AATGACAATG ATAACATTTT CCTGTCACCC 240 CTGAGTATCT CCACGGCTTT TGCTATGACC AAGCTGGGTG CCTGTAATGA CACCCTCCAG 300
  • AAATTGTGAC AAATCACTTC ATACCCTTTT TGGAGACAAA TTATGCACAG TTGCAACTCT 240
  • ATCCTCGAAG GGCTGAACTT CAACCTGACG GAGATCCCGG AGGCGCAGAT CCACGAGGGC 360 TTCCAGGAGC TGCTCAGGAC GCTCAACCAG CCGGACTCCC AGCTCCAGCT CACCACCGGC 420
  • GAGACCTACC AGGACATCAG TGAGTTGGTA TATGGAGCCA AGCTCCAGCC CCTGGACTTC 600

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • General Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Reproductive Health (AREA)
  • Pregnancy & Childbirth (AREA)
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  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Fertilizers (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

L'invention concerne un procédé servant à la production de l'une des protéines suivantes dans des cellules de plantes monocotylédones transgéniques: (1) α1-antitrypsine (AAT) glycosylée mature comportant la même séquence d'acides aminés N-terminaux que l'AAT mature produite par l'homme, et un motif de glycosylation permettant d'accroître sensiblement la demi-vie de sérum par rapport à celle d'AAT non glycosylée mature; (2) antithrombine III glycosylée (ATIII) mature comportant la même séquence d'acides aminés N-terminaux que l'ATIII produite par l'homme; (3) albumine sérique humaine (HSA) mature comportant la même séquence d'acides aminés N-terminaux que l'HSA mature produite par l'homme, et présentant le même motif de repliement que l'HSA mature native, comme le montrent ses caractéristiques de fixation de bilirubine; et (4) subtilisine BPN' (BPN') active mature présentant la même séquence d'acides aminés N-terminaux que la BPN' produite par Bacillus. Des cellules de plantes monocotylédones sont transformées à l'aide d'un gène chimère comportant une séquence codante d'ADN codant pour une protéine de fusion possédant (1) une fraction N-terminale correspondant à un peptide signal d'α-amylase de riz, et (2) immédiatement adjacente à l'acide aminé C-terminal dudit peptide, une fraction de protéine correspondant à la protéine mature à produire.
PCT/US1998/003068 1997-02-13 1998-02-13 Production de proteines matures dans des plantes WO1998036085A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
CA002280894A CA2280894A1 (fr) 1997-02-13 1998-02-13 Production de proteines matures dans des plantes
EP98906507A EP0981635A1 (fr) 1997-02-13 1998-02-13 Production de proteines matures dans des plantes
AU61716/98A AU746826B2 (en) 1997-02-13 1998-02-13 Production of mature proteins in plants
JP53599798A JP2001512318A (ja) 1997-02-13 1998-02-13 植物における成熟タンパク質の産生

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US3816997P 1997-02-13 1997-02-13
US3817097P 1997-02-13 1997-02-13
US3816897P 1997-02-13 1997-02-13
US3799197P 1997-02-13 1997-02-13
US60/038,168 1997-02-13
US60/037,991 1997-02-13
US60/038,169 1997-02-13
US60/038,170 1997-02-13

Publications (1)

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WO1998036085A1 true WO1998036085A1 (fr) 1998-08-20

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EP (1) EP0981635A1 (fr)
JP (1) JP2001512318A (fr)
AU (1) AU746826B2 (fr)
CA (1) CA2280894A1 (fr)
WO (1) WO1998036085A1 (fr)

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WO1999038987A1 (fr) * 1998-01-30 1999-08-05 Meristem Therapeutics PROCEDE DE PRODUCTION, PAR DES CELLULES VEGETALES, D'α1-ANTITRYPSINE ET DE SES VARIANTES, ET PRODUITS CONTENANT L'α1-ANTITRYPSINE AINSI OBTENUE
WO2000005384A1 (fr) * 1998-07-22 2000-02-03 Prodigene, Inc. Production commerciale de proteases dans des vegetaux
WO2001025456A3 (fr) * 1999-10-01 2001-12-27 Greenovation Pflanzenbiotechno Procede de production de substances proteiques
WO2002010414A3 (fr) * 2000-07-31 2002-12-27 Biolex Inc Expression de polypeptides biologiquement actifs dans une lenticule mineure
US6905688B2 (en) 2000-04-12 2005-06-14 Human Genome Sciences, Inc. Albumin fusion proteins
US6946134B1 (en) 2000-04-12 2005-09-20 Human Genome Sciences, Inc. Albumin fusion proteins
US6972322B2 (en) 1992-01-31 2005-12-06 Aventis Behring L.L.C. Interferon and albumin fusion protein
US7045318B2 (en) 1995-12-30 2006-05-16 Delta Biotechnology Limited Recombinant fusion proteins to growth hormone and serum albumin
US7141547B2 (en) 2001-12-21 2006-11-28 Human Genome Sciences, Inc. Albumin fusion proteins comprising GLP-1 polypeptides
WO2006108830A3 (fr) * 2005-04-13 2006-12-07 Bayer Cropscience Sa Vegetaux transplastomiques exprimant l'$g(a)1-antitrypsine
US7320887B2 (en) 2001-10-31 2008-01-22 Henkel Kommanditgesellschaft Auf Aktien Alkaline protease variants
US7507413B2 (en) 2001-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
US7521424B2 (en) 2003-01-22 2009-04-21 Human Genome Sciences, Inc. Albumin fusion proteins
US7632983B2 (en) 2000-07-31 2009-12-15 Biolex Therapeutics, Inc. Expression of monoclonal antibodies in duckweed
EP2230311A1 (fr) * 2005-06-28 2010-09-22 Ventria Bioscience Composants de milieux de culture de cellules produits à partir de cellules végétales
US8022270B2 (en) 2000-07-31 2011-09-20 Biolex Therapeutics, Inc. Expression of biologically active polypeptides in duckweed
US8067198B2 (en) 2003-06-25 2011-11-29 Prolume Ltd. Protein expression system
US8552256B2 (en) 2008-04-11 2013-10-08 National Institute Of Agrobiological Sciences Gene capable of being expressed specifically in endosperm of plant, promoter for the gene, and use of the gene and the promoter
EP2655396A4 (fr) * 2010-12-24 2014-10-15 Healthgen Biotechnology Co Ltd Procédé pour purifier la sérum-albumine humaine à partir de grain de riz transgénique
US10618951B1 (en) 2009-02-20 2020-04-14 Ventria Biosciences Inc. Cell culture media containing combinations of proteins
US20240199721A1 (en) * 2021-06-29 2024-06-20 Genecell Biotech Inc. Plant cell line for producing albumin at high efficiency and use thereof

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JP2007151435A (ja) * 2005-12-02 2007-06-21 Niigata Univ デンプン集積能力の高い形質転換植物およびその製造方法

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US6972322B2 (en) 1992-01-31 2005-12-06 Aventis Behring L.L.C. Interferon and albumin fusion protein
US7041478B2 (en) 1992-01-31 2006-05-09 Aventis Behring L.L.C. G-CSF and albumin fusion protein
US6989365B2 (en) 1992-01-31 2006-01-24 Aventis Behring L.L.C. Methods of treatment with erythropoietin and albumin fusion protein
US6987006B2 (en) 1992-01-31 2006-01-17 Aventis Behring L.L.C. Erythropoietin and albumin fusion protein, nucleic acids, and methods thereof
US7056701B2 (en) 1992-01-31 2006-06-06 Aventis Behring L.L.C. Hormone and albumin fusion protein
US7435410B2 (en) 1992-01-31 2008-10-14 Novozymes Biopharma Uk Limited Methods of treatment with interferson and albumin fusion protein
US7410779B2 (en) 1992-01-31 2008-08-12 Novozymes Biopharma Uk Limited Fusion polypeptides of human serum albumin and a therapeutically active polypeptide
US7094577B2 (en) 1992-01-31 2006-08-22 Aventis Behring L.L.C. Insulin and albumin fusion protein
US7081354B2 (en) 1992-01-31 2006-07-25 Aventis Behring L.L.C. Interleukin and albumin fusion protein
US7550432B2 (en) 1995-12-30 2009-06-23 Novozymes Biopharma Uk Limited Recombinant fusion proteins to growth hormone and serum albumin
US7045318B2 (en) 1995-12-30 2006-05-16 Delta Biotechnology Limited Recombinant fusion proteins to growth hormone and serum albumin
WO1999038987A1 (fr) * 1998-01-30 1999-08-05 Meristem Therapeutics PROCEDE DE PRODUCTION, PAR DES CELLULES VEGETALES, D'α1-ANTITRYPSINE ET DE SES VARIANTES, ET PRODUITS CONTENANT L'α1-ANTITRYPSINE AINSI OBTENUE
US6087558A (en) * 1998-07-22 2000-07-11 Prodigene, Inc. Commercial production of proteases in plants
WO2000005384A1 (fr) * 1998-07-22 2000-02-03 Prodigene, Inc. Production commerciale de proteases dans des vegetaux
US7049484B2 (en) 1998-07-22 2006-05-23 Prodi Gene, Inc. Commercial production of proteases in plants
WO2001025456A3 (fr) * 1999-10-01 2001-12-27 Greenovation Pflanzenbiotechno Procede de production de substances proteiques
US6994857B2 (en) 2000-04-12 2006-02-07 Human Genome Sciences, Inc. Albumin fusion proteins
US6905688B2 (en) 2000-04-12 2005-06-14 Human Genome Sciences, Inc. Albumin fusion proteins
US6926898B2 (en) 2000-04-12 2005-08-09 Human Genome Sciences, Inc. Albumin fusion proteins
US6946134B1 (en) 2000-04-12 2005-09-20 Human Genome Sciences, Inc. Albumin fusion proteins
US7507414B2 (en) 2000-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
US7482013B2 (en) 2000-04-12 2009-01-27 Human Genome Sciences, Inc. Albumin fusion proteins
WO2002010414A3 (fr) * 2000-07-31 2002-12-27 Biolex Inc Expression de polypeptides biologiquement actifs dans une lenticule mineure
US6815184B2 (en) 2000-07-31 2004-11-09 Biolex, Inc. Expression of biologically active polypeptide in duckweed
JP4795619B2 (ja) * 2000-07-31 2011-10-19 バイオレックス・セラピューティクス インコーポレイテッド ウキクサにおける生物学的に活性なポリペプチド類の発現
US8022270B2 (en) 2000-07-31 2011-09-20 Biolex Therapeutics, Inc. Expression of biologically active polypeptides in duckweed
US7632983B2 (en) 2000-07-31 2009-12-15 Biolex Therapeutics, Inc. Expression of monoclonal antibodies in duckweed
US7507413B2 (en) 2001-04-12 2009-03-24 Human Genome Sciences, Inc. Albumin fusion proteins
US7320887B2 (en) 2001-10-31 2008-01-22 Henkel Kommanditgesellschaft Auf Aktien Alkaline protease variants
US8993517B2 (en) 2001-12-21 2015-03-31 Human Genome Sciences, Inc. Albumin fusion proteins
US7592010B2 (en) 2001-12-21 2009-09-22 Human Genome Sciences, Inc. Albumin fusion proteins
US9221896B2 (en) 2001-12-21 2015-12-29 Human Genome Sciences, Inc. Albumin fusion proteins
US7141547B2 (en) 2001-12-21 2006-11-28 Human Genome Sciences, Inc. Albumin fusion proteins comprising GLP-1 polypeptides
US9296809B2 (en) 2001-12-21 2016-03-29 Human Genome Sciences, Inc. Albumin fusion proteins
US7238667B2 (en) 2001-12-21 2007-07-03 Human Genome Sciences, Inc. Albumin fusion proteins
US7521424B2 (en) 2003-01-22 2009-04-21 Human Genome Sciences, Inc. Albumin fusion proteins
US9115364B2 (en) 2003-06-25 2015-08-25 Prolume Ltd. Protein expression system
US8067198B2 (en) 2003-06-25 2011-11-29 Prolume Ltd. Protein expression system
WO2006108830A3 (fr) * 2005-04-13 2006-12-07 Bayer Cropscience Sa Vegetaux transplastomiques exprimant l'$g(a)1-antitrypsine
AU2006261687B8 (en) * 2005-06-28 2012-01-12 Invitria, Inc. Components of cell culture media produced from plant cells
AU2006261687B2 (en) * 2005-06-28 2011-09-01 Invitria, Inc. Components of cell culture media produced from plant cells
EP2230311A1 (fr) * 2005-06-28 2010-09-22 Ventria Bioscience Composants de milieux de culture de cellules produits à partir de cellules végétales
US8552256B2 (en) 2008-04-11 2013-10-08 National Institute Of Agrobiological Sciences Gene capable of being expressed specifically in endosperm of plant, promoter for the gene, and use of the gene and the promoter
US10618951B1 (en) 2009-02-20 2020-04-14 Ventria Biosciences Inc. Cell culture media containing combinations of proteins
US10981974B2 (en) 2009-02-20 2021-04-20 Ventria Bioscience Inc. Cell culture media containing combinations of proteins
US11492389B1 (en) 2009-02-20 2022-11-08 Ventria Biosciences Inc. Cell culture media containing combinations of proteins
US12286467B2 (en) 2009-02-20 2025-04-29 Invitria, Inc. Cell culture media containing combinations of proteins
EP2655396A4 (fr) * 2010-12-24 2014-10-15 Healthgen Biotechnology Co Ltd Procédé pour purifier la sérum-albumine humaine à partir de grain de riz transgénique
US20240199721A1 (en) * 2021-06-29 2024-06-20 Genecell Biotech Inc. Plant cell line for producing albumin at high efficiency and use thereof

Also Published As

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AU6171698A (en) 1998-09-08
CA2280894A1 (fr) 1998-08-20
AU746826B2 (en) 2002-05-02
JP2001512318A (ja) 2001-08-21
EP0981635A1 (fr) 2000-03-01

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