WO1999055890A1 - A method for increasing the protein content of plants - Google Patents
A method for increasing the protein content of plants Download PDFInfo
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- WO1999055890A1 WO1999055890A1 PCT/US1999/009067 US9909067W WO9955890A1 WO 1999055890 A1 WO1999055890 A1 WO 1999055890A1 US 9909067 W US9909067 W US 9909067W WO 9955890 A1 WO9955890 A1 WO 9955890A1
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- 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
- C12N15/8243—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 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
- C12N15/8253—Methionine or cysteine
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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)
- C12N15/8241—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
- C12N15/8243—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 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
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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
- C12N15/8243—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 involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
- C12N15/8254—Tryptophan or lysine
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Definitions
- composition of plant storage proteins determines the nutritional value of plants and grains when they are used as foods for man and domestic animals.
- the amount of protein varies with genotype or cultivar, but in general, cereals contain 10% of the dry weight of the seed as protein, while in legumes, the protein content varies between 20% and 30% of the dry weight. In many seeds, the storage proteins account for 50% or more of the total protein and thus determine the protein quality of seeds.
- Each year the total world cereal harvest amounts to some 1 ,700 million tons of grain (Keris et al. 1985). This yields about 85 million tons of cereal storage proteins harvested each year and contributes a majority ofthe total protein intake of humans and animals.
- AMINO ACID REQUIREMENTS The biosynthesis of amino acids from simpler precursors is a process vital to all forms of life as these amino acids are the building blocks of proteins. Organisms differ markedly with respect to their ability to synthesize amino acids. In fact, virtually all members of the animal kingdom are incapable of manufacturing some amino acids. There are twenty common amino acids which are utilized in the fabrication of proteins and essential amino acids are those protein building blocks which cannot be synthesized by the animal. It is generally agreed that humans require eight ofthe twenty common amino acids in their diet. Protein deficiencies can usually be ascribed to a diet which is deficient in one or more of the essential amino acids. A 2 nutritionally adequate diet must include a minimum daily consumption of these amino acids
- Seed storage proteins can be characterized by several main features (Pernollet and Mosse 1983): 1) their main function is to provide amino acids or nitrogen to the young seedling; 2) the general absence of any other known function; 3) their peculiar amino acid composition in cereal and legume seeds; and 4) their localization within storage organelles called protein bodies, at least during seed development.
- Several classes of storage proteins are generally recognized based on their solubilities in different solvents. Proteins soluble in water are called “albumins”; proteins 3 soluble in 5% saline, “globulins”; and proteins soluble in 70% ethanol, “prolamins”. The proteins that remain following these extractions are treated further with dilute acid or alkali, and are named “glutelins”.
- prolamin type proteins Most cereals contain primarily prolamin type proteins and can be classified into different groups on the basis of the relative proportions of prolamins, glutelins, and globulins, and the subcellular location of these proteins in the mature seed.
- the first group corresponds to the Panicoideae sub-family, the second group the Triticeae tribe, and the last one to oat and rice storage proteins.
- the principal members of the Panicoideae sub-family are maize, sorghum, and millet. Their major storage proteins are prolamins (50 to 60% of seed protein) and glutelins (35 to 40% of seed protein) (Pernollet and Mosse 1983). Prolamins are stored within protein bodies, but glutelins are located both inside and outside these organelles.
- the Triticeae tribe which includes wheat, barley, and rye, differ from the Panicoideae mainly in storage protein localization and structure. In the starchy endosperm ofthe seeds belonging to this tribe, no protein bodies are left at maturity. Clusters of proteins are then deposited between starch granules, but are no longer surrounded by a membrane.
- the major storage proteins are salt-soluble globulins (80%)) and prolamins (10-15%). Globulins can be divided into vicillins and legumins (Agros, 1985), based on their sedimentation coefficient (7S/11S), oligomeric organization (trimeric/hexameric), and polypeptide chain structure (single chain/disulphide-linked pair of chains).
- protein bodies are embedded between starch granules
- phaseolin tetramer of trimer
- Glycinin the soybean legumin
- the soybean legumin has a quaternary structure that was suggested by Badley et al. (1975) to be twelve subunits packed in two identical hexagons.
- the legumin molecule is a polymer formed by the association of six monomers. Each monomer consists of two subunits, acidic and basic. Sometimes, these subunits are associated by disulf ⁇ de linkages.
- arachin the peanut legumin, was found to consist of different kinds of subunits. The arachin hexamer association does not need different kinds of subunits, which suggests that the subunits have a very similar structure.
- zeins The most studied storage proteins, in terms of structure, are the corn prolamines called zeins. These proteins perform no known enzymatic function.
- Three types of zeins ( , ⁇ and ⁇ ) (Esen 1986) are synthesized on rough endoplasmic reticulum and aggregate within this membrane as protein bodies. The zein protein readily self-associates to form protein bodies and is insoluble in water even in low concentrations of salt.
- the presence of all types of zeins is not necessary for the formation of a protein body as a single type of zein can aggregate into a dense structure and is generally found at the surface of protein bodies (Lending et al. 1988; Wallace et al. 1988).
- Circular dichroic measurements, amino acid sequence analysis, and electron microscopy of a zein protein suggests that zein secondary structure is primarily helical with nine adjacent, topologically antiparallel helices clustered within a distorted cylinder (Agros et al., 1982; Larkins, 1983; Larkins, et al., 1984).
- Polar and hydrophobic residues are appropriately distributed along the helical surfaces allowing intra- and intermolecular hydrogen bonds and van der Waals interactions among neighboring helices, such that rod-shaped zein molecules can aggregate and then stack through glutamate interactions at the cylindrical caps. Because of this structure, zein is much less soluble under physiological conditions than the globulin phaseolin, and precipitation of insoluble zein in the tightly packed protein body may make them less available for proteolytic degradation (Greenwood and Chrispeels 1985).
- the storage protein structures are adapted to a maximal packing within protein bodies (Pernollet and Mosse 1983). Maximal packing is achieved in at least one of two ways. The folding of the polypeptide chain may favor the maximal packing of amino acids within the 5 protein molecule, or the compacting of proteins is increased by the formation of closely packed quaternary structure. High degrees of polymerization can be observed in pearl millet pennisetin (Pernollet and Mosse 1983) or zein (Lending et al. 1988; Wallace et al. 1988). Also, wheat prolamins and glutelin associate into aggregates arising in the formation of insoluble gluten. These insoluble forms of protein deposits are osmotically inactive and stable during the long period of storage between the time of seed maturation and germination.
- Cis-acting DNA sequences involved in developmental and/or tissue-specific regulation of gene expression can be defined by introducing plant storage protein gene regulatory regions coupled to bacterial reporter genes (Twell and Ooms 1987; Wenzler et al. 1989, Marries et al. 1988; Chen et al. 1988), or by introducing entire or dissected genes (Colot et al. 1987; Chen et al. 1986) into a transgenic environment.
- bacterial reporter genes Twell and Ooms 1987; Wenzler et al. 1989, Marries et al. 1988; Chen et al. 1988
- Colot et al. 1987; Chen et al. 1986 a transformation system for the nutritionally important cereal species has not yet been well established. Therefore, most regulation mechanisms have been studied with transgenic dicot plants.
- gene expression is controlled, at least partly, by the interaction between regulatory molecules and short sequences that are present in the 5' flanking region ofthe gene.
- the regulatory sequences of potato storage protein were investigated using transgenic potato plants.
- a 2.5 kb 5' flanking DNA fragment containing the promoter and the patatin gene was used to construct a transcriptional fusion gene with chloramphenicol acetyl transferase
- CAT ⁇ -glucuronidase
- GUS ⁇ -glucuronidase
- the expression pattern of storage protein genes of cereals is retained in tobacco, not only with respect to tissue, but also to temporal expression.
- the 5' upstream regions of wheat glutenin genes possess regulatory sequences that determine endosperm-specific expression in transgenic tobacco (Colot et al. 1987).
- Deletion analysis of the low molecular weight (LMW) glutenin sequence indicated that sequences present between 326 bp and 160 bp upstream of the transcriptional start point are necessary to confer endosperm-specific expression.
- cis-acting elements determining the regulation of each gene in the cluster are recognized by the tobacco trans-acting factor but also that cis-acting elements directing expression of one gene do not affect expression of neighboring genes.
- VSP Vegetative storage protein
- Agrobacterium tumefaciens Ti plasmid As a vector system for the transformation of plants.
- A. tumefaciens infects most dicotyledonous and some monocotyledonous plants by entry through wound sites.
- the bacteria bind to cells in the wound and are stimulated by phenolic compounds released from these cells to transfer a portion of their endogenous, 200 kb Ti plasmid into the plant cell (Weiler and Schroder 1987).
- T-DNA Ti plasmid
- the transferred portion ofthe Ti plasmid, (T-DNA) becomes covalently integrated into the plant genome, where it directs the biosynthesis of phytohormones using enzymes which it encodes.
- the vir gene in the bacterial genome is known to be responsible for this process.
- directly repeating sequences of 25 bases called "border" sequences are essential, but only the right terminus has been shown to be used for T-DNA transfer and integration.
- Ti plasmids from which these disease-producing genes have been removed or replaced, are referred to as "disarmed” and can be used for the introduction of foreign genes into plants.
- intermediate vectors such as pMON237 or pBI121 can be used to introduce genes into the Ti plasmid.
- cointegrating vectors and binary vectors are available as intermediate vectors.
- a cointegrating transformation vector must include a region of homology between the vector plasmid and the Ti plasmid. Once recombination occurs, the cointegrated plasmid is replicated by the Ti plasmid origin of replication.
- the cointegrate system while more difficult to use, does offer advantages. Once the cointegrate has been formed, the plasmid is stable in Agrobacterium.
- a binary vector contains an origin of replication from a broad host-range plasmid instead of a region of homology with the Ti plasmid. Since the plasmid does not need to form a cointegrate, these plasmids are considerably easier to introduce into Agrobacterium.
- the other advantage to binary vectors is that this vector can be introduced into any Agrobacterium host 8 containing any Ti or Ri plasmid, as long as the vir helper function is provided. Using these systems, the gene regulation mechanism of storage proteins has been elucidated.
- the amino acid composition ofthe cereal endosperm protein is characterized by a high content of proline and glutamine while the amount of essential amino acids, lysine and tryptophan in particular, is a limiting factor (Pernollet and Mosse 1983).
- sulfur containing amino acids such as methionine and cysteine are the major limiting essential amino acids for the efficient utilization of plant protein as animal or human food while roots and tubers are deficient in almost all of the essential amino acids.
- the modified proteins are typically susceptible to proteolytic attack in the plant. Because a natural storage protein is a highly evolved structure, artificial modifications to it are likely to destabilize it. For example, a stabilizing glutamic acid-lysine salt bridge might be broken. (3) Multiple copies of genes.
- Naturally occurring storage proteins are typically encoded by multiple gene copies. A mutation in just one ofthe copies ofthe gene will likely have only a limited effect.
- Lysine and tryptophan-encoding oligonucleotides were introduced at several positions into a 19 kD ⁇ -type zein complementary DNA by oligonucleotide-mediated mutagenesis (Wallace et al. 1988).
- Messenger RNA for the modified zein was synthesized in vitro and injected into Xenopus laevis oocytes.
- the modified zein aggregated into structures similar to membrane-bound protein bodies. This experiment suggested the possibility of creating high- lysine corn by genetic engineering.
- the maize 15 kD zein structural gene was placed under the regulation of French bean ⁇ - phaseolin gene flanking regions and expressed in tobacco (Hoffmann et al. 1987). Zein accumulation was obtained as high as 1.6%> of the total seed protein. Zein was found in roots, 10 hypocotyls, and cotyledons ofthe germinating transgenic tobacco seeds. Zein was deposited and accumulates in the vacuolar protein bodies ofthe tobacco embryo and endosperm. The storage proteins of legume seeds such as the common bean (Phaseolus vulgaris) and soybean (Glycine max) are deficient in sulfur-containing amino acids. The nutritional quality of soybean could be improved by introducing and expressing the gene encoding methionine-rich 15 kD zein
- HAAE I High Essential Amino Acid Encoding
- Protein design has two components: the design of activity and the design of structure. This review will concentrate on the design of structurally stable storage protein-like proteins.
- the stability of this peptide is 1000-fold greater than the value calculated from the Zimm-Bragg equation.
- Specific side-chain interactions, factors that are not considered in the Zimm-Bragg model, are responsible, at least in part, for the fact that the C-peptide is much more helical than predicted (Scheraga, 1985).
- Medium-range interactions are responsible for the additional stabilization of secondary structures (DeGrado et al. 1989). Interaction between the side-chains are regarded as important medium range interactions (Shoemaker et al. 1987; Marqusee and Baldwin 1987). These include electrostatic interactions, hydrogen bonding, and the perpendicular stacking of aromatic residues (Blundell et al. 1986).
- An -helix possesses a dipole moment as a result ofthe alignment of its peptide bonds. The positive and negative ends ofthe amide group dipole point toward the helix NH 2 -terminus and COOH-terminus, respectively, giving rise to a significant macrodipole.
- Protein structures contain several long-range stabilizing interactions which include hydrophobic and packing interactions, and hydrogen bonds. Among these, the hydrophobic effect is a prime contributor to the folding and stabilizing of protein structures.
- the driving force for helix formation in RNase A arises from long-range interactions between C-peptide and S-protein, a large fragment ofthe protein from which C-peptide was excised ( Komoriya and Chaiken 1985).
- Hydrophobic residues often repeat every three to four residues in an -helix and form an amphiphilic structure (DeGrado et al. 1989). Amphiphihcity is important for the stabilization of the secondary structures of peptides and proteins which bind in aqueous solution to extrinsic apolar surfaces, including phospholipid membranes, air, and the hydrophobic binding sites of regulatory proteins (Degrade and Lear 1985). This amphiphilic secondary structure can be stabilized relative to other conformations by self-association. Therefore, short peptides often form the -helix in water only because the helix is amphiphilic and is stabilized by peptide aggregation along the hydrophobic surface.
- Natural globular proteins are folded by a similar mechanism, involving hydrophobic interaction between neighboring segments of secondary structure (Presnell and Cohen 1989).
- DeGrado and coworkers have successfully built peptide-hormone analogs with minimal homology to the native sequences. These peptides, like the native ones, are not helical in solution but do form helices at the hydrophobic surfaces of membranes.
- Designed synthetic peptides have been used to show how hydrophobic periodicity in a protein sequence stabilizes the formation of simple secondary structures such as an amphiphilic ⁇ -helix (Ho and DeGrado 1987).
- the strategies used in the design ofthe helices in the four-helix bundles are: 1) the helices should be composed of strong helix forming amino acids and 2) the helices should be amphiphilic; i.e., they should have an apolar face to interact with neighboring helices and a polar face to maintain water solubility ofthe ensuing aggregates.
- the results show that hydrophobic periodicity can determine the structure of a peptide. Therefore, the peptides 13 tend to have random conformations in very dilute solution, but form secondary structures when they self-associate (at high concentration) or bind to the air- water surface.
- the free energy associated with dimerization or tetramerization ofthe designed peptides could be experimentally determined from the concentration dependence ofthe CD spectra for the peptides (DeGrado et al. 1989; Lear et al. 1988; DeGrado and Lear 1985).
- the peptides were found to be monomeric and have low helical contents, whereas at high concentration they could self-associate and stabilize the secondary structure. Therefore, possible hairpin loops between helices can affect the stability ofthe secondary structure by enhancing the self-association between the helical monomers.
- a strong helix breaker (Chou and Fasman 1978; Kabsch and Sander 1983, Sueki et al.
- proline residue was included as the first and last residue to set the stage for adding a hairpin loop between the helices.
- a single proline residue appeared capable of serving as a suitable link if the C and N terminal glycine residue are slightly unwound.
- Glycine lacks a ⁇ -carbon, which is essential for the reverse turn where positive dihedral angles are required.
- the pyrrolidine ring of proline constrains its f dihedral angle -60°.
- proline should be destabilizing at positions where significantly different backbone torsion angles are required.
- This amino acid, as well as glycine has a high tendency to break helices and occurs frequently at turns (Creighton 1987).
- Structural stability of proteins is directly related to in vivo proteo lysis (Parasell and Sauer 1989). Proteolysis depends on the accessibility of the scissile peptide bonds to the attacking protease. The sites of proteolytic processing are generally in relatively flexible interdomain segments or on the surface of the loops, in contrast to the less accessible interdomain peptide 14 bonds (Neurath 1989). This suggests that the stability ofthe folded state ofthe protein is the most important determinant for its proteolytic degradation rate. The effect of a folded structure on the proteolytic degradation has been proven by several experiments. First, proteins that contain amino acid analogs or are prematurely terminated are often degraded rapidly in the cells (Goldberg and St. John 1976).
- Metabolic stability is another factor influencing the in vivo stability of proteins.
- damaged and abnormal proteins are metabolically unstable in vivo (Finley and Varshavsky 1985; Pontremoli and Melloni 1986).
- covalent conjugation of ubiquitin with proteins is essential for the selective degradation of short-lived proteins (Finley and Varshavsky. 1985).
- the amino acid at the amino-terminus of the protein determined the rate of ubiquitination (Bachmair et al. 1986).
- Both prokaryotic and eukaryotic long-lived proteins have stabilizing amino acids such as methionine, serine, alanine, glycine, threonine, and valine at the amino terminus end.
- amino acids such as leucine, phenylalanine, aspartic acid, lysine, and arginine destabilize the target proteins.
- transgenic plants which produce higher levels of essential amino acids.
- plants were made transgenic with a synthetic nucleic acid construct which encoded a protein containing high levels of essential amino acids. Resulting transgenic plants produced not only higher levels of essential amino acids, but unexpectedly these plants also produced higher levels of protein in general.
- This increase in total protein content ranged from approximately 2-fold to 5-fold.
- One aspect of the invention is a transgenic plant comprising a gene which encodes a protein which causes the transgenic plant to overproduce total protein as compared to a nontransgenic plant.
- a second aspect ofthe invention is a gene encoding a protein wherein plants which are transgenic for this gene overproduce total protein as compared to a nontransgenic plant.
- a third aspect ofthe invention is a protein wherein if a plant is made transgenic for a gene encoding said protein said transgenic plant will overproduce total plant protein as compared to the plant when it is not transgenic.
- This protein may comprise an amphiphilic ⁇ -helical sequence, a ⁇ -pleated sheet sequence, or a combination of -helix and ⁇ -pleated sheet.
- Another aspect ofthe invention is a transgenic plant cell which contains a gene encoding a protein which causes the plant cell to overproduce total protein as compared to a nontransgenic cell.
- Yet another aspect ofthe invention is a method for increasing the production of a specific protein in a plant or plant cell by transforming the plant or plant cell with a gene which encodes a protein which causes the overproduction of total protein in the transgenic plant or plant cell.
- Still another aspect of the invention is a method for increasing the production of a nonprotein product in a plant or plant cell by transforming the plant or plant cell with a gene encoding a protein which causes the overproduction of total protein in the transgenic plant or plant cell and thereby results in the increased synthesis of nonproteinaceous material.
- Yet another aspect ofthe invention is a method for enhancing the production of a specific protein or nonprotein in a plant or plant cell by cotransforming the plant or plant cell with 1) a gene encoding the specific protein or a protein involved as an enzyme in the synthetic pathway of the nonprotein product and 2) a gene encoding a protein which results in the generalized overproduction of total plant or plant cell protein.
- Figure 1 shows the average essential amino acid requirement for both children and adults in mg per kg body weight.
- Figure 2 shows the amounts of foodstuffs which must be consumed in grams per day in order to meet the minimum daily requirement of all essential amino acids.
- Figure 3 illustrates how the amino acid composition of the ASPl monomer was chosen.
- Figure 4 shows the percentage of essential amino acids (EAA) and percentage of most limiting essential amino acids (MLEAA) in ASP 1 tetramer compared with natural proteins.
- Figure 5 is a depiction ofthe amphiphihcity ofthe ASPl monomer where hydrophobic amino acids are in the white rectangle and hydrophilic amino acids are in the shaded rectangle.
- Figure 6 shows the amino acid sequence of the ASPl tetramer (SEQ ID NO:2). Hydrophilic amino acids are underlined and ⁇ -turns are indicated.
- Figures 7A-7B show the protein content of plants.
- Figure 7A shows the overall protein content determined by amino acid analysis.
- P-2 TC is a control plant and P-7 T, P-l 1 T, P-17 T and P-29 T are plants transformed with ASPl tetramer.
- Figure 7B shows the % increase of protein content in the transformed plants as compared to the control plant. These data were derived from seedlings obtained from transformed mother plants. A minimum of four separate assays were used and the variation was no more than 30%. 17
- Figure 8A depicts the overall protein content of leaves from control and ASPl tetramer seedlings. The plants are labeled as for Figures 7A-7B.
- Figure 8B shows the % increase in protein content for the transformed plants as compared to the control plant.
- the present invention uses quite a different approach. Rather than mutate or transfer a gene for a naturally occurring protein, an artificial protein has been constructed de novo. This de novo protein has nutritionally balanced proportions of the essential amino acids, is stable following expression in a plant, and shares some of the characteristics of naturally occurring plant storage proteins. Transgenic plants have been produced which contain such a gene. These plants not only produce more essential amino acids compared to controls, but surprisingly the total amount of protein produced by these plants is also increased. Furthermore, the total amount of nonproteinaceous components can also be increased via these methods.
- Val 1.23 1.31 1.19 1.01 1.36 19 these values and derived a set of numbers we call the 'Average Ratio for All Crops Idealized to the DNP 1 Monomer' ( Figure 4). This set of numbers represents the ratio of essential amino acids necessary to complement the deficiencies found in all 10 crops for all human age groups.
- ASPl nutritional protein for humans
- the amino acid sequence for ASPl is shown in Figure 6 and is SEQ ID NO:2.
- the DNA sequence used to encode this protein is shown as SEQ ID NO:l. It has 1.8 times more of the essential amino acids compared to zein or phaseolin. The difference in MLEAA is much higher, containing 3 times more than phaseolin and 6.5 times more than zein.
- the helical region of ASPl is amphipathic (hydrophobic residues clustered on one face of the helix while hydrophilic residues are found on the other face) and is stabilized by several GLU - LYS salt bridges ( Figure 6
- the helix breaker Gly-Pro-Gly-Arg (SEQ ID NO: 8) has been used as a turn sequence.
- the design results in an antiparallel tetramer which achieves an extraordinarily stable secondary and tertiary structure even at low concentration.
- ASPl has been designed to have a stable storage protein-like structure in plants. Its design is based on the structurally well-studied corn storage zein proteins (Z19 and Z22), which are comprised of 9 repeated helical units (Agros et al. 1982).
- Each helical unit, 16 to 26 amino acids long, of zein is flanked by turn regions and forms an antiparallel helical bundle. Most of the amino acids in the helices are hydrophobic residues.
- ASPl is comprised of 4 helical repeating units, each 20 amino acids long ( Figure 6). Increased gene copy number by concatenation can increase the protein yields. At the same time, gene concatenation gives the increased molecular mass ofthe encoded protein.
- the gene encoding this novel peptide was chemically synthesized and cloned into an E. coli expression vector.
- This gene contains plant consensus sequences at the 5' end of the translation initiation site to optimize the expression of proteins in vivo. It was placed under the control ofthe 35S cauliflower mosaic virus (CaMV) promoter in order to permit the constitutive 20 expression of this gene in tobacco.
- CaMV 35S cauliflower mosaic virus
- the gene can also be cloned into other microorganisms, such as yeasts, through standard means known in the art.
- ASPl is intended to encompass any one or more of the following: (1) the peptide whose sequence is SEQ ID NO:3; (2) the peptide whose sequence is SEQ ID NO:4; (3) any polymer, copolymer, oligomer or co-oligomer of one or both of SEQ ID NO:3 and SEQ ID NO:4, such as the tetrameric ASPl whose sequence is SEQ ID NO:2; or (4) any peptide or protein having substantially the same amino acid sequence as any ofthe above, and substantially the same stability upon expression in at least one plant, but whose amino acid sequence has been modified in a manner which will naturally occur to one of skill in the art, such as by insertions, deletions, and/or transpositions which are not substantially detrimental to the stability of, or to the nutritionally balanced essential amino acid composition of, the protein.
- the protein should also be designed for ready digestibility by the proteases of the intended consumer. For example, frequent lysine (or arginine) sites will promote proteolytic attack by trypsin. Frequent phenylalanine (or tyrosine) sites will promote proteolytic attack by chymotrypsin.
- an artificial storage protein to be expressed in maize might have one composition if the maize is intended for 21 human consumption, and a somewhat different composition if the maize is intended for feeding pigs.
- amphipathic peptide or protein is one in which the hydrophobic amino acid residues are predominantly on one side, while the hydrophobic amino acid residues are predominantly on the opposite side, resulting in a peptide or protein which is predominantly hydrophobic on one face, and predominantly hydrophilic on the opposite face.
- PREDICT-SECONDARY in ⁇ -SYBYL.
- the percentage of -helix content predicted by information- theory showed a higher -helix content compared to the other two prediction methods (Bayes-statistic and neural-net) in PREDICT-SECONDARY.
- the predicted secondary structures by information-theory gave 100%> helical content for the monomer and 74% for the tetramer.
- CD spectra of ASPl -monomer showed the typical pattern of alpha helical proteins with double minima at 208 and 222 nm in aqueous solution (data not shown).
- the stability of the secondary structure can be induced by the inter-molecular interaction between the helical chains (DeGrado et al. 1989). Therefore, stable aggregation between monomers, presumably through hydrophobic interactions, could stabilize the helical structure.
- ⁇ -turn Gly-Pro-Gly-Arg (SEQ ID NO:8) sequences were inserted between four monomers for the ASPl -tetramer construction.
- the ⁇ -turn could play an important role for structural stability of the ASPl -tetramer when it is expressed in vivo. It can also help stabilize tertiary structure formation.
- the interactions between the helical monomers might be much faster due to the proximate effect when they are connected. This proximate effect might be critical for folding at the low concentrations of ASP 1 -tetramer that are possible when they are expressed in vivo.
- the stability ofthe secondary structure is increased by the hydrophobic interactions between helical monomers.
- this ⁇ -turn sequence has a tryptic digestion site (Gly- Arg) which can increase the digestibility of this protein when it is consumed by animals.
- the stability of the folded structure of a protein has a close relation to its proteolytic degradation rate (Pace and Barret 1984; Pakula and Sauer 1986; Parasell and Sauer 1989; Pakula 23 and Sauer 1989).
- Stable quaternary structure is essential for the formation of protein bodies of storage proteins in zein or phaseolin (Lawrence et al. 1990). These higher order structures can be achieved through the interaction and close packing ofthe stable tertiary structures.
- the major driving force for this quaternary structure formation is also hydrophobic interaction between the tertiary structures.
- Leaf discs transformed with LBA4404 carrying the ASPl gene, gave about 5 to 7 shoots two to three weeks after infection. A total of 565 kanamycin-resistant shoots were regenerated from 120 leaf discs. These shoots were excised from the leaf discs and transferred to new media to grow several more weeks, and then transferred to rooting media. After three weeks in rooting medium, 126 rooted shoots were analyzed for ⁇ -glucuronidase (GUS). Root tips of 56 out of 126 plants showed various levels of GUS activity. Not all the kanamycin-resistant shoots showed the GUS positive result.
- GUS ⁇ -glucuronidase
- kanamycin resistance was due to the expression of neomycin phosphotransferase (NPT II gene), regeneration of nontransgenic shoots in the presence of kanamycin has been reported. Therefore, escapes from the screening based on kanamycin sensitivity might have occurred in the nontransformed plants, making them kanamycin resistant.
- NPT II gene neomycin phosphotransferase
- nuclease hypersensitive sites correlate to active transcription (Gross and Garrard 1987).
- the degree of methylation of DNA is inversely related to gene expression.
- the gene is located near the plant's endogenous promoter or enhancer sites, the level of expression of this gene will be increased by these near-by enhancing factors. Therefore, the difference in the levels of GUS activity between the transformed plants might be due to this positional effect, which was determined by the sites of incorporation of this gene into the tobacco genome.
- Efficient transcription of inserted ASPl genes in the tobacco plants was tested by Northern blot analysis.
- the polyA RNA was analyzed using the ASPl -tetramer probe.
- the correct gene size transcribed was about 490 bases, which consisted of 30 bases upstream and 170 bases downstream of the ASPl -tetramer gene.
- eukaryotic mRNA contains different sizes of polyA. Therefore, the expected size ofthe ASPl -tetramer message should be around 600 plus -100 bases long. Bands were observed which corresponded to this expected size from all the samples which were analyzed. However, the levels of transcription of the ASPl genes were dramatically different among the different transformed plants.
- Transformed plant #17 accumulated 5- to 50-fold more transcripts than the other transformed plants. Such differences in accumulation could be explained by the effect of position, or by the effect of multiple copy insertion.
- the expression levels of the ASPl gene and its neighboring GUS gene correlated with each other in some transformed plants (such as in plant #17), but not in all. These results suggested that the level of expression of two closely connected genes can be dramatically different. Multiple transcripts with different sized bands (500-700 bases) were observed from several transformed plants. This result might be due to multiple insertion ofthe ASPl gene into the tobacco genome. These inserted genes may be rearranged, but still produce transcripts. Another possibility might be strong secondary structure which could be formed due to the four directly repeated sequences ofthe tetrameric ASPl transcripts. Different mobilities could result, depending on the secondary structure. Expression of ASPl
- Standard means known in the art were used to raise polyclonal antibody against synthetic ASPl monomer. This antibody was used to detect the production of stable ASPl protein in tobacco. If desired, standard means known in the art can also be used to prepare monoclonal antibodies against ASPl . High levels ofthe tetrameric form (11.2 kD) ofthe ASPl protein were detected from plant #17 by Western blot analysis (data not shown). Therefore, direct correlation was found between gene copy number, number of genetic NPTII loci, GUS expression, accumulation of ASPl transcript and protein expression level in the case of plant #17. Some heterologous seed proteins undergo specific degradation when expressed in transgenic plants.
- ASPl in addition to being a very stable protein in a plant cell, ASPl must function as a general 'protein-stabilizer' and reduces overall protein turnover without apparent deleterious effects to the plants, since there is no observable difference in growth characteristics in the plants producing high amounts of ASPl as compared to control plants.
- Table 7 lists the percentage of total protein, as a function of dry weight, ofthe transformed controls and ASPl transformants of sweet potato. The numbers are the average of 5 separate assays.
- Table 8 indicates the amount of essential amino acid in mg/100 grams edible portion ofthe sweet potato and the numbers are the average of 3 separate assays.
- Table 9 illustrates the percentage of these essential amino acids compared to the transformed control, the numbers being the average of 3 separate assays.
- Table 10 shows data 29 for a repeat of experiments as done in Table 8 but with the content of more ofthe amino acids determined.
- Table 10 The numbers in Table 10 are the average of 3 separate assays.
- Table 11 shows the increase in transformant #5.
- Table 12 shows the %> protein (wet weight basis) of roots and leaves, with the numbers being the average of at least 3 separate assays, while Table 13 depicts the overall protein content ofthe roots of transformed plants on a dry weight basis and percent dry matter and overall moisture content.
- Methionine 30 143 190 173 165 197
- HDNP1 which has the following monomeric amino acid sequence:
- MLEEIFKKMTE IEKVLKTM (SEQIDNO:6) hhHHhhHHhHHhhHhh (SEQ ID NO:31)
- Hydrophobic amino acids comprise: isoleucine, methionine, phenylalanine, tryptophan, valine, leucine, alanine and cysteine.
- Hydrophilic amino acids comprise: arginine, glutamic acid, histidine, lysine, asparagine, aspartic acid, glutamine, tyrosine and proline. Glycine, threonine and serine can act as either hydrophilic or hydrophobic amino acid residues depending upon their immediate environment.
- the HDNP1 monomer is composed of 20 amino acids in the structural motif to render an amphiphilic -helix.
- the tetrameric form is: MLEEIFKKMTE WIEKVLKTMgpgrMLEEIFKKMTE WIEKVLKTMgpgrMLEEIFKKMTE
- This tetrameric form shows the 4 -helices interspaced with the ⁇ -turn gpgr (SEQ ID NO:8).
- the tetramer is composed of 92 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic -helix.
- HDNPl is quite similar to ASPl except that the Leu in position 5 ofthe monomer has been changed to He and also the He in position 17 ofthe monomer for ASPl has been changed to a Leu in HDNPl .
- these changes are made throughout the protein as can be seen by comparing the amino acid sequences.
- This monomer is composed of 20 amino acids in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- the tetrameric form shown below has each stretch of ⁇ -pleated sheet interspaced with the ⁇ -turn gpgr (SEQ ID NO:8).
- the sequence ofthe tetrameric form is: MTIEWKVELKFEMKIELKMTgpgrMTIEWKVELKFEMKIELKMTgpgrMTIEWKVELKF
- the tetramer is composed of 92 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- the proteins ASPl, HDNPl and HDNP2 were designed to yield high levels of essential amino acids especially suitable for humans. Each type of animal has its own set of required essential amino acids and these sets of essential amino acids, while usually overlapping, are different from each other. Other proteins can be designed which yield higher levels of essential amino acids more suitable for organisms other than humans. For example, pigs have one set of essential amino acids, chickens have a different set, and fish have yet a different set. Transgenic plants can be engineered to be designed to be fed to one particular species of animal.
- transgenic corn plants can be produced wherein one transgenic form is most suitable for humans, a second transgenic form will produce a high level of those essential amino acids suited for pigs, and a third transgenic form can be made which is most suited for chickens.
- This monomer is composed of 41 amino acids in the structural motif to render an amphiphilic -helix.
- the tetrameric form is: MFETI VKLVEETMHKWEEVIKKFVTMVEETLKKFEEITKKMgpgrMFETIVKLVEETM
- HKWEEVIKKFVTMVEETLKKFEEITKKMgpgrMFETIVKLVEETMHKWEEVIKKFVT MVEETLKKFEEITKKMgpgrMFETIVKLVEETMHKWEEVIKKFVTMVEETLKKFEEIT KKM (SEQ ID NO: 12).
- This tetrameric form shows the 4 -helices interspaced with the ⁇ -turn gpgr (SEQ ID NO:8).
- the tetramer is composed of 176 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic ⁇ -helix.
- a second protein for swine is SDNP2 and has the monomeric amino acid sequence MTIEFKVELKVETH EMKIEVKFETKIEVKTEMKLEVKFTM (SEQ ID NO: 13) hHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhH
- sequence ofthe tetrameric form is: MTIEFKVELKVETHWEMKIEVKFETKIEVKTEMKLEVKFTMgpgrMTIEFKVELKVET HWEMKIEVKFETKIEVKTEMKLEVKFTMgpgrMTIEFKVELKVETHWEMKIEVKFETK IEVKTEMKLEVKFTMgpgrMTIEFKVELKVETHWEMKIEVKFETKIEVKTEMKLEVKFTM
- the tetramer is composed of 176 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- a protein directed to use with poultry is PDNP1 which has the amino acid sequence: MFEGLVKIMEEVLRHWTEVFGKIFE GTRFLEGFTKM (SEQIDNO:15) hhHHhhHhhHHhhHHhhHHhhhHhhHHhhHHhHHh (SEQ ID NO:33) 36
- This monomer is composed of 37 amino acids in the structural motif to render an amphiphilic -helix.
- the tetrameric form is:
- This tetrameric form shows the 4 -helices interspaced with the ⁇ -turn gpgr (SEQ ID NO: 8).
- the tetramer is composed of 160 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic a -helix.
- a second protein for poultry is PDNP2 and has the monomeric amino acid sequence MEFKVGIELRFT EMHVGFELKIGFTVEMRLGFETKM (SEQIDNO:17) hHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhH
- This monomer is composed of 37 amino acids in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- the tetrameric form shown below has each stretch of ⁇ -pleated sheet interspaced with the ⁇ -turn gpgr (SEQ ID NO:8).
- the sequence ofthe tetrameric form is: MEFKVGIELRFTWEMHVGFELKIGFTVEMRLGFETKMgpgrMEFKVGIELRFTWEMH
- the tetramer is composed of 160 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- a protein directed to use with fish is FDNP1 which has the amino acid sequence: FEELVRTIEELMKK EEVFKRVLHILEEFVRKFEETMRK (SEQ ID NO:19) hhHHhhHhhHHhHHhHHhhhHhhHHhHHhHHhHHhhHH (SEQ ID NO:34)
- This monomer is composed of 40 amino acids in the structural motif to render an amphiphilic ⁇ -helix.
- the tetrameric form is: MFEELVRTIEELMKKWEEVFKRVLHILEEFVRKFEETMRKgpgrMFEELVRTIEELMK
- This tetrameric form shows the 4 -helices interspaced with the ⁇ -turn gpgr (SEQ ID NO:8).
- the tertramer is composed of 172 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic -helix.
- a second protein for fish is FDNP2 and has the monomeric amino acid sequence 37
- MEIKLEVRFETKVELKVEWRIEFHTELKMELRVELRFEMK (SEQIDNO:21) hHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhH
- This monomer is composed of 40 amino acids in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- the tetrameric form shown below has each stretch of ⁇ -pleated sheet interspaced with the ⁇ -turn gpgr (SEQ ID NO:8).
- the sequence ofthe tetrameric form is:
- the tetramer is composed of 172 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- DDNP1 A protein directed to use with dogs is DDNP1 which has the amino acid sequence:
- the tertramer is composed of 164 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic -helix.
- a second protein for dogs is DDNP2 and has the monomeric amino acid sequence
- This monomer is composed of 38 amino acids in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- the tetrameric form shown below has each stretch of ⁇ -pleated sheet interspaced with the ⁇ -turn gpgr (SEQ ID NO:8).
- the sequence ofthe tetrameric form is:
- the tetramer is composed of 164 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- CDNP1 A protein directed to use with cats is CDNP1 which has the amino acid sequence:
- This monomer is composed of 38 amino acids in the structural motif to render an amphiphilic ⁇ -helix.
- the tetrameric form is:
- This tetrameric form shows the 4 ⁇ -helices interspaced with the ⁇ -turn gpgr (SEQ ID NO:8).
- the tertramer is composed of 164 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic -helix.
- a second protein for cats is CDNP2 and has the monomeric amino acid sequence MTLEFKLTMELH EIKVELKTEVRIEMKFEVRLEFRMT (SEQ ID NO:29) hHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhHhH
- This monomer is composed of 38 amino acids in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- the tetrameric form shown below has each stretch of ⁇ -pleated sheet interspaced with the ⁇ -turn gpgr (SEQ ID NO:8).
- the sequence ofthe tetrameric form is: MTLEFKLTMELHWEIKVELKTEVRIEMKFEVRLEFRMTgpgrMTLEFKLTMELHWEIK
- the tetramer is composed of 164 amino acids, including the 12 amino acids comprising the 3 ⁇ -turns, in the structural motif to render an amphiphilic ⁇ -pleated sheet.
- the generally enhanced levels of protein production can be useful in expressing other valuable proteins. For example, if a gene coding for insulin were cloned into a plant expressing the ASPl gene, it is expected that levels of insulin production will be higher, as compared to control plants having the insulin gene, but lacking the ASPl gene. Therefore plants which are transgenic for both the ASPl gene or similar gene which also results in increased total protein 39 production and for a second gene which encodes a protein of interest will make more of the protein of interest than if the plant were transformed solely with the gene encoding the protein of interest and not transformed with the ASPl or similar gene. It is irrelevant whether the plant is first transformed with ASPl or a similar gene and later transformed with a gene of interest, or whether the plant is first transformed with the gene of interest and then is later transformed with
- ASPl or similar gene a transformation can be performed using both genes simultaneously.
- Plants and plant cells which have been made transgenic for ASPl or similar amphipathic proteins produce greater amounts of all protein than do nontransgenic plants or cells. As a result of this generally higher level of protein, higher levels of nonprotein products will also be made. This result is expected because there will be an increase in the levels of enzymes which are used in the synthesis of such products. For example, taxol is naturally synthesized by certain plants and the synthesis of taxol is dependent on enzymes. Increased levels of those enzymes will lead to increased levels of taxol. Similarly, many plants produce sugars, e.g., sugarcane. Again, the synthesis of sugars is dependent on enzymes within the plant. Increased levels of these enzymes will yield increased levels ofthe sugars.
- Sweetpotato was transformed with ASPl and two transformed lines were assayed for sugar content and overall amount of dry matter versus moisture content. Results are shown in Table 14. 40 Table 14
- Transformant 1 had an increased production of sucrose but normal production of both glucose and fructose whereas Transformant 2 had increased production of all 3 sugars as compared to the control plant.
- Table 13 indicates that the overall amount of dry matter is increased from 17% in the control to roughly 22% in the transformants. This is approximately a 30% increase in dry matter as a percent of the total weight ofthe plant.
- ASPl or related genes can also be performed, in a manner generally analogous to that described above for tobacco and sweet potato, in certain economically important plants such as rice, wheat, barley, sorghum, maize, potato, plantain, cassava, taro, soybean, alfalfa, or a forage grass. It is desirable to incorporate suitable promoters or other regulatory sequences to encourage expression (preferably constitutive expression) primarily in the part of the plant intended as a foodstuff. For example, in rice or maize, expression is desired primarily in the seeds; while in potato or sweet potato, expression is desired primarily in the tuber.
- transformation protocols known in the art other than the Agrobacterium protocol will be used, such as transformation through DNA particle gun or via plant protoplasts. See, e.g., Klein et al. (1987) and Croughan et al. (1989). These plants can be transformed with vectors encoding not only ASPl , but for any such similar proteins including any ofthe proteins disclosed above.
- Plant cells can be made transgenic with a gene encoding ASPl or other amphipathic protein and these transgenic cells can be grown in culture or in a bioreactor. This avoids the necessity of having to regenerate 41 a plant. These transgenic cells will produce enhanced levels of protein and other products as was seen in the transgenic plants. These cells can be cotransformed with any genes of interest, for example a gene encoding insulin. The desired product will be overproduced as compared to a nontransgenic plant cell or a cell not transformed with a gene encoding ASPl or other amphipathic protein. The desired product can be purified from the cultured cells.
- the term “higher plant” is intended to encompass gymnosperms, monocotyledons, and dicotyledons; as well as any cells, tissues, or organs taken or derived from any of the above, including without limitation any seeds, leaves, stems, flowers, roots, tubers, single cells, gametes, or protoplasts taken or derived from any gymnosperm, monocotyledon, or dicotyledon. Also, the term “higher plant” is intended to encompass gymnosperms, monocotyledons, and dicotyledons; as well as any cells, tissues, or organs taken or derived from any of the above, including without limitation any seeds, leaves, stems, flowers, roots, tubers, single cells, gametes, or protoplasts taken or derived from any gymnosperm, monocotyledon, or dicotyledon. Also, the term “higher plant” is intended to encompass gymnosperms, monocotyledons, and dicotyledons; as well as any cells, tissues,
- protein is meant to include peptides such as dipeptides or any longer peptide as well as proteins.
- Bacteriophage 1 Cro mutation effect on activity and intracellular degradation. Proc. Natl. Acad. Sci. U.S.A. 82: 8829-8833.
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CA002325463A CA2325463A1 (en) | 1998-04-27 | 1999-04-27 | A method for increasing the protein content of plants |
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WO2004111244A2 (en) * | 2003-06-17 | 2004-12-23 | Sembiosys Genetics Inc. | Methods for the production of insulin in plants |
Citations (3)
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WO1989004371A1 (en) * | 1987-11-02 | 1989-05-18 | Louisiana State University Agricultural And Mechan | Plants genetically enhanced for disease resistance |
WO1993003160A1 (en) * | 1991-08-09 | 1993-02-18 | E.I. Du Pont De Nemours And Company | Synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants |
WO1997028247A2 (en) * | 1996-01-29 | 1997-08-07 | Biocem | AMINO ACID-ENRICHED PLANT PROTEIN RESERVES, PARTICULARLY LYSINE-ENRICHED MAIZE η-ZEIN, AND PLANTS EXPRESSING SUCH PROTEINS |
-
1999
- 1999-04-27 AU AU38681/99A patent/AU3868199A/en not_active Abandoned
- 1999-04-27 AR ARP990101938A patent/AR016229A1/en unknown
- 1999-04-27 WO PCT/US1999/009067 patent/WO1999055890A1/en active Application Filing
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Publication number | Priority date | Publication date | Assignee | Title |
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WO1989004371A1 (en) * | 1987-11-02 | 1989-05-18 | Louisiana State University Agricultural And Mechan | Plants genetically enhanced for disease resistance |
WO1993003160A1 (en) * | 1991-08-09 | 1993-02-18 | E.I. Du Pont De Nemours And Company | Synthetic storage proteins with defined structure containing programmable levels of essential amino acids for improvement of the nutritional value of plants |
WO1997028247A2 (en) * | 1996-01-29 | 1997-08-07 | Biocem | AMINO ACID-ENRICHED PLANT PROTEIN RESERVES, PARTICULARLY LYSINE-ENRICHED MAIZE η-ZEIN, AND PLANTS EXPRESSING SUCH PROTEINS |
Non-Patent Citations (2)
Title |
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JAYNES, J M: "De novo designed synthetic plant storage proteins: Enhancing protein quality of plants for improved human and animal nutrition", BIOTECHNOL. FEED IND., PROC. ALLTECH'S ANNU. SYMP., 10TH (1994), 129-53. EDITORS: LYONS, T. P.; JACQUES, K. A.; PUBLISHER: NOTTINGHAM UNIVERSITY PRESS, LOUGHBOROUGH, UK., XP002115146 * |
KIM, JAE HO ET AL: "Enhancing the nutritional quality of crop plants: design, construction, and expression of an artificial plasnt torage protein gene.", MOL. APPROACHES IMPROV. FOOD QUAL. SAF. (1992), 1-36. EDITORS: BHATNAGAR, D. ;CLEVELAND, T. E.; PUBLISHER: VAN NOSTRAND REINHOLD, NEW YORK, USA, XP002115145 * |
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WO2004111244A2 (en) * | 2003-06-17 | 2004-12-23 | Sembiosys Genetics Inc. | Methods for the production of insulin in plants |
WO2004111244A3 (en) * | 2003-06-17 | 2005-02-03 | Sembiosys Genetics Inc | Methods for the production of insulin in plants |
EP1959017A1 (en) * | 2003-06-17 | 2008-08-20 | SemBioSys Genetics Inc. | Methods for the production of insulin in plants |
US7547821B2 (en) | 2003-06-17 | 2009-06-16 | Sembiosys Genetics Inc. | Methods for the production of insulin in plants |
EA014887B1 (en) * | 2003-06-17 | 2011-02-28 | Сембайосиз Джинетикс Инк. | Method for the expression of insulin in plant seeds, a method for obtaining plant seeds comprising insulin and a plant capable of setting seeds comprising insulin |
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