JP2006522585A - Human blood proteins expressed in monocotyledonous seeds. - Google Patents

Abstract

Human blood protein is produced at a high rate in monocotyledonous seeds.
A monocotyledonous plant cell is transformed with a chimeric gene, and the monocotyledonous plant from the transformed monocotyledonous cell is transformed into a sufficient time for seeds containing human blood protein to be produced. Grow and harvest seeds from plants. The resulting human blood protein constitutes at least about 3.0% of the total soluble protein in the harvested seed.

Description

  The present invention relates to human blood proteins produced in monocotyledonous seeds for use in producing human and veterinary topical compositions and human therapeutic compositions.

Many human blood proteins are needed due to the large amount of human blood protein that provides a favorable therapeutic effect, or because the amount of human blood protein required by the worldwide patient population suffering from a specific disease is high. Insufficient or limited supply. It is also desirable to produce blood protein, usually extracted from blood products, using alternative sources such as crop plants. Producing blood proteins from plants reduces contamination by human viruses in the blood protein fraction and other mediators that cause other diseases present in the human or animal blood product fraction.
Blood proteins such as hemoglobin, alpha-1-antitrypsin (“AAT”), fibrinogen, human serum albumin, thrombin, antibodies and blood clotting factors (eg Factor V-XIII) have therapeutic efficacy in a number of human diseases. It is known to have.
Hemoglobin is the main blood component molecule that transports oxygen into cells. Mammalian hemoglobin is a tetrameric protein composed of two α-like polypeptide subunits and two non-α (usually β, γ or δ) subunits. Their subunits differ in major amino acid sequence, but their secondary and tertiary structures are similar. Each globin subunit is bound by non-covalent interaction to a Fe ²⁺ -porphyrin complex known as a heme group to which oxygen is bound. The main hemoglobin in adult erythrocytes is α2β2, known as hemoglobin A ₁ (HbA). The molecular weight of each hemoglobin tetramer is 64 kD, and the molecular weight of each α-like and β-like chain is about 15.7 kD (141 amino acids) and about 16.5 kD (146 amino acids), respectively.

  AAT belongs to the class of serpin inhibitors and is one of the major protease inhibitors present in human plasma. AAT is a single polypeptide with 394 amino acids and a molecular mass of about 52 kD, and the natural human form of this molecule contains about 15% carbohydrate. AAT concentrations in human plasma range from 1000 to 3000 mg / L, and concentrations in human milk range from 100 to 400 mg / L. A major physiological role of AAT is the inhibition of neutrophil elastase, but when deficient it leads to the development of emphysema. Excessive activity of elastase results in emphysema, hepatitis and various skin diseases. Although AAT has the greatest binding affinity for human neutrophil elastase, AAT also has an affinity for pancreatic proteases such as chymotrypsin and trypsin. The main starting point currently in the treatment of AAT deficiency is the isolation of AAT from human plasma.

  Fibrinogen is involved in the blood coagulation cascade and changes to fibrin by interacting with thrombin, a natural coagulant. Fibrin is the main blood clotting component. Mature human fibrinogen is composed of two pairs of three independent polypeptide chains (α, β and γ), which are linked together by 29 intramolecular and intermolecular disulfide bonds. Thus, a natural protein of 340 kD is formed and is present in human plasma at a concentration of about 2500 mg / L. Three-dimensional structural analysis of individual fibrinogen domains has discovered detailed structural features that provide important clues for the multi-functional role of human fibrinogen. The polypeptides of fibrinogen are about 72 kD (α), 52 kD (β) and 48 kD (γ), respectively, and the β polypeptide chain determines the natural molecular assembly. The structure of fibrinogen facilitates interactions with fibrinogen, other proteins, specific cell types, and many important physiological processes that fibrinogen encompasses blood clotting, inflammation, angiogenesis, wound closure, arteriogenesis and tumorigenesis It is characterized by having a large number of structural and functional domains containing multiple binding sites that allow it to participate in. Fibrin formation from the coagulation point of view is mediated by the interaction of natural fibrinogen with its natural coagulants factor XIII and thrombin, which occurs in the presence of calcium soluble in blood.

  Albumin serves a variety of functions in mammalian serum biology, notably other functions that carry hormones and other soluble ligands from site to site, and that contribute primarily to the general biochemistry of mammals. It is a transport protein molecule that performs activities. Human serum albumin (“HSA”) is also a major protein component of blood, which is actively present at serum concentrations of about 30,000 to 50,000 mg / L. HSA is a 66.5 kD single polypeptide chain that is first synthesized in the liver as a preproalbumin molecule and released from the endoplasmic reticulum after the N- and C-termini are Golgi processed. The resulting mature protein is 585 amino acids long. It has been shown that the natural preprosequence of HSA can function in precise protein targeting / processing across plant cell membranes in transgenic tobacco leaves (Non-Patent Document 1).

  Prothrombin, a plasma glycoprotein, is the zymogen of the serine protease thrombin and not only catalyzes the conversion of fibrinogen to fibrin but also catalyzes several other reactions that may be important for blood clotting . Prothrombin is a single polypeptide chain having a molecular weight of about 72,000. The complete human thrombin cDNA is composed of 622 amino acid residues and includes a leader sequence made up of 36 amino acid residues. The apparent molecular weight of active thrombin is 36,000, which is composed of two disulfide-linked polypeptide chains that result from prothrombin cleavage. It is very well characterized that when proteolysis occurs, human prothrombin is activated in vitro and converted to active thrombin.

  Factor V-XIII is a protein (most proteases are active) that are involved in the “inherent pathway” of the classical cascade mechanism of blood clotting. Such molecules are mostly present as precursors, which are processed in an orderly transformation order, from an inactive form to a catalytically active form. Factor V is proacelelin (promoting globulin), while factor VI is the active form of factor V. Factor VII is proconvertin, a plasma thromboplastin component, while both factor VIII (antihemophilic factor) and factor IX (Christmas antihemophilic factor) are both associated with hemophilia conditions . Factor X (Stuart-Power factor), Factor XI (plasma thromboplastin precursor) and Factor XII (Hageman factor) are all involved in thrombin maturation / stabilization. Factor XIII (fibrin stabilizing factor) is a plasma transglutaminase that acts directly on fibrin during the clotting process. All of these factors are present in serum plasma at relatively low concentrations (0.001 to 50 mg / L). Other protein factors involved in the blood coagulation cascade include Fletcher factor (pre-kallikrein), Fitzgerald factor (kininogen) and von Willebrand factor.

Immunoglobulins (antibodies) present in humans act to confer resistance to various pathogens that the patient may have contacted. Immunoglobulin molecules account for 15-20% of the mass in human serum and are mainly composed of IgG, IgM and IgA type antibodies that are found in the blood system and potentially in the rest of the body. Involved in the fight against various infectious diseases that invade. IgG type antibodies are most prevalent and are present at serum concentrations of 6-18 g / L. The blood system also serves as a carrier that directs such molecules to specific areas of the body for the purpose of combating the resulting infection and potential tumor targets. Mature antibodies are composed of two polypeptides (light and heavy chains) that need to be expressed and brought together in equimolar amounts to form a functional entity. The light chain (˜25 kD) is a protein having a length of ˜210-240 amino acids, while the heavy chain (˜50 kD) is a protein having a length of ˜450-460 amino acids. Both the light and heavy chains have a signal peptide that is processed and secreted into the blood stream. Of particular interest in the expression of monoclonal antibodies that occur in plants because two genes are expressed, the two proteins are synthesized and the tetrameric protein must be assembled correctly to result in a functional antibody. Is held.
Early studies on antibodies in plants focused on the types of IgG antibodies (Non-Patent Documents 2 and 3), but more recent studies have shown that complex antibody molecules such as secretory IgA antibodies (4 genes) ) And more complex antibody forms in plants have been studied (Non-Patent Documents 4 and 5).

Patent Documents 1, 2, 3 and 4 (all related patents) disclose the production of antibody molecules in the leaves of transgenic tobacco plants.
Patent Document 5 discloses that an immunoglobulin-containing protective protein is produced in leaves, stems, flowers and roots of tobacco plants.
Patent Document 6 discloses the production of antibodies in the plastids of tobacco plants.
An environmental agricultural bioreactor controlled to be suitable for commercial production of heterologous proteins in transgenic plants is disclosed in US Pat. Patent Document 7 discloses that production of protein in mammalian blood can be achieved. Example 7 discloses the production of human blood factors in the leaves of potato, tobacco and alfalfa plants.
Patent Document 8 discloses that hemoglobin and myoglobin are produced in the leaves of tobacco plants. Example X discloses that recombinant hemoglobin is extracted from tobacco seeds and purified to some extent. Such expression is obtained by transformation of the co-expression plasmid pBIOC59, which is constructed to target in the chloroplast and for that purpose, Pisum sativum L. ) Ribulose 1,5-diphosphate carboxylase small subunit precursor transit peptide. In seeds, it was reported that expression occurs to the extent that the maximum concentration of recombinant hemoglobin is 0.05% based on the total soluble protein extracted.

Example XI of Patent Document 8 discloses the construction of a plasmid containing one of the α or β chains of hemoglobin so that constitutive expression or expression in albumin occurs in corn seeds. According to Patent Document 8, the following regulatory sequences were required to cause constitutive or albumin-specific expression of hemoglobin chains. Three promoters allowing constitutive expression [(i) rice actin intron following rice actin promoter contained in plasmid pAct1-F4, (ii) cauliflower mosaic virus 35S double constitutive promoter, or ( iii) one and two of the terminators [(i) 35S poly A terminator or (ii) NOS poly A terminator] in the corn y zein gene promoter contained in plasmid py63] It is. No experiments or data have been shown regarding the transformation or expression of such plasmids in maize or maize seeds.
The use of a seed-specific promoter obtained from ACP of Brassica napus for the purpose of influencing and changing the expression of seed oil occurring in rapeseed and tobacco plants is disclosed in US Pat. . U.S. Pat. No. 6,057,086 generally discloses that such seed-specific promoters can be used for the expression of pharmaceutical proteins such as blood factors or human serum albumin, however, any experiment relating thereto No data is shown.

Non-Patent Document 6 is a general review discussing recent developments in the field of medical molecular aquaculture involving the production of antibodies and proteins in plants.
Production of human blood protein in monocotyledonous plant seeds in high yield is not disclosed in either the patent or the publication. When trying to treat humans that are treatable by administering a special blood protein, there is no source of contamination so that such protein can be supplied to the patient population in sufficient supply It is desirable to produce human blood protein in high yield.
US Pat. No. 6,417,429 US Pat. No. 5,959,177 US Pat. No. 5,639,947 US Pat. No. 5,202,422 US Pat. No. 6,303,341 Published US Patent Application 2002/0174453 Published US Patent Application 2002/0046418 US Pat. No. 6,344,600 US Pat. No. 5,767,363 Sismons et al., 1990 Hiatt et al., 1989 Hiatt and Ma, 1992 Ma (Ma) et al., 1995 Vine et al., 2001 Daniel et al., 2001

One aspect of the present invention encompasses a method for producing recombinant human blood protein in monocot seeds, comprising:
(A) a monocotyledonous plant cell (i) a promoter derived from a gene of a ripening-specific monocotyledonous plant storage protein,
(Ii) encodes a monocotyledon seed-specific signal sequence that is functionally linked to the promoter and allows the polypeptide linked to it to target the endosperm cells of monocotyledonous seeds 1 A second DNA sequence linked in a translation frame with the first DNA sequence and encoding a human blood protein;
The first DNA sequence and the second DNA sequence together are transformed with a chimeric gene encoding a fusion protein comprising an N-terminal signal sequence and a human blood protein,
(B) growing monocotyledonous plants from the transformed monocotyledonous cells for a time sufficient to produce seeds containing the human blood protein; and (c) harvesting the seeds from the plants. To
And wherein the human blood protein constitutes at least about 3.0% of the total soluble protein in the harvested seed.

The present invention also includes a purified human blood protein obtained by this method. The human blood protein preferably contains one or more plant glycosyl groups.
The present invention also provides monocotyledon seed products prepared from harvested seeds obtained by the method of the present invention, preferably monocotyledon seed products selected from whole seeds, flour, extracts and malts. Preferably, the human blood protein comprises at least about 3.0% of the total soluble protein in the seed product.
The present invention further comprises a composition comprising purified human blood protein, preferably purified human blood protein containing at least one plant glycosyl group, and at least one pharmaceutically acceptable excipient or nutrient. The human blood protein is produced in a monocotyledonous plant containing a nucleic acid sequence encoding the human blood protein and purified from seed harvested from the monocotyledonous plant. The nutrient is obtained from a raw material other than the monocotyledonous plant. Such formulations can be used for parenteral, enteral, inhalation, intranasal or topical delivery purposes.
The foregoing and other objects and features of the invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings and claims.

  Unless otherwise stated, all terms used herein have the meanings given below or generally correspond to the meanings of the terms for those skilled in the art. The practitioner is concerned with technical definitions and representations, in particular Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2nd edition), Cold Spring Harbor Press, Plains, NY View (Cold Spring Harbor Press, Plainview, NY), Ausubel FM et al. (1993) Current Protocols in Molecular Biology, New York State, New York John Wiley & Sons, N W York, NY) and edited by Helvin and Schilperot (1997) Plant Molecular Biology Manual, Kluwer Academic Publishers, Kluwer Academy, Netherlands Will be helpful.

The polynucleotides of the present invention may be in the form of RNA or in the form of DNA, including messenger RNA, synthetic RNA and DNA, cDNA and genomic DNA. Such DNA may be double stranded or single stranded, and if single stranded may be the coding strand or the non-coding (antisense, complementary) strand.
The term “stablely transformed” when referring to a plant cell means that the plant cell has an exogenous (heterologous) nucleic acid sequence integrated into the genome and that has been for more than two generations. It means that it is retained.
“Host cell” means a cell that contains a vector and supports the replication and / or transcription and / or expression of heterologous nucleic acid sequences. The host cell according to the present invention is preferably a monocotyledonous plant cell. Other host cells such as bacteria, yeast, insect, amphibian or mammalian cells can be used as secondary hosts to transfer the DNA to the desired plant host cell.
“Plant cell” refers to any cell derived from a plant and includes not only undifferentiated tissue (eg, callus) but also plant seeds, pollen, pearls, embryos, suspension cultures, division regions, leaves, roots, buds. , Gametophytes, spores and microspores.

The term “mature plant” refers to a fully differentiated plant.
The term “seed product” includes, but is not limited to, seed fractions such as whole seeds that have been peeled, flour (seed that has been peeled and ground to a powder by milling), seed extraction And preferably a protein extract (when the protein fraction of the flour is separated from the carbohydrate fraction), malt (including malt extract or malt syrup) and / or purified protein fraction obtained from genetically modified cereals included.
The term “biological activity” refers to any biological activity that one skilled in the art would typically attribute to the protein.
The term “blood protein” refers to one or more proteins or biologically active fragments thereof present in normal human blood and includes hemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin, prothrombin / Thrombin, antibody, blood coagulation factor (factor V, factor VI, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, Fletcher factor, Fitzgerald factor And von Willebrand factor) and biologically active fragments thereof.
The term “non-nutritive” refers to a pharmaceutically acceptable excipient that does not feed the recipient as a major effect. Preferably, the excipient provides one of the following roles in the formulation delivered to the intestine: Quality that is useful for acting as a carrier for therapeutic proteins, protecting the protein from acid in the digestive tract, providing sustained release of the active material to be delivered, or administering the blood protein to the patient To the formulation.

“Monocotyl plant seed component” refers to carbohydrate, protein and lipid components that can be extracted from monocot plant seeds, typically mature monocot plant seeds.
“Seed maturation” refers to various tissues in the seed (cereals), such as endosperm, seed coat, aleurone layer, and the like, where metabolic stores such as sugars, oligosaccharides, starch, phenolic components, amino acids and proteins, etc. start from fertilization. It refers to the period that ends with seed growth, ripening and seed drying as a result of accumulating in the squamous epithelium with or without vacuolar targets.
“Maturation-specific protein promoter” refers to a promoter that exhibits substantially up-regulated activity (to a degree greater than 25%) during seed maturation.
"Heterologous DNA" is introduced into a plant cell from another source or is under the control of a promoter that is derived from one plant source (including the same plant source) but does not normally regulate the expression of the heterologous DNA. Refers to DNA.
A “heterologous protein” is a protein encoded by a heterologous DNA.
A “signal sequence” is an N-terminal or C-terminal polypeptide sequence and is effective to specify that the peptide or protein to be bound is in a selected intracellular or extracellular region. According to the present invention, preferably the signal sequence binds a peptide or protein bound to an endosperm cell, more preferably an endoplasmic organelle such as an intracellular vacuole or other protein reservoir, chloroplast, mitochondrion or endoplasmic reticulum. Alternatively, the target is to be placed in a position such as an extracellular space after secretion from the host cell.

Expression vectors suitable for use in the present invention are chimeric nucleic acid constructs (or expression vectors or cassettes) designed to function with associated upstream and downstream sequences in plants.
In general, the expression vector used in the practice of the present invention includes the following functionally linked components that constitute a chimeric gene. Promoter derived from the gene of ripening-specific monocotyledonous storage protein, operably linked to said promoter, and the linked polypeptide targets endosperm cells, preferably organelles of endosperm cells such as protein reservoirs A first DNA sequence encoding a monocotyledonous plant seed-specific signal sequence (eg, an N-terminal leader sequence or a C-terminal trailer sequence), and linked in the translation frame together with said first DNA sequence It is the second DNA sequence encoding a human blood protein. Preferably, the signal sequence is cleaved from the human blood protein in plant cells.
Next, the chimeric gene is usually placed in a suitable plant transformation vector. The vector comprises (i) a companion sequence located upstream and / or downstream of the chimeric gene (which is derived from a plasmid or virus and that the vector transfers DNA from bacteria to the desired plant host. (Ii) a selectable marker sequence, and (iii) a transcription termination region (which is generally located at the opposite end of the vector from the transcription initiation regulatory region). ).

  Numerous types of expression vectors and suitable regulatory sequences are known in the art that are suitable for a variety of plant host cells. The promoter region is selected to be regulated in an inducible manner under seed maturation conditions. In one aspect of such an embodiment of the invention, the expression construct comprises a promoter that exhibits activity that is specifically upregulated during seed maturation. The promoter used in the present invention is usually a promoter derived from cereals such as rice, barley, wheat, oats, rye, corn, straw, rye wheat or sorghum. Examples of such promoters include a maturation-specific promoter region associated with one of the maturation-specific monocotyledonous storage proteins described below. They are rice glutelin, origin and prolamin, barley hordein, wheat gliadin and glutelin, corn zein and glutelin, oats glutelin, and sorghum kafilin, strawberry penisetin and rye secarin. Examples of representative regulatory regions derived from such genes are SEQ ID NOS: 6-14. Particularly when such promoters are used in conjunction with transcription factors, other promoters suitable for expression in seed maturation include barley endosperm-specific B1-hordein promoter, GluB-2 promoter, Bx7 promoter, Gt3 promoter, GluB- 1 promoter and Rp-6 promoter are included.

  Of particular interest is the expression of nucleic acids encoding human blood proteins using promoters that are preferentially expressed in plant seed tissue. Examples of such promoter sequences include sequences derived from sequences encoding plant storage protein genes or sequences derived from genes involved in fatty acid biosynthesis occurring in oil seeds. Preferred typical promoters include the glutelin (Gt1) promoter that causes gene expression in the outer layer of endosperm as exemplified by SEQ ID NO: 6, and the endosperm center as exemplified by SEQ ID NO: 7 A globulin (Glb) promoter that causes gene expression is included. A promoter sequence controls transcription of a gene coding sequence operably linked to itself, including a naturally occurring promoter or a region of a natural promoter that can direct seed-specific transcription, and two or more types. Hybrid promoters combined with promoter elements. Methods for constructing such hybrid promoters are well known in the art.

In some cases, the promoter is derived from the same plant species as the plant cell into which the chimeric nucleic acid construct is to be introduced. In another embodiment, the promoter is heterologous to the plant host cell.
Alternatively, seed-specific promoters from certain types of monocots can be used to regulate transcription of nucleic acid coding sequences from different monocots or monocots other than cereals.
In addition to encoding an important protein, the expression cassette or heterologous nucleic acid construct optionally contains DNA that encodes a signal peptide that allows the protein to be processed and translocated. Typical signal sequences are those associated with monocotyledon maturation specific genes, which are glutelin, prolamin, hordein, gliadin, glutenin, zein, albumin, globulin, ADP glucose pyrophosphorylase, starch synthase, Branch enzyme, Em and lea. A typical sequence encoding a signal peptide for a protein reservoir is identified herein as SEQ ID NOS: 15-21.

  In one preferred embodiment, the method localizes a protein in an endosperm cell, preferably an endosperm organelle such as a protein reservoir, mitochondria, endoplasmic reticulum, vacuole, chloroplast or other plastid compartment. It is intended to make it. For example, when the protein targets a plastid such as a chloroplast, the construct also uses a sequence that directs the gene product to the plastid for the purpose of causing expression. Such sequences are referred to herein as chloroplast transport peptides (CTP) or plastid transport peptides (PTP). Thus, when an important gene is not inserted directly into the chloroplast, the expression construct additionally contains a gene encoding a carrier peptide that directs the important gene to the chloroplast. Chloroplast transit peptides are derived from important genes or heterologous sequences with CTP and are known in the art. See, for example, Smekens et al., 1986; Wasmann et al., 1986, Von Heijne et al., 1991, US Pat. Nos. 4,940,835 and 5,728,925. . In addition, additional delivery peptides suitable for translocating the protein to the endoplasmic reticulum (ER) (Chrispeels, 1991; Vitale and Chrispeels, 1992), nuclear retention signals ( 1993; Varagona et al., 1992) or vacuoles (Raikhel and Chrispeels 1992; Bednarek and Raikel, 1992; also the United States) No. 5,360,726) can also be used in the constructs of the present invention.

Another typical type of signal sequence is a sequence that is effective in helping Aleurone cells secrete heterologous proteins during seed germination, including alpha-amylase, protease, carboxypeptidase, endoprotease, ribonuclease , DNase / RNase, (1-3) -beta-glucanase, (1-3) (1-4) -beta-glucanase, esterase, acid phosphatase, pentosamine, endoxylanase, β-xylopyranosidase, arabi Signal sequences associated with nofuranosidase, beta-glucosidase, (1-6) -beta-glucanase, peroxidase and lysophospholipase are included.
Since various protein storage proteins are under the control of a maturation-specific promoter and such promoters are operably linked to signal sequences that target protein bodies, promoter sequences are constructed when constructing this chimeric gene. And a signal sequence may be isolated from a single protein storage gene and then functionally linked to a blood protein. One preferred exemplary promoter-signal sequence is a sequence derived from the rice Gt1 gene, which has a typical sequence identified by SEQ ID NO: 6. Alternatively, the promoter sequence and leader sequence can be obtained from different genes. One preferred exemplary promoter-signal sequence combination is the rice Glb promoter linked to the rice Gt1 leader sequence, an example of which is SEQ ID NO: 7.
Preferably, an expression vector or heterologous nucleic acid construct designed to function in plants contains companion sequences upstream and downstream of the expression cassette. The companion sequence is a plasmid or virus source sequence, and the vector has the necessary features to enable transfer of DNA, such as a sequence containing a replication origin and a selectable marker, from a secondary host to a plant host. Give to the vector. Typical secondary hosts include bacteria and yeast.
In one example, the secondary host is E. coli, the origin of replication is ColE1 type, and the selectable marker is a gene encoding ampicillin resistance. Such sequences are well known in the art and are also commercially available [eg, Clontech, Palo Alto, Calif .; Stratagene, La Jolla, California ( Stratagene, La Jolla, CA)].

The transcription termination region can be taken from a gene that is normally associated with the transcription initiation region or from a different gene. Typical transcription termination regions include the NOS terminator derived from the Agrobacterium Ti plasmid and the rice α-amylase terminator.
It is also possible to add a polyadenylation tail to the expression cassette for the purpose of optimizing each of the high levels of transcription and reasonable transcription termination. Polyadenylation sequences include, but are not limited to, an Agrobacterium octopine synthase signal or the same species of nopaline synthase.
Selectable markers suitable for selection in plant cells include, but are not limited to, antibiotic resistance genes such as kanamycin (nptll), G418, bleomycin, hygromycin, chloramphenicol, ampicillin, tetracycline. Additional selectable markers include the bar gene encoding bialaphos resistance, the mutant EPSP synthase gene encoding glyphosate resistance, the nitrilase gene conferring bromoxynil resistance, the mutant acetolactate synthase gene (ALS conferring imidazolinone or sulfonylurea resistance) ), And the methotrexate resistant DHFR gene.
The individual marker gene used is a marker gene that allows selection of transformed cells compared to cells into which the DNA has not been introduced. Preferably, they are selectable marker genes that facilitate selection at the tissue culture stage, such as kanamycin, hygromycin or ampicillin resistance genes.

In addition, the vector of the present invention is modified to include a region showing homology to an Agrobacterium tumefaciens vector, a T-DNA border region derived from Agrobacterium tumefaciens, a chimeric gene or an expression cassette (described above). It is also possible to contain an intermediate plant transformation plasmid containing In addition, the vectors of the present invention may contain a disarmed plant tumor-inducing plasmid of Agrobacterium tumefaciens.
In general, the selected nucleic acid sequence is inserted into a portion or sites of an appropriate restriction endonuclease in the vector. When constructing the vectors used in the present invention, standard methods of cleavage, ligation and transformation into secondary host cells known to those skilled in the art are used (generally, Maniatis et al. (See Ausubel et al., And Helvin et al.).
Plant cells or tissues are transformed using a variety of standard techniques with expression constructs [eg, heterologous nucleic acid constructs such as plasmid DNA into which the gene of interest has been inserted]. It is an important aspect of the present invention to effectively introduce a vector for the purpose of promoting the improvement of plant gene expression. It is preferred that the sequence of such vectors be stably transformed, preferably integrated into the host genome.

The method of transformation of the host plant cell is not critical to the present invention. Those skilled in the art will recognize that there are a variety of transformation techniques in the art and that new techniques will be available on an ongoing basis. Any technique suitable for the target host plant can be used within the scope of the present invention. For example, the construct can be introduced in a variety of forms, including but not limited to DNA strands, plasmids or artificial chromosomes. Introduction of this construct into target plant cells can be accomplished using a variety of techniques including, but not limited to, calcium phosphate-DNA coprecipitation, electroporation, microinjection, Agrobacterium-mediated transformation, liposome-mediated transformation, protoplast fusion or microprojectile bombardment (Christou, 1992; Sanford et al., 1993). One skilled in the art can refer to the literature for details and select a technique suitable for use in the method of the present invention.
When Agrobacterium is used for transformation of plant cells, the vector is introduced into the Agrobacterium host to cause homologous recombination with T-DNA or Ti or Ri plasmid present in the Agrobacterium host. Let Ti or Ri plasmids containing T-DNA for recombination can be armed (allows hump formation) or disarmed (makes hump formation impossible), but transformed agro The latter is allowed as long as the vir gene is present in the bacterial host. Using armed plasmids can result in a mixture of normal plant cells and humps.

When Agrobacterium is used as a vehicle for transformation of host plant cells, expression or transcriptional constructs adjacent to one or more T-DNA border regions replicate in E. coli and Agrobacterium. Examples of broad host range vectors are described in the literature, such as pRK2 or derivatives thereof. See Ditta et al., 1980 and EPA 0 120 515. Alternatively, sequences expressed in plant cells can be inserted into vectors containing individual replication sequences, one of the individual sequences stabilizing the vector in E. coli and the other being Agrobacterium. Stabilize the vector in the um. Referring to McBride and Summerfeld 1990, the replication origin pRiHRI (Jouannin et al., 1985) was used to improve the stability of plant expression vectors in host Agrobacterium cells. increase.
The T-DNA incorporated into the expression construct is one or more selectable marker coding sequences that allow selection of transformed Agrobacterium and transformed plant cells. A number of antibiotic resistance markers suitable for use with plant cells have been developed, including genes that inactivate antibiotics such as kanamycin, aminoglycoside G418, hygromycin. The particular marker used is not essential to the invention and the preferred individual marker will depend on the particular host and the mode of construction.

In Agrobacterium-mediated transformation of plant cells, explants are incubated with Agrobacterium for a time sufficient to result in infection, after killing the bacteria, the plant cells are cultured in an appropriate selection medium. To do. After callus formation, bud formation can be promoted by using an appropriate plant hormone according to a known method, and then the shoot is transferred to a rooting medium to regenerate the plant. Next, the plant is grown and fruited, then the seeds are used to establish generational repetition and the recombinant protein produced by the plant is isolated.
There are many ways to obtain plant cells containing two or more expression constructs. As one strategy, plant cells contain the first and second constructs in a single transformation vector, or one of them uses a separate vector that expresses the desired gene. , Co-transformed with both expression constructs. The second construct is introduced into a plant that has already been transformed with the first expression construct, or, alternatively, a transgenic plant having one first and one second cross is crossed. It is possible to bring the construct together into the same plant.

In a preferred embodiment, the plant used in the method of the invention is a plant derived from a member of the taxonomic family known as Gramineae. It includes all members of the grass family, among which edible varieties are known as cereals. Such cereals include very diverse species such as wheat (Triticum sps.), Rice (Oryza sps.), Barley (Hordeum sps.), Oats (Avena sps.), Rye (Secale sps.), Corn ( maize) (Zea sps.) and cocoon (Pennisqtum sps.). Cereals suitable for the practice of the present invention are rice, wheat, corn, barley, rye and rye, with rice being most preferred.
For the purpose of producing transgenic plants that express human blood proteins in seeds, monocotyledonous cells or tissues derived therefrom are transformed with an expression vector comprising the human blood protein coding sequence. . By culturing the genetically modified plant cell in a medium containing an appropriate selection agent, a plant cell expressing a heterologous nucleic acid sequence is identified and selected. After selecting a plant cell that expresses the heterologous nucleic acid sequence, the whole plant is regenerated from the selected genetically modified plant cell. Techniques for regenerating whole plants from transformed plant cells are generally known in the art. Genetically modified plant strains such as rice, wheat, corn or barley may be grown and genetically crossed using conventional plant propagation techniques.

Transformed plant cells are selected to determine whether they can be cultured in a selective medium having a selective agent concentration as a threshold. Usually, plant cells grown on or in the selective medium are transferred to the same freshly supplied medium and cultured again. The explants are then cultured under regeneration conditions to produce regenerated plant buds. After bud formation, the shoots are transferred to selective rooting media to produce complete plants. Next, the transformed plant can also be propagated by growing, growing, and cutting the plant. Such a method allows efficient transformation of plant cells and regeneration of transgenic plants capable of producing recombinant human blood proteins.
In addition to using standard analytical techniques such as Western blot, ELISA, PCR, HPLC, NMR or mass spectrometry, the expression of recombinant human blood proteins is biologically specific to the individual protein to be expressed. It can be verified by assaying the activity.
The present invention also provides purified blood protein recombinantly produced in plant cells, preferably blood that is substantially free of host plant cells and preferably contains at least one plant glycosyl group. Medium protein is also provided. The plant glycosyl group indicates that blood protein has been produced in the plant and does not significantly impair the biological activity of the blood protein in any applied therapeutic context (no recombination). The degree of activity loss when compared with human blood protein is preferably less than 25%, more preferably less than 10%). According to some embodiments of the present invention, typically human blood protein is at least about 0.5%, at least about 1.0% or at least about total of soluble protein in seeds harvested from genetically modified plants. Make up 2.0%. However, the protein expression in the preferred embodiment is much higher than previously reported, i.e. at least about 3.0%, thus the commercial production is very feasible. Protein expression is preferably at least about 5.0%, at least about 10%, at least about 15%, at least about 20%, at least about 30% or even at least about 40% of the total soluble protein.

The invention also encompasses plant seed products made from the harvested seeds. Preferably, human blood protein comprises at least about 3.0%, more preferably at least about 5.0%, and most preferably at least about 10.0% of the total soluble protein in the seed product. As shown in this figure, the expression of human blood proteins, three types of fibrinogen polypeptides and HSA in rice grains represented by AAT account for at least about 10% of the total soluble proteins.
The present invention also provides a composition comprising a recombinantly produced human blood protein in the seed of a monocotyledonous plant, and a method for producing the composition. In the practice of the present invention, human blood proteins are produced in the seeds or grains of transgenic plants that express the nucleic acid coding sequence. After expression, the blood protein is provided to the patient in a substantially unpurified form (ie, at least 20% of the composition comprises plant material) or the blood protein is a mature seed product. It can also be provided to the patient by isolating or purifying from (eg, flour, extract, malt or whole seed) and formulating.
Such compositions include formulations suitable for the intended type of delivery. Types of delivery include parenteral, enteral, inhalation, intranasal or topical delivery. Parenteral delivery includes intravenous, intramuscular or suppositories, etc., and enteral delivery includes formulations made with pills, capsules, or other non-nutritive pharmaceutically acceptable excipients, Or, for example, orally administering a composition containing a nutrient derived from a genetically modified plant, such as in a cereal extract from which a protein is produced, or a nutrient derived from a source other than the genetically modified plant. Such nutrients include salts, sugars, vitamins, minerals, amino acids, peptides, and proteins other than human blood proteins. Intranasal and inhalation delivery systems include sprays or aerosols that enter the nostrils or mouth. Topical delivery includes creams, topical sprays or ointments. Preferably, such compositions are substantially free of genetically modified plant contaminants and preferably have a plant material content of less than 20%, more preferably less than 10%, most preferably less than 5%. To. The preferred route of administration is in the intestine and the composition is preferably non-nutritive.
The protein in blood can be purified from the seed product by a method such as grinding, filtration, heating, pressurization, salt extraction, evaporation or chromatography.

Human blood proteins produced according to the present invention also include all variants, whether allelic or synthetic variants. The nucleic acid sequence encoding a “mutant” human blood protein can encode a mutated human blood protein amino acid sequence that is altered by one or more amino acids from a native blood protein sequence, wherein at least one amino acid is It is preferably substituted, deleted or inserted. Nucleic acid substitutions, insertions or deletions resulting in a variant are any sequence within the sequence, as long as the encoded amino acid sequence maintains substantially the same biological activity as the native human blood protein. It can occur at residues. In another example, the mutant human blood protein nucleic acid sequence may encode the same polypeptide as the native sequence, but due to the degeneracy of the genetic code, the variant is derived from the native polynucleotide sequence. It has a nucleic acid sequence that is altered by one or more bases.
A variant nucleic acid sequence can encode a variant amino acid sequence that includes “conservative” substitutions, where the substituted amino acid has the same structural or chemical properties as the amino acid prior to substitution, and is found in nature. Physicochemical amino acid side chain properties and high degree of substitution in the resulting homologous protein (measured with a standard Dayhoff frequency exchange matrix or BLOSUM matrix, etc.). In addition, or alternatively, the mutated nucleic acid sequence can encode a mutated amino acid sequence that includes “non-conservative” substitutions, in which the substituted amino acid has a different structure than the amino acid before substitution. Has chemical or chemical properties. As is generally known to those skilled in the art, standard substitution types include six amino acids based on the common side chain properties and maximum degree of substitution exhibited by homologous proteins found in nature, and mutations It is used to develop nucleic acid sequences that encode human blood proteins.

  As will be appreciated by those skilled in the art, in some cases it may be advantageous to use nucleotide sequences encoding human blood proteins with non-naturally occurring codons. Suitable codons for individual eukaryotic hosts are recombinant RNAs with desirable properties such as, for example, to increase the rate of protein expression in human blood or to have a longer half-life than transcripts produced by naturally occurring sequences. Selected to produce a transcript. As an example, codons for genes expressed in rice have been shown to be rich in guanine (G) or cytosine (C) in the third codon position (Huang et al., 1990 ). Changing from a low G + C content to a high G + C content has been shown to increase the expression level of foreign protein genes in barley grains (Horvath et al., 2000). The gene encoding blood protein used in the present invention is an operon technology based on the use of rice gene codon bias (Huang et al., 1990) with appropriate restriction sites for gene cloning [Alameda, Calif. ( Alameda, CA)]. After ligating these “codon optimized” genes with regulatory / secretory sequences for seed-directed monocotyledonous expression, these chimeric genes were inserted into the appropriate plant transformation vector.

Nucleotide sequences encoding human blood proteins include, but are not limited to, human blood protein coding sequences for a variety of reasons, including changes that modify the cloning, processing and / or expression of human blood proteins by cells. Manipulated to change.
The heterologous nucleic acid construct can comprise a coding sequence for a given human blood protein, which is (i) isolated, (ii) a fusion protein or signal in which the human blood protein coding sequence is the major coding sequence. Combined with an additional coding sequence such as a peptide, (iii) an intron, or a promoter or terminator element or 5 ′ and / or 3 ′ untranslated, effective to express the coding sequence in a suitable host Can be included in combination with non-coding sequences such as regulatory elements such as regions, and / or (iv) in the presence of a vector or human blood protein coding sequence in a host environment that is a heterologous gene It is.
The expression construct can contain a nucleic acid sequence that encodes the entire human blood protein, or a portion thereof, depending on the intended use. For example, when a human blood protein sequence is used in a construct used as a probe, a special human blood protein encoding sequence such as a sequence that has been confirmed to encode a highly conserved human blood protein region. It would be advantageous to prepare a construct that contains only a small portion.

The present invention provides, in one embodiment, a seed composition comprising, in an unpurified form, flour, extract or malt obtained from mature monocot seed and one or more human blood proteins produced by the seed. To do. In connection with the isolation of blood proteins from such flour, the seeds are ground to flour, the flour is extracted with a buffered aqueous solution and optionally the extract is partially concentrated and / or unwanted components It is possible to form an extraction composition by removing. In a preferred method, mature monocotyledonous seeds, such as rice seeds, are ground to flour and the flour is then saline or phosphate buffered saline (“PBS”), ammonium bicarbonate buffer, ammonium acetate. Suspend in a buffer such as buffer or Tris buffer. Volatile buffers or salts such as ammonium bicarbonate or ammonium acetate do not require a salt removal step, thus simplifying the extraction process.
The powder suspension is incubated with shaking, typically at a temperature in the range of 20-55 ° C. for 30 minutes to 4 hours. The resulting homogenate is clarified by either filtration or centrifugation. Further processing such as ultrafiltration or dialysis or both may be added to the clarified filtrate or supernatant to remove contaminants such as lipids, sugars and salts. Finally, the material may be dried, such as by lyophilization, to produce a dried cake or powder. Such extracts have the advantage that, in addition to the high yield of protein in the blood, the losses associated with protein purification are essentially limited.
In general, once a protein is produced in a mature seed product, the protein may be purified using standard methods such as filtration, affinity columns, gel electrophoresis and other standard procedures known in the art. The protein may be further purified. The purified protein may then be formulated in a manner desired for delivery to a human patient. A therapeutic formulation can bind two or more proteins. In biomedical applications that require administration of a protein for purposes other than food, the protein may be purified and used.
The following examples illustrate the invention and are not intended to limit the invention in any way.

Production of transgenic rice encoding AAT and fibrinogen polypeptides The basic procedure for particle bombardment-mediated rice transformation and plant regeneration was performed as described by Huang et al. (2001). The rice mutant TP309 seeds were peeled and sterilized by placing them in a 50% (volume / volume) commercial bleach for 25 minutes, followed by washing with sterile water. The sterilized seeds were placed on a rice callus induction medium (RCI) plate containing N6 salt (Sigma), B5 vitamin (Sigma), 2,4-D 2 mg / l and sucrose 3%. Callus formation was induced by incubating the rice seeds for 10 days. Primary callus was cut from the seeds and placed on RCI for 3 weeks. By performing this twice more, secondary and tertiary calluses used in bombardment were formed, and the secondary culture was continued. Prior to bombardment, a callus having a diameter of 1 to 4 mm was placed on an RCI containing 0.3 M mannitol and 0.3 M sorbitol in a 4 cm circle for 5 to 24 hours. Microprojectile bombardment was performed using a Biolistic PDC-1000 / He instrument (Bio-Rad). This procedure requires coating 1.5 mg of gold particles (60 ug / ml) with 2.5 ug of DNA. Gold particles that had been coated with DNA were bombarded on rice callus using a He pressure of 1100 pounds per square inch (about 7.58423 × 10 ⁶ Pascals). After the bombardment callus was recovered over 48 hours, it was transferred to RCI containing 30 mg / l of hygromycin B for selection and incubated in the dark at 26 ° C. for 45 days. Transformed callus was selected and transferred to RCI (except 2,4-D) containing 5 mg / l ABA, 2 mg / l BAP, 1 mg / l NAA and 30 mg / l hygromycin B Set a day. The transformed callus was transferred to a regeneration medium consisting of RCI (excluding 2,4-D) without hygromycin B and containing 3 mg / l BAP and 0.5 mg / l NAA. Cultured for ~ 4 weeks. The regenerated plant body (height 1 to 3 cm) was transferred to a rooting medium having a concentration half that of the MS medium (Sigma) and 1% sucrose and 0.05 mg / l NAA. The plants were rooted after 2 weeks on the rooting medium and grown until the buds were about 10 cm. The plants were transferred to a 6.5 x 6.5 cm pot mixed with 50% commercial soil (Sunshine # 1) and 50% rice soil. Plants were covered with plastic containers to maintain approximately 100% humidity and grown for 1 week under continuous light. The clear plastic cover was slowly moved over the course of the day to gradually reduce humidity and water and add fertilizer as needed. When the transgenic R0 plants were about 20 cm in height, they were transferred to the greenhouse and grown therein until they matured.
Individual R1 seed grains obtained from individual R0 regenerated plants were cut into embryos and endosperm. The expression level of recombinant blood proteins (AAT and fibrinogen polypeptide) in the isolated rice endosperm was examined. Embryos obtained from individual R1 grains having a high level of recombinant protein expression were placed in a 50% bleach solution and sterilized for 25 minutes, and then washed with sterile distilled water. The sterilized embryos were placed in a tissue culture tube containing 1/2 MS basal salt supplemented with 1% sucrose and 0.05 mg / l NAA. In about 2 weeks, the embryos germinated and a plant body having a shoot of up to 7 cm and a healthy root system was obtained. Mature R1 plants were obtained as regenerated bodies.

Manufacture of rice extract containing recombinant blood protein, and further use of said extract in parenteral and enteral preparations General rice extract manufacturing procedure Dry rice of genetically modified rice containing heterologous polypeptide It can be converted to rice extract by either milling or wet milling methods. In the dry milling method, the skin of genetically modified rice seeds containing a heterologous polypeptide was peeled off with a peeling machine. For example, in one experiment, the rice was ground in a dry milling process, such as using K-TEC model 91 Kitchen Mill at a speed of 3, to obtain a fine powder. Phosphate buffered saline (“PBS”) is added to the rice flour, which is 0.135 N NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 1.7 mM KH ₂ PO ₄ , pH 7.4. And optionally additional NaCl such as 0.35N NaCl. In some experiments, about 10 ml of extraction buffer was used per gram of flour. In other experiments, the initial flour / buffer ratio was varied from 1 g / 40 ml to 1 g / 10 ml. The mixture was incubated for 1 hour at room temperature with gentle shaking. In another experiment, the incubation temperature was lowered or raised, such as from about 22 ° C. to about 60 ° C., and the incubation time was lengthened or shortened, eg, from about 10 minutes to about 24 hours. The particles were kept in suspension by using a Thermolyne VariMix platform mixer set at high speed.
In some experiments, other buffers were used instead of PBS, such as ammonium bicarbonate. In one example, 10 liters of 0.5M ammonium bicarbonate was added to 1 kg of rice flour.
The resulting homogenate was clarified by either filtration or centrifugation. As a method of filtration, the mixture was allowed to settle for about 30 minutes at room temperature, and then the homogenate was collected and filtered. Three different forms of filters purchased from Pall Gemansciences were used: 3 μm pleated capsules, 1.2 μm serum capsules and Supercap capsule 50 (0.2 μm). Centrifugation was carried out using a Beckman J2-HC centrifuge, and the mixture was centrifuged at 30,000 g at 4 ° C. for about 1 hour. The supernatant was retained and the precipitate was discarded.
In one example, the filtrate and supernatant were further processed, such as by ultrafiltration and / or dialysis, to remove components such as lipids, sugars and salts.
Next, the filtrate obtained by the above filtration procedure (also called purified extract) was concentrated using a spiral wound tangential flow filter operated in a batch circulation mode. In one example, a PES (polyethersulfone) 3000-4000 molecular weight cut-off membrane was used at this stage. The concentrated final extract was kept in a cold room overnight.
Next, the concentrated extract was lyophilized to a powder. The freeze-dried material was scooped from the freeze-dryer tray and placed together in a plastic bag. After compressing the dried material by applying a vacuum to the bag, the material was mixed by hand kneading through the plastic to reduce the particle size.
It is also possible to produce rice extract using a wet milling procedure. Genetically modified rice seeds containing recombinant human blood protein may be rehydrated at 30 ° C. for 0 to 288 hours. The rehydrated seed is ground in PBS buffer for extraction. The initial seed / buffer ratio may be varied, for example, in the range of 1 g / 40 ml to 1 g / 10 ml.
Using such a wet milling method, human blood protein can be recovered to a degree exceeding 20%. As a result of wet milling, depending on the stability of the protein target in the blood, the initial extract can be kept cold (4 ° C) or can be stored frozen until use Become. The manner in which the initial extract is processed to obtain a dried extract is similar to that described in this chapter for dry milling.

Concentration and diafiltration of recombinant blood protein and control rice extract The conditions used for concentration and diafiltration vary depending on volume, speed, cost, etc. These conditions are standard in the art for the disclosure herein. The frozen initial extract was thawed in a cold room (about 2-8 ° C.) for 6 hours. The thawed material was purified by passing through a 0.45 μm filter, and then concentrated using a polyethersulfone 5000 nominal molecular weight cut-off membrane.
After concentrating the 90 ml control extract filtrate to 10 ml, an additional 10 ml of deionized water may be added to the concentrated filtrate. Dialysis filtration using water may be performed once again on the diluted filtrate. Precipitates begin to form at 16 mS and increase with decreasing ionic strength. A 1.0 M ammonium bicarbonate solution was added to the retentate to increase ionic strength. The haze did not disappear completely but decreased. The material was subjected to several diafiltrations with water several times in one example and three times with 0.1M ammonium bicarbonate, three times in one example. After the material was concentrated to 9 ml, the membrane was rinsed with 0.1 M ammonium bicarbonate. The concentrate was filtered through several 0.2 μm button filters. In one example, 2.3 ml of the filtrate is lyophilized as is, 2.3 ml of the filtrate is diluted with deionized water to 12 ml and lyophilized, and 2.0 ml of the filtrate is deionized water. The solution was diluted to 25 ml and freeze-dried. All of these filtrates remained clear.
A total of 89 ml of the recombinant protein extract filtrate is concentrated to 10 ml, followed by an additional 10 ml of 0.1M ammonium bicarbonate. The resulting mixture is concentrated back to 10 ml and an additional 10 ml of 0.1M ammonium bicarbonate is added. The retentate begins to cloud. The material was subjected to several diafiltrations with 0.1M ammonium bicarbonate, three times in one example. After concentrating the material to 9 ml, the membrane was rinsed with 0.1 M ammonium bicarbonate. The concentrate was filtered through several 0.45 μm button filters. In one example, 2.0 ml of the filtrate was lyophilized as it was, and when 2.0 ml of the filtrate was diluted with deionized water to 12 ml, clouding occurred, followed by lyophilization and 2.0 ml. When the filtrate was diluted with 0.1 M ammonium bicarbonate to 12 ml, it remained clear and then lyophilized.

Comparison of trials for extraction of recombinant protein rice with PBS and ammonium bicarbonate Conditions used for concentration and diafiltration vary according to volume, speed, cost, etc. These conditions are all standard in the technical field related to the disclosure herein. Recombinant protein rice flour and extraction buffer are mixed at a rate of about 100 g / L for about 1 hour using a magnetic stir bar. The extraction buffer contained in one 2 L beaker was PBS (pH 7.4), and 0.35 M NaCl was added thereto. The extraction buffer in another 2L beaker is 0.5M ammonium bicarbonate. After pre-covering a 15 cm Buchner funnel with about 6 g of Cel-pure C300, add another 20 g of Cel-pure C300. The mixture is filtered at about 3-4 Hg. Next, the filtered material is washed twice with about 100 ml of extraction buffer. The extracted filtrate was collected and concentrated with an ultrafiltration cartridge, namely 5K Regenerate Cellulose, 5K PES and 1K Regenerated Cellulose. The concentrate was freeze-dried and analyzed for the protein activity content of recombinant blood. Both ammonium bicarbonate and PBS (pH 7.4) plus 0.35 M NaCl extract approximately equal amounts of rAAT. There is almost no loss of recombinant protein units in the permeate when using any of the ultrafiltration equipment used.
It is also possible to use other extraction buffers for the purpose of extracting recombinant proteins expressed in genetically modified rice grains. For example, Tris buffer, ammonium acetate or the like may be used depending on the application. Is also possible.

Production of rice extract containing recombinant blood protein The conditions used for concentration and diafiltration vary according to volume, speed, cost, etc. These conditions are all standard in the technical field related to the disclosure herein. All equipment is immersed in hot 0.1M NaOH with a starting temperature of about 55 ° C. Rice flour is added at a rate of 95-105 g / L to an approximately 250-500 gallon (approximately 946.4-1892.7 cubic decimeter) stainless steel tank containing 0.5M ammonium bicarbonate. And mix at about 9 ° C. for about 60-80 minutes.
Twelve 36 inch filter presses C300 were pre-covered with about 3-6 kg Cel-pure C300. Add approximately 19-26 g / L of Cel-pure to the extract and mix thoroughly. The mixture is compressed at a pressure of about 22 pounds per square inch (about 151,658 Pascals) at a flow rate of about 82 liters / minute. The filtrate is collected and placed in a 250 gallon (approximately 946.4 cubic decimeter) stainless steel tank and then washed with 0.5 M ammonium bicarbonate. The compressed product is blown dry. This process is carried out at about 10 ° C.
A 300 NMW cutoff membrane [Polysulfone] that has been washed and stored with 0.1 M NaOH after the control experiment is thoroughly rinsed with deionized water. After concentrating the extract, it is placed in a 100 gallon (about 378.5 cubic decimeter) stainless steel tank. The membrane and the concentration tank were flushed with 0.1M ammonium bicarbonate to recover all remaining extract. The product was covered with plastic and left at room temperature overnight in a 100 gallon (about 378.5 cubic decimeter) tank. The concentrate is filtered through a 1 μm spiral filter and then placed in a 5 gallon (about 18.9 cubic decimeter) plastic container.

Mixing rice extract containing recombinant protein into parenteral, inhalation, intranasal and topical preparations. For the purpose of using recombinant blood protein (eg AAT) for medical / pharmaceutical applications, Highly refined grains can be obtained. A protocol was developed to purify rice seed extract expressing human AAT (Huang et al., 2002), which prepared a rice seed extract according to the above-described Examples and Con-A, Further purification of the extract preparation using each of DEAE and Octyl Sepharose chromatography. Using this procedure, AAT can be purified to a degree of homogeneity greater than 90% (Huang et al., 2002). Purified AAT can be used in potential pharmaceutical / medical applications for the following indications: AAT augmentation / replacement therapy (Sandhaus, 1993; Hubbard et al., 1989, Cystic fibrosis (McElvaney et al., 1991; Allen, 1996), psoriasis , Panniculitis and dermatovasculitis (O'Riordan et al., 1997; Dowd et al., 1995) and lung inflammation (Bingle and Tetley, 1996). In some such indications, the purified AAT protein preparation may be administered intravenously (iv) in the form of a 0.09% saline solution, alternatively, the serum solution may contain 0.5% serum albumin. Buffer used or buffered with some other pharmaceutically acceptable protein carrier molecule AAT dosage is typically about 60 mg / kg For aerosol delivery, an aerosol suitable for droplet size to adhere to the lower respiratory tract (aerodynamic diameter <3 um) is used. Aerosol generators using compressed air propelled atomizers selected based on their ability to generate can be used (Hubbard et al., 1989) Again, the protein can be supplied in sterile water or a pharmaceutically acceptable protein carrier. Alternatively, the dried protein powder containing the purified protein component can be used as a dispersant, and the dried protein powder contains an AAT component. It may be an extract based on at least 50% by weight of rice.
In another case, recombinant rice that expresses and extracts human blood proteins such as AAT and fibrinogen can be used locally. The use of fibrin sealants / bandages has been widely accepted and used in the medical community. Fibrin sealants are effective hemostatic agents (Mankad and Codispotti, 2001), a means to achieve tissue adhesion, prevent fluid accumulation and promote wound healing (Spotnitz ( Spotnitz), 2001). Fibrin sealant can also be used as a drug sustained-release means and includes antibiotics, growth factors and other drugs (Spotnitz, 1997). Rice expressing fibrinogen can also be a low cost, animal virus free source that is effective for their adaptation.

References

1. Allen, ED (1996). Opportunities for the use of aerosolized α1-antitrypsin for the treatment of cystic fibrosis. CHEST 110: 256S-260S.
2. Ausubel FM et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,
John Wiley & Sons, New York, NY, 1993.
3. Beatty, K., Beath, J. and Travis, J. (1980). Kinetics of association of serine proteinase with native and oxidized α-1-proteinase inhibitor and α-1-antichymotrypsin. Jour Biol Chem 255: 3931-34.
4). Bednarek, SY and Raikel, NV (1992) .Intracellular trafficking of secretory proteins.Plant Mol Biol 20: 133-50.
5. Bhagavan, NV (2002). “Biochemistry of Hemostatis” in Medical Biochemistry, Academic Press Inc, Harcourt Inc, Orlando, Fla.
6). Bhagavan, NV (2002). “Molecular Immunology” in Medical Biochemistry, Academic Press Inc, Harcourt Inc, Orlando, Fla.
7). Bingle, L. and Tetley, TD (1996) .Secretory leukoprotease inhibitor; partnering α1-proteinase inhibitor to combat pulmonary inflammation. Thorax 51: 1273-4.
8). Brennan, SO, Owen, MC, Boswell, DR, Lewis, JH and Carrell, RW (1984). Circulating proalbumin associated with a variant proteinase inhibitor. Biochem Biophys Acta 802: 24-48.
9. Brown, JH, Volkmann, N., Jun, G., Henschen-Edman, AH and Cohen, C. (2000) .The crystal structure of modified bovine fibrinogen. Proc Natl Acad Sci 97: 85-90.
10. Carrell, RW, Jeppsson, JO, Vaughan, L., Brennen, S., Owen, MC and Boswell, DR (1981) .Human α-1-antitrypsin: Carbohydrate attachment and sequence homology.FEBS Lett 135: 301-3.
11. Carrell, RW, Jeppson, JO, Laurrell, CB, Brennan, SO, Owen, MC, Vaughan, L. and Boswell, DR (1982) .Structure and variation of human alpha-1-antitrypsin.Nature 298: 329-34.
12 Chrispeels, MJ (1991) .Sorting proteins in the secretory system.Annu Rev Plant Physiol Plant Mol Biol 42: 21-53.
13. Chrispeels, MJ and Raikhel, NV (1992) .Short peptide domains target proteins to plant vacuoles. Cell 68: 613-16.
14 Christou, P. (1992). Genetic transformation of crop plants using microprojectile bombardment. Plant Jour 2: 275-281.
15. Daniell, H., Streatfield, SJ and Wycoff, K. (2001) .Medical molecular farming: production of antibodies, biopharmaceuticals and edible vaccines in plants.TRENDS Plant Sci 6: 219-226.
16. Davidson LA and Lonnerdal, B. (1987). Persistence of human milk proteins in the breast-fed infant. Act Pediatric Scand 76: 733-40.
17. Ditta, G., Stansfield, S., Corbin, D. and Helinski, DR (1980) .Broad host ramge DNA cloning system from gram-negative bacteria: Construcyionof a gene bank from Rhizobium meliloti.Proc Natl Acad Sci USA 77: 7347 -51.
18. Dowd, SK, Rodgers, GC and Callen, JP (1995). Effective treqatment with α1-protease inhibitor of chronic cutaneous vasculitis associated with α1-antitrypsin deficiency. Jour Am Acad Dermatol 33: 913-6.
19. Eriksson, S. (1964). Acta Medic Scand 175: 197-205.
20. Everse, SJ, Spraggon, G., Veerapandian, L. and Doolittle, RF (1999) Conformational changes in fragments D and doubl-D from human fibrinogen upon binding the peptide ligan Gly-His-Pro-amide. Biochemistry 38: 2941- 2946.
21. Friezner-Degen SJ, MacGillivray, RTA and Davie, EW (1983). Characterization of the complementary deoxyribonucleic acid and gene coding for human prothrombin. Biochemistry 22: 2087-97.
22. Holmberg. N., Gosta, L, Bailey, JE and Bulow, L. (1997). Transgenic tobacco expressing Vitreoscilla hemoglobin exhibits enhanced growth and altered metabolite production. Nature Biotech 15: 244-7.
23. Hiatt, A., Cafferkey, R. and Bowdish, K. (1989). Production of antibodies in transgenic plants. Nature 342: 76-8.
24. Hiatt, A. and Ma, JKC (1992) .Monoclonal antibody engineering in plants.FEBS Letters 307: 71-5.
25. Horvath, H., Huang, J., Wong. O., Kohl E., Okita, T., Kannangara, CG and vonWettstein, D. (2000). The production of recombinant proteins in transgenic barley grains.Proc Natl Acad Sci USA 97: 1914-19.
26. Huang, N., Simmons, CR and Rodriguez, RL (1990). Codon usage patterns in plant genes. J CAASS 1: 73-86.
27. Huang, N., Wu, I., Nandi, S., Bowman, E., Huang, J., Sutliff, T. and Rodriguez, RL (2001). The tissue-specific activity of a rice B-glucanase promoter ( Gns9) is used to select rice transformants.Plant Sci 161: 589-95.
28. Huang, J., Sutliff, TD, Wu, L., Nandi, S., Benge, K., Terashima, M., Ralston, AH, Drohan, W., Huang, N. and Rodriguez, RL (2001). Expression and purification of functional human α-1-antitrypsin from cultured plant cells.Biotechnol Prog 17: 126-33.
29. Hubbard, RC, McElvaney, NG, Sellers, SE, Healy, JT, Czerski, DB and Crystal, RG (1989) .Recombinant DNA-produced α1-antitrypsin administered by aerosol augments lower respiratory tract antineutrophil elastase defenses in individuals with α1-antitrypsin deficiency. Jour Clinical Invest 84: 1349-54.
30. Jouanin, L., Vilaine, F., d'Enfert, C. and Casse-Delbart, F. (1985) .Localization and restriction maps of the replication origin regions of the plasmids of Agrobacterium rhizogenes strain A4. Mol Gen Genet 201: 370-4.
31. Judah, JD, Gamble, M. and Steadman, JH (1973) .Biosynthesis of serum albumin in rat liver, evidence for the exsistance of 'proalbumin.' Biochem J 134: 1083-91.
32. Kurachi, K., Chandra, T., Degen, SJ, White, TT, Marchioro, TL, Woo, SL and Davie, EW (1981) .Cloning and sequence of a cDNA coding for α-1-antitrypsin.Proc Natl Acad Sci 78: 6826-30.
33. Ma, JKC, Hiatt, A., Hein, M., Vine, ND, Wang, F., Stabila, P., van Dolleweerd, C., Mostov, K. and Lehner, T. (1995). Generation and assembly of Secretory Antibodies in plants.Science 268: 716-9.
34. Madrazo, J., Bzrown, JH, Litvinovich, S., Dominguez, R., Yakovlev, S., Medved, L. and Cohen, C. (2001) Crystal structure of the central region of bovine fibrinogen E5 fragment at 1.4A resolution.Proc Natl Acad Sci 98: 11967-72.
35. Mankad, PS and Codispoti, M. (2001) .The role of fibrin sealants in hemostasis. Amer Jour Surg 182: 21S-28S.
36. McBride, KE and Summerfelt, KR (1990). Improved binary vectors for Agrobacterium mediated plant transformation.Plant Mol Biol 14: 269-76.
37. McElvaney, NG, Hubbard, R, C., Birrer, P., Chernick, MS, Caplan, DB, Frank, MM and Crystal, RG (1991). Aerosol α1-antitrypsin treatrment for cystic fibrosis. The Lancet 337: 392- Four.
38. McGilligan, KM, Thomas, DW and Eckert, CD (1987). Alpha-1-antitrypsin concentration in human milk. Pediatr Res 22: 268-70.
39. O'Riordan, K., Blei, A., Rao, MS and Abecassis, M. (1997) .α1-antitrypsin deficiency-associated panniculitis. Transplant 63: 480-2.
40. Peters, TJ (1996) .All about Albumin: Biochemistry, genetics and medical applications.Academic Press Inc, Harcourt Brace & Company, Orlando, Fla.
41. Rabiet, MJ, Blashill, A., Furie, B. and Furie, BC (1986) .Prothrombin fragment 1 * 2 * 3; a major product of prothrombin activation in human plasma.Jour Biol Chem 261: 13210-15.
42. Rosenberg, JS, Beeler, DL and Rosenberg, RD (1975) .Activation of human prothrombin by highly purified human factors V and Xa in the presence of human antithrombin.Jour Biol Chem 250: 1607-17.
43. Travis, J. and Salvesen, GS (1983) .Human plasma proteinase inhibitors. Annu Rev Biochem 655-709.
44. Sambrook J. et al., MOLECULAR CLONING: A LABORATORY MANUAL (Second Edition), Cold Spring arbor Press, Plainview, N. Y, 1989.
45. Sandhaus, RA (1993). “Alpha-1-antitrypsin augmentation therapy” in Proteases, Protease Inhibitors and Protease-Derived Peptides, Birkhauser Verlag, Basel, pp97-102 Sanford, J., Smith, FD and Russell, JA (1993) Optimizing the biolistic process for different biological applications. Meth Enzymol 217: 83-509.
46. Shieh, MW, Wessler, SR and Raikel, NV (1993) .Nuclear targeting of the maize R protein requires two nuclear localization sequences.Plant Physiol 101: 353-61.
47. Sijmons, PC, Dekker, BM, Schrammeijer, B., Verwoerd, TC, van den Elzen, PJM and Hoekema, A. (1990). Production of correctly processed human serum albumin in transgenic plants.Bio/Technology 8: 217-21 .
48. Smeekens, S., Bauerle, C., Hageman, J., Keegstra, K. and Weisbeek, P. (1986) The role of the transit peptide in the routing of precursors towards different chloroplast compartments.Cell 46: 365-76.
49. Spotnitz, WD (1997) .New developments in the use of fibrin sealants: a surgeon's perspective.Jour Long Term Effects Med Implants 7: 243-53.
50. Spotnitz, WD (2001). Commercial fibrin sealants in surgical care. Amer Jour Surg 182: 8S-14S. Vine, ND, Drake, P., Hiatt, A. and Ma., JKC (2001). Assembly and plasma membrane targeting of recombinant immunoglobin chains in plants with a murine immunoglobin transmembrane sequence.Plant Molec Biol 45: 159-67.
51. Tietz, NW (1995). Clinical Guide to Laboratory Tests, WB Saunders, Philadelphia.
52. Varagona, MJ, Schmidt, RJ and Raikhel, NV (1992) .Nuclear localization signals required for nuclear targeting of the maize regulatory protein, Opaque-2, Plant Cell 4: 1213-27.
53. Vitale, A. and Chrispeels, MJ (1992) .Sorting of proteins to the vacuoles of plant cells.BioEssays 14: 151-60.
54. Wassmen, CC, Reiss, B., Bartlett, SG and Bohnert, HJ (1986) .The importance of the transit peptides and the transported protein for protein import into chloroplasts. Mol Gen Genet 205: 446-53.
55. Weeke, B. and Krasilnikoff (1971) .A polynomial expression for the serum concentrations of 21 serum proteins from 1 to 93 years of age in normal males and females.Protein Biol Fluids 18: 173-9.
56. Yakovlev, S., Litvinovich, S., Loukinov, D. and Medved, L. (2000) .Role of the beta-strand in the central domain of fibrinogen gamma module.Biochemistry 39: 15721-9.
57. Yang. Z., Mochalkin, I., Veerapandian, L., Riley, M. and Doolittle, RF (2000) Crystal structure of native chicken fibrinogen at 5.5A resolution.Proc Natl Acad Sci 97: 3907-12.

All publications cited herein are expressly incorporated herein.

FIG. 1 shows three codon optimized genes (SEQ ID NO.) Encoding fibrinogen polypeptides α (pAPI 398), β (pAPI 417) and γ (pAPI 327), each under the control of the rice glutelin promoter Gt1. : Shows a plasmid with a construct containing 1-3). A gene set that expresses the three genes in mature rice seeds by colliding the plasmid including a plasmid (not shown) containing a hygromycin selectable marker with an embryogenic rice callus. Produced a replacement rice plant. FIG. 2 shows a Western blot analysis of transgenic rice strains expressing individual subunits of human fibrinogen. Lane 1 is a positive control purified natural human fibrinogen (obtained from Red Cross), which shows all three polypeptide chains, and lane 2 is a non-GMO rice cultivar Tapel. Lane 3 is a molecular weight standard, lane 4 is an extract of rice seeds expressing fibrinogen α chain, and lane 5 is an extraction of rice seeds expressing fibrinogen β chain. Lane 6 is an extract of rice seeds expressing fibrinogen γ chain. All protein extraction of rice seeds was performed in 2% SDS, 1M urea, 1% βMe and PBS (pH 7.4). Fibrinogen polypeptides were detected using antibodies that recognize all three chains or only individual chains. FIG. 3 shows the three fibrinogen polypeptide chains (α, β and γ) coexpressed in transgenic rice seeds and analyzed by Western blot analysis. Fibrinogen polypeptides and protein aggregates were detected using antibodies that recognize all three chains. FIG. 3A shows that from rice seeds by running on a non-denaturing 10% acrylamide gel under non-denaturing conditions [350 mM NaCl, PBS (pH 7.4), 0.01% Tween-20 / Trition X-100 / CHAPS]. The total protein extracted is shown. Lane 1 is 1 μg of purified human fibrinogen, lanes 2 and 3 are extracts from non-recombinant rice cultivar Tapel 309, lane 4 is a molecular weight marker, lanes 5 and 7 is an extract obtained from two types of transgenic rice strains containing 1.0% βMe in the extraction buffer, and lanes 6 and 8 do not contain βMe in the extraction buffer 2 It is an extract obtained from various types of transgenic rice strains. Lanes 6 and 8 show large protein aggregates extracted by running the recombinant strains at approximate locations in the complex natural human fibrinogen under non-denaturing conditions. FIG. 3B shows the total protein extracted by putting rice seeds in 2% SDS, 1M urea, 1% βMe and PBS (pH 7.4) and running on SDS-PAGE. Lane 1 is the positive control natural human fibrinogen (obtained from the Red Cross), which shows all three polypeptide chains, lane 2 is the molecular weight standard, lanes 3-5 are 3 Each of the three types of transgenic rice strains that express all of the types of fibrinogen peptides. FIG. 4 shows plasmid pAPI 250 that expresses the codon optimized gene (SEQ ID NO: 5) of alpha-1-antitrypsin (AAT) under the control of rice glutelin promoter Gt1. This plasmid, together with a plasmid (not shown) containing a hygromycin selectable marker gene, is struck into the embryogenic rice callus to create a transgenic rice plant that expresses AAT in mature rice seeds. It was. FIG. 5 shows Coomassie brilliant blue staining of an aqueous phase extract of a genetically modified rice grain expressing human recombinant AAT. Grinding seeds of both untransformed rice (rice cultivar Kitake) and transformed rice (10 R1 seeds pooled from individual 5 transgenic plants) with PBS (pH 7.4) buffer did. The resulting extract was spun at 14,000 rpm for 10 minutes at 4 ° C. After the supernatant was collected, ˜20 μg of the soluble protein extract was put into a sample filling buffer and resuspended, and then loaded onto a pre-shaped SDS-PAGE gel. Lane 1 is a molecular weight protein marker, lane 2 is purified non-recombinant human AAT, and lane 3 is an extract obtained from a non-transformed control Kitaake variety. The results obtained using the extracts of individual 5 transgenic plants are shown between lanes 2 and 3. FIG. 6 shows a Western blot analysis of recombinant human AAT expressed in transgenic rice grains. A soluble protein extract of R1 seed pooled from individual 7 transgenic rice transformants (-10 μg of total protein) was prepared as shown in FIG. 5 and separated using an SDS-PAGE gel. Blotted onto a nitrocellulose filter. Identification of AAT expressed in rice seeds was performed by Western analysis using an anti-AAT antibody. Lane 1 is a molecular weight protein marker, lanes 2 and 3 are 1 μg and 2 μg of purified non-recombinant human AAT, respectively, and lanes 4 and 5 are extractions of untransformed control rice (Kitaake variety). It is a liquid. The final 7 lanes show the results obtained using the extracts of individual 7 transgenic plants. Extracts obtained from two of the seven genetic recombination systems did not express AAT. There was a shift in gel mobility between the non-recombinant human form and the form in which the recombinant rice was expressed, but this was due to differences in the type and glycosylation of the human protein and the protein expressed by the recombinant rice. Because there is. FIG. 7 shows the activity of purified recombinant AAT (rAAT) obtained from rice extract against purified porcine pancreatic elastase (PPE) as measured by Coomassie staining and Western blot analysis. A band shift assay using elastase, a substrate for AAT protease, is used to demonstrate the activity exhibited by rAAT. AAT samples obtained from human and rice extracts were incubated with equimolar PPE for 15 minutes at 37 ° C. The negative control for the band shift assay was prepared by incubating the AAT sample with PPE added to it in an equal volume. Lane MW refers to a molecular weight marker. Lane 1 in FIG. 7A is purified non-recombinant AAT obtained from human plasma, lane 2 is purified AAT obtained from human plasma + PPE, and lane 3 contains AAT obtained from transgenic rice seeds. A soluble protein extract, lane 4 is an AAT-containing protein extract obtained from transgenic rice seed + PPE, lane 5 is an untransformed rice seed extract, and lane 6 Untransformed rice seed extract + PPE. FIG. 7B shows that band shifts are seen in lanes 1, 2 and 3. Western blot analysis demonstrates that such a band shift, ie, the complex between the PPE and the AAT fragment contains AAT. The lanes shown in FIG. 7B are similar to those shown in FIG. 7A. FIG. 8 shows AAT obtained by purification by first passing the rice cell extract through each of Con-A and DEAE Sepharose and then packing it onto an octyl Sepharose column. Octyl sepharose is the final purification step, thereby separating active AAT from the inactive form of the protein. Lane 1 is a molecular weight marker, lane 2 is 2 μg of purified non-recombinant human AAT as a standard, and lane 3 is an eluent obtained by pooling solutions eluted from a DEAE Sepharose column. The remaining column shows the liquid and eluent that has passed through the octyl Sepharose column. Approximately 50 μL of each column fraction was loaded onto an SDS-PAGE gel and the protein was visualized by Coomassie staining. The solution that has passed through octyl Sepharose shows inactive AAT protein, while the eluent elutes active AAT. FIG. 9A shows the AAT binding rate constant for the activity of purified recombinant AAT against PPE when measured as shown in the procedure in FIG. 7 using non-recombinant human AAT as a control. Data were obtained using Coomassie protein staining and Western blot analysis as described in FIG. FIG. 9B shows the thermal stability of the plant-derived recombinant AAT as compared to natural human AAT as measured by the PPE inhibition assay. FIG. 10 shows a plasmid pAP19 that expresses codon-optimized human serum albumin (HSA) (SEQ ID NO: 4) under the control of a rice Amy1A promoter / signal peptide. This plasmid is useful for expressing HSA in germinated rice seeds. FIG. 11 shows HSA expression that occurs in transgenic rice seeds. After seeding pooled seeds from the transgenic rice strain 3-11-2 in water for 24 hours, 2 μM of gibberellic acid (GA) was added. For the purpose of performing Western analysis, seed samples at 24, 48, 72 and 120 hours were taken out after GA was added, and soluble proteins were extracted. Each lane was loaded with 15 μg of soluble protein along with the protein isolated from the untransformed negative control strain TP309. Blots were examined with monoclonal antibodies prepared against HSA.

Claims (18)

A method for producing recombinant human blood protein in the seeds of monocotyledons,
(A) a monocotyledonous plant cell (i) a promoter derived from a gene of a ripening-specific monocotyledonous plant storage protein,
(Ii) encodes a monocotyledon seed-specific signal sequence that is operably linked to the promoter and allows the polypeptide linked to it to target the endosperm cells of monocotyledonous seeds 1 A second DNA sequence linked in a translation frame with the first DNA sequence and encoding a human blood protein;
The first DNA sequence and the second DNA sequence together are transformed with a chimeric gene encoding a fusion protein comprising an N-terminal signal sequence and a human blood protein,
(B) growing monocotyledonous plants from the transformed monocotyledonous cells for a time sufficient to produce seeds containing the human blood protein; and (c) harvesting the seeds from the plants. To
And wherein the human blood protein constitutes at least about 3.0% of the total soluble protein in the harvested seed.   The method of claim 1 wherein the human blood protein comprises at least about 5.0% of the total soluble protein in the harvested seed.   2. The method of claim 1 wherein the human blood protein comprises at least about 10.0% of the total soluble protein in the harvested seed.   The production method according to claim 1, comprising purifying the human blood protein from the harvested seeds.   The production method according to claim 1, wherein the human blood protein is selected from the group consisting of hemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin, thrombin, antibody and blood coagulation factor.   The production method according to claim 1, wherein the human blood protein produced by the method comprises one or more plant glycosyl groups.   A purified human blood protein obtained by the method according to claim 1, wherein the human blood protein contains one or more plant glycosyl groups.   The human blood protein according to claim 7, which is selected from the group consisting of hemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin, thrombin, antibody and blood coagulation factor.   A monocotyledon seed product selected from the group consisting of whole seed, flour, extract and malt prepared from harvested seed obtained by the method of claim 1, wherein said human blood protein is said seed product A seed product comprising at least about 3.0% of the total soluble protein therein.   The seed product of claim 9, wherein the human blood protein comprises at least about 5.0% of the total soluble protein in the seed product.   The seed product of claim 9, wherein the human blood protein comprises at least about 10.0% of the total soluble protein in the seed product.   10. The seed product of claim 9, wherein the human blood protein is selected from the group consisting of hemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin, thrombin, antibody and blood clotting factor.   A composition comprising a purified human blood protein comprising at least one plant glycosyl group and at least one pharmaceutically acceptable excipient or nutrient, wherein the human blood protein is a human blood protein. A composition produced in a monocotyledonous plant containing a nucleic acid sequence encoding and purified from seed harvested from said monocotyledonous plant, wherein said at least one nutrient is derived from a source other than said monocotyledonous plant .   14. The composition of claim 13, wherein the human blood protein is selected from the group consisting of hemoglobin, alpha-1-antitrypsin, fibrinogen, human serum albumin, thrombin, antibody and blood clotting factor.   14. The composition of claim 13, wherein said composition is substantially free of said monocotyledonous contaminants.   14. The composition of claim 13, wherein the at least one nutrient is selected from the group consisting of a salt, sugar, vitamin, mineral, amino acid, peptide, and protein other than the human blood protein.   14. The composition of claim 13, wherein the composition is formulated for parenteral, enteral, inhalation, nasal or topical delivery. 14. The composition of claim 13, wherein the composition is formulated for enteral delivery, contains at least one pharmaceutically acceptable excipient, and is non-nutritive.

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