JP2001512318A - Production of mature proteins in plants - Google Patents

Abstract

(57) SUMMARY Disclosed are methods of producing one of the following proteins in transgenic monocotyledonous plant cells: (i) mature glycosylated α1-antitrypsin (AAT), which is produced in humans. Mature glycosylated AAT having the same N-terminal amino acid sequence as mature AAT and a glycosylation pattern that substantially increases the half-life of serum over the half-life of mature non-glycosylated AAT; (ii) mature glycosylated antithrombin III (ATIII), a mature glycosylated ATIII having the same N-terminal amino acid sequence as mature ATIII produced in humans; (iii) mature human serum albumin (HSA), Fall of native mature HSA having the same N-terminal amino acid sequence and as evidenced by its bilirubin binding properties And (iv) mature active subtilisin BPN '(BPN'), which has the same N-terminal amino acid sequence as BPN 'produced in Bacillus. Monocot plant cells are transformed with a chimeric gene comprising a DNA coding sequence encoding a fusion protein having: (i) an N-terminal portion corresponding to the rice α-amylase signal sequence peptide; The protein portion immediately adjacent to the C-terminal amino acid corresponding to the mature protein to be produced.

Description

DETAILED DESCRIPTION OF THE INVENTION                     Production of mature proteins in plantsField of the invention   The present invention relates to the production of mature proteins in plant cells, and in particular to mature secreted forms. Regarding the production of proteins.Background of the Invention   The major commercial focus of biotechnology is industrial enzymes and important therapeutic applications Is a recombinant production of a protein containing both proteins having   Therapeutic proteins are commonly used in microbial expression systems (eg, E. coli systems and yeast). It is produced recombinantly by the strain S. cerevisiae). To date, the microbial host The cost of recombinant proteins produced in a variety of therapeutically important proteins (eg, For example, human serum albumin (HSA) and α₁Antitrypsin (AAT)) available Sex was limited to some extent by an inadequate supply of this protein.   Some therapeutic proteins are glycosylated for optimal activity or stability. Seems to depend on And, generally, microbial systems glycemic mammalian proteins. Because of the inability to cosylate or properly glycosylate, these recombinant expression systems Utility has also been limited. In some cases, a mammal-specific folder Or other folding conditions are required to ensure proper protein folding. I can't.   To some extent, cultured mammalian cells or transgenic animals Protein expression can overcome the limitations of microbial expression systems. However, the weight of the protein The cost of spill is still high in mammalian expression systems. And mammals Danger of protein contamination by irs can be a serious issue to be controlled . Protein production by transgenic animals can also occur from one generation to another. Risk of genetic change to The associated danger is that the recombinant Changes in protein, for example, resulting in naturally occurring active proteins with different N-terminal residues To Changes in protein processing.   Therefore, a tool that greatly overcomes the problems associated with microbial and mammalian cell systems. Produce selected therapeutic or industrial proteins in protein expression systems It is desired to do so. In particular, protein production is low cost, large capacity And should be properly processed and glycosylated Should allow the production of the engineered protein. Production systems are also relatively inexpensive for generations. Should have a certain genetic trait. These goals are based on the therapeutic protein AA T, HSA, and antithrombin III (ATIII) and the industrial enzyme subtilis This is achieved in the present invention for thin BPN '.Human α ₁ antitrypsin   Human α₁Antitrypsin (AAT) is a monomer having a molecular weight of about 52 kD. Through A normal AAT contains 394 residues and binds to asparagine 46, 83, and 247, and It has three complexed oligosaccharide units exposed on the surface (Carrell, P. et al., Nature (1 982) 298: 329).   AAT is mainly responsible for trypsin, elastase, and chymotrypsin in plasma. A major plasma proteinase in which regulates the proteolytic activity of Hibiter. In particular, this protein is a potent inhibitor of neutrophil elastase. Bitter, and AAT deficiency is observed in many patients with chronic emphysema Can be A proportion of patients with AAT serum deficiency develop cirrhosis and liver failure (Eg, Wu, Y. et al., BioEssays 13 (4): 163 (1991)).   Because of the important role of AAT as an elastase inhibitor, and for AAT serum levels AAT for human therapeutic use due to the prevalence of genetic disorders resulting in There is great interest in recombinant synthesis. To date, this approach has For the reasons discussed above, we are not satisfied with AAT produced by recombinant methods. He was not in good condition.Human antithrombin III   Antithrombin III (ATIII) is the major inhibitor of thrombin and factor Xa. Bitter, and to a lesser extent, produced during the clotting process Inhibitors of other serine proteases (eg, factor IXa, XIa Factor, and factor XIIa). The inhibitory effect of ATIII is dramatically enhanced by heparin Is done. Patients with a history of deep vein thrombosis and pulmonary embolism have ATIII deficiency. The morbidity is 2-3%.   ATIII proteins are useful for treating hereditary ATIII deficiency and When used in combination with phosphorus, for example, high risks such as surgery and labor For the prevention of thrombosis in concomitant situations and to treat acute thrombotic episodes There are a wide range of clinical applications.   ATIII is a glycoprotein having a molecular weight of 58,200 and 432 amino acids. Contains disulfide bonds and four asparagine-linked biantennary sugar chains . Due to the important role of ATIII as an antithrombin factor, and antithrombotic treatment Recombinant ATIII for human therapeutic use due to its broad clinical potential in therapy There is a great interest in success. To date, this approach has been discussed above. AA produced by microbial or mammalian recombinant methods for the reasons discussed I was not satisfied with TIII.Human serum albumin   Serum albumin is a major protein component of plasma. Its main function is blood The regulation of the colloid osmotic pressure in the stream. Serum albumin is Ca²⁺, Na⁺, K⁺,fat Numerous ions and acids, including acids, hormones, bilirubin and certain drugs Binds with small molecules.   Human serum albumin (HSA) is expressed as a 609 amino acid preproprotein. This means that one amino-terminal peptide and six additional amino acid residues Removal further processes it to form the mature protein. In human serum The mature protein found is a monomeric, 585 amino acid long (66 kDal) Uncosylated protein maintained by 17 disulfide bonds It has a spherical structure. The disulfide bond pattern is high affinity bilirubin One small disulfide-bonded and two large dissociation to form a joining site Form a structural unit of a sulfide-bonded double loop (Geisow, M.J. et al. (1977) Bioch em. J. 163: 477-484).   HSA is used to increase blood volume, and to reduce shock, trauma, and postoperative recovery. In some cases, it is used to raise low blood protein levels. HSA then It is often given in emergency situations to stabilize blood pressure.   Due to the important role of HSA as an osmotic stabilizer, and for example, plasma replacement therapy Of HSA for human therapeutic applications due to its broad clinical potential in Japan There is great interest in synthesis. This approach is discussed above For HSA produced by microbial or recombinant mammalian methods for I was not in a satisfactory state.Subtilisin BPN '   Subtilisin BPN '(BPN') is an industrial grade that is particularly important for use as detergent enzymes. It is an enzyme. Some groups include enzyme activity, optimal pH, stability and / or Reports amino acid substitution modifications of enzymes that are effective in enhancing therapeutic use I have.   BPN 'is expressed as a 381 amino acid preproenzyme and has 35 amino acids required for secretion. No acid sequence and a 77 amino acid moiety that acts as a chaperone to facilitate folding including. Studies have shown that this pro moiety acts extracellularly in trans ing.   To date, large-scale production of BPN 'has been mainly through microbial fermentation. This creature Relatively high costs are required for product fermentation. In addition, this enzyme It tends to degrade automatically in fermentation growth medium conditions.Summary of the Invention   In one aspect, the present invention relates to a monocot plant cell selected from the group consisting of: Including methods of producing the mature heterologous protein of choice: (i) maturation produced in humans N-terminal amino acid sequence identical to α1 antitrypsin (AAT), and non-glycosylated Glycosylated proteins increase serum half-life substantially beyond the serum half-life of mature AAT A mature glycosylated AAT having turns; (ii) a mature Mature glycoprotein having the same N-terminal amino acid sequence as anti-thrombin III (ATIII) (III) N-terminal amino acid sequence identical to mature HSA produced in humans Natural maturation as has a sequence and is evidenced by its bilirubin binding properties Mature HSA having a folding pattern of human serum albumin (HSA); and (iv) Baci has the same N-terminal amino acid sequence as subtilisin BPN '(BPN') produced in llus Mature, active BPN ', glycosylated or unglycosylated.   This method obtains a monocot plant cell transformed with a chimeric gene having the following: (I) by addition or removal of small molecules, or by seed maturation A monocotyledonous plant transcriptional regulatory region inducible there between; (ii) a first encoding a heterologous protein; And (iii) a second DNA sequence encoding a signal peptide. Second D The NA sequence is operably linked to the transcription regulatory region and to the first DNA sequence. No. One DNA sequence, together with the second DNA sequence, is in the translation frame, and the two Sequence encodes the fusion protein. Transformed cells induce transcriptional regulatory regions Cultivated under conditions effective to effect expression and expression of the fusion protein. And the secretion of mature heterologous proteins from transformed cells. Then the transformation This mature heterologous protein produced by the cell is isolated.   In one embodiment of this method, the first DNA sequence is prosubtilisin BPN '(proBPN'), and the culturing step comprises culturing the transformed cells at pH 5-6. The step of including and isolating automatically converts proBPN 'to active mature BPN' Incubating proBPN 'under conditions effective for Another embodiment In one embodiment, the first DNA sequence encodes mature BPN 'and the cell comprises (I) induction by addition or removal of small molecules (Ii) a third DNA sequence encoding the propeptide portion of BPN '; Sequence, and (iii) a fourth DNA sequence encoding a signal polypeptide. 4th DNA The sequence is operably linked to the transcription regulatory region and to a third DNA sequence. Soshi The signal polypeptide is in the translation frame with the propeptide portion, Effective for facilitating secretion of the propeptide moiety expressed from transformed cells is there. The culturing step involves culturing the transformed cells at pH 5-6, and Isolation step allows conversion of BPN 'to a more active mature BPN' by the pro moiety Involves incubating mature BPN 'and pro moieties under conditions effective for I do.   In another embodiment of the method, the signal peptide is a RAmy3D signal pair. It is a peptide (SEQ ID NO: 1) or a RAmy1A signal peptide (SEQ ID NO: 4). Shi The coding sequence of the signal peptide is, for example, the codon sequence identified as SEQ ID NO: 3. It can be a codon-optimized sequence such as an optimized RAmy3D sequence. The first DNA sequence also Codons can be optimized. Exemplary codon-optimized signal peptide-heterologous tamper 3D-AAT (SEQ ID NO: 18), 3D-ATIII (SEQ ID NO: No. 19), and 3D-HSA (SEQ ID NO: 20). The first DNA sequence is, for example, A heterologous protein, such as a codon-optimized sequence encoding 3D-proBPN '(SEQ ID NO: 21). One or more potential glycosylation sites present in the natural amino acid sequence of the protein It may further include codon substitutions that are eliminated.   In another embodiment of the method, the transcriptional regulatory region is RAmy1A, RAmy1B, RAmy1B. 2A, RAmy3A, RAmy3B, RAmy3C, RAmy3D, RAmy3E, pM / C, gKAmy141, gKAmy155, Am Promoters derived from rice or barley α-amylase gene, including y32b or HV18 Data. The chimeric gene contains a transcription regulatory region and a fusion protein coding region. Between the rice and barley α-amylase genes described above. It may further include a 5 'untranslated region (5'UTR) of a monocotyledonous plant gene. One preferred 5 'UTR is derived from the RAmy1A gene, which enhances gene transcript stability It is effective to do. The chimeric gene is located downstream of the coding sequence, Is derived from an inducible monocot gene such as one of the barley alpha amylase genes It may further include a 3 'untranslated region (3'UTR). One preferred 3 'UTR is the RAmy1A gene Origin.   This method is preferred when used for protein production in monocot cell cultures. A new promoter is the RAmy3D and RAmy3E gene promoters, In cell culture, it is upregulated by sugar depletion. This gene When used for protein production in bud seeds, a preferred promoter is RAmy1A Gene promoter, which is up-regulated by gibberellic acid during seed germination Regulated. Genes are up-regulated during seed maturation A preferred promoter is the barley endosperm-specific B1 hordein promoter. You.   The invention also includes the mature heterologous protein produced by the above method. This Is a glycosyl that is characteristic of monocotyledonous plants in which the protein is produced It has a chemical pattern. This glycosylation pattern is selected from the group consisting of: (I) N-terminal amino acid identical to mature α1 antitrypsin (AAT) produced in humans Serum halving of mature glycosylated and non-glycosylated mature AAT with no acid sequence Mature group with a glycosylation pattern that increases serum half-life substantially beyond the Lycosylated AAT; (ii) identical to mature antithrombin III (ATIII) produced in humans Mature glycosylated ATIII having the N-terminal amino acid sequence of: (iii) produced in Bacillus Glycosylated BP with an N-terminal amino acid sequence identical to subtilisin BPN ' N '.   The present invention provides a plant cell and a plant cell capable of producing a mature heterologous protein according to the above method. And seeds.   These and other objects and features of the invention are set forth in the following Detailed Description of the Invention. It will be more fully understood when read in conjunction with the accompanying drawings.                             BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 is used in the present invention, identified in the bottom row as SEQ ID NO: 1 The amino acid sequence of the signal sequence of RAmy3D is shown; The corresponding natural coding sequence identified in the upper row; and Shows the corresponding codon optimized sequence identified as No. 3;   FIG. 2 shows the components of the chimeric gene constructed according to an embodiment of the present invention;   3A and 3B show mature proteins in cell culture according to one embodiment of the present invention. Exemplary forms for use in transforming monocotyledonous plants to produce quality 1 shows the construction of a transduction vector (under the control of the RAmy3D promoter and signal sequence). Natural mature AAT coding sequence);   Figure 4 shows the factors of metabolic regulation of AAT production in rice cell culture;   FIG. 5 shows immunodetection of AAT using an antibody raised against the C-terminal region of AAT. You;   FIG. 6 shows AAT produced by transformed rice cell lines 18F, 11B, and 27F. Shows Western blot analysis of;   FIG. 7 shows elastase: AAT complex forms of human-produced form AAT and rice-produced form AAT. Showing the time course of the formation;   FIG. 8 is produced according to the present invention and is identified herein as SEQ ID NO: 22 Shows the N-terminal sequence of mature α1 antitrypsin (AAT);   FIG. 9 shows a Western blot of ATIII produced according to the present invention;   FIG. 10 compares expression from the codon-optimized sequence and the native coding sequence. Figure 5 shows a Western blot of plant-produced BPN ';   FIG. 11 shows BPN ′ codon optimization (AP106) versus BPN ′ native (AP101) in rice callus cell culture. Compare the specific activity of expression; and   FIG. 12 shows a Western blot of HSA produced in germinated seeds according to the present invention. You.                             A brief description of the array   SEQ ID NO: 1 is the amino acid sequence of a RAmy3D signal peptide;   SEQ ID NO: 2 is the native sequence encoding the RAmy3D signal peptide;   SEQ ID NO: 3 is the codon optimized sequence encoding the RAmy3D signal peptide ;   SEQ ID NO: 4 is the amino acid sequence of the RAmy1A signal peptide;   SEQ ID NO: 5 is the 5 'UTR from the RAmy1A gene;   SEQ ID NO: 6 is the 3′UTR from the RAmy1A gene;   SEQ ID NO: 7 is the amino acid sequence of mature α1-antitrypsin (AAT);   SEQ ID NO: 8 is the native DNA coding sequence for the sequence of mature AAT;   SEQ ID NO: 9 is the amino acid sequence of mature antithrombin III (ATIII);   SEQ ID NO: 10 is the native DNA coding sequence for the sequence of mature ATIII;   SEQ ID NO: 11 is the amino acid sequence of mature human serum albumin (HSA);   SEQ ID NO: 12 is the native DNA coding sequence for the sequence of mature HSA;   SEQ ID NO: 13 is the amino acid sequence of native pro BPN ';   SEQ ID NO: 14 is the native DNA coding sequence for pro-BPN ';   SEQ ID NO: 15 is the amino acid sequence of the "pro" portion of BPN ';   SEQ ID NO: 16 is the amino acid sequence of native mature BPN ';   SEQ ID NO: 17 shows that all strong N-glycosylation sites have been removed according to Table 2. The amino acid sequence of a mature BPN 'variant;   SEQ ID NO: 18 is a RAmy3D signal sequence / mature α1-antitrypsin fusion protein A codon-optimized sequence encoding a protein;   SEQ ID NO: 19 was fused to a native mature antithrombin III coding sequence RAmy3D signal sequence / mature ATIII fusion with codon optimized RAmy3D coding sequence Sequence encoding a protein;   SEQ ID NO: 20 is a codon optimized RAmy3 fused to a native mature HSA coding sequence RAmy3D signal sequence / mature human serum albumin fusion tan with D coding sequence A sequence encoding a protein;   SEQ ID NO: 21 is a RAmy3D signal sequence / prosubtilisin BPN ′ fusion protein A codon-optimized sequence encoding   SEQ ID NO: 22 is the N-terminus of mature α1-antitrypsin made in accordance with the present invention. End sequence;   SEQ ID NO: 23 shows the orientation used to prepare the intermediate p3DProSig construct of Example 1. Gonucleotides;   SEQ ID NO: 24 is the complement of SEQ ID NO: 23;   SEQ ID NO: 25 was used to prepare the intermediate p3DProSigENDlink construct of Example 1. Oligonucleotides;   SEQ ID NO: 26 is the complement of SEQ ID NO: 25;   SEQ ID NO: 27 shows the six amino acids used to prepare the intermediate p1AProSig construct of Example 1. One of the oligonucleotides of   SEQ ID NO: 28 shows the six amino acids used to prepare the intermediate p1AProSig construct of Example 1. One of the oligonucleotides of   SEQ ID NO: 29 is the six amino acids used to prepare the intermediate p1AProSig construct of Example 1. One of the oligonucleotides of   SEQ ID NO: 30 is the six amino acids used to prepare the intermediate p1AProSig construct of Example 1. One of the oligonucleotides of   SEQ ID NO: 31 shows the six amino acids used to prepare the intermediate p1AProSig construct of Example 1. One of the oligonucleotides of   SEQ ID NO: 32 shows the six amino acids used to prepare the intermediate p1AProSig construct of Example 1. One of the oligonucleotides of   SEQ ID NO: 33 is the N-terminus used for PCR amplification of the AAT coding sequence according to Example 1. A primer; and   SEQ ID NO: 34 is the C-terminus used for PCR amplification of the AAT coding sequence according to Example 1. Is a primer;                             Detailed description of the invention I.Definition:   The following terms have the following meanings unless otherwise indicated in the specification.   "Cell culture" is grown or suspended in a suitable growth medium , A cell and a population of cells, typically a callus cell.   "Sprouting" is the destruction of the dormant state of the seed and the effective Restoration of metabolic activity of seeds, including the production of enzymes that degrade.   "Inducible" is up-regulated by the presence or absence of small molecules Means a promoter. It includes both indirect and direct leads. No.   "Inducible during germination" is substantially silent prior to germination, but overall Not silent, but substantially during germination and emergence in the seed ( (Greater than 25%) A promoter that is turned on. Inducible during germination Examples of promoters are shown below.   "Small molecule" refers to small molecules, typically less than 1 kDal, in the context of promoter induction. They are organic matter or biological organic molecules. Examples of such small molecules are sugars, sugar derivatives (Including phosphate derivatives), and plant hormones (eg, gibberellic acid or Cisidic acid).   "Specifically regulatable" refers to, for example, an increase in the overall transcription within a cell by a small molecule. Contrary to non-specific effects, such as addition or reduction, one promoter or For transcription from a group of promoters (eg, the α-amylase gene family) Refers to the ability of small molecules to influence preferentially.   "Seed maturation" or "kernel development" is initiated by fertilization, where it can be metabolized Storage (eg sugars, oligosaccharides, starches, phenols, amino acids, And proteins), with or without targeting to vacuoles, Tissue (eg, endosperm, seed coat, aleurone layer, and scutellar epithelium) )) Accumulates, leads to grain expansion, grain filling, and ends by grain drying The period during which   "Inducible during seed maturation" is substantially active (greater than 25%) during seed maturation Promoter.   “Heterologous DNA” or “foreign DNA” refers to plant cells from another source or the same plant. Source of the plant, including the source of the product, but does not normally regulate the expression of the heterologous DNA. DNA introduced into plant cells under the control of a promoter or terminator Say.   "Heterologous protein" refers to a protein, including a polypeptide, encoded by heterologous DNA. It is protein. The “transcription regulatory region” or “promoter” influences the start of transcription. A nucleic acid sequence that affects and / or promotes. Promoters are typical Contains regulatory regions such as enhancer or inducer elements Conceivable.   A “chimeric gene” is, in the context of the present invention, typically a heterologous gene product. Encoding DNA sequence, eg, selectable marker gene or fusion protein gene A operably linked promoter sequence. Chimeric genes are also Transcriptional regulatory elements, such as transcription termination signals, and stop codons It may include translational control signals.   "Operably linked" functions as a unit for expressing a heterologous protein , A component of a chimeric gene or an expression cassette. For example, the protein ー The promoter operably linked to the heterologous DNA to be loaded To promote efficient mRNA production.   "Products" encoded by DNA molecules include, for example, RNA molecules and polypeptides. including.   "Removal" in the context of metabolites refers to depletion of metabolites through washing and absorption Physical removal, as well as metabolite metabolism by cells.   "Substantially isolated" is used in some contexts, and is typically Low protein or polypeptide to separate unrelated or mixed components At least refers to partial purification. Methods of isolating or purifying proteins or polypeptides And procedures are known in the art.   As used herein, "stably transformed" refers to a gene that has been transmitted through many generations. Cereal cells with foreign nucleic acid molecules stably integrated into their genome. Or plant.   “Α1-antitrypsin” or “AAT” is an AAT identified by SEQ ID NO: 7. A protein having an amino acid sequence substantially identical or homologous to a protein; Zein inhibitor.   "Antithrombin III" or "ATIII" is a combination of thrombin and factor Xa. ATIII inhibitor which is a palin activation inhibitor and is identified by SEQ ID NO: 9 A protein having an amino acid sequence substantially identical or homologous to a protein.   “Human serum albumin” or “HSA” is a component identified by SEQ ID NO: 11. A protein having an amino acid sequence substantially identical or homologous to the mature HSA protein Quality.   “Subtilisin” or “Subtilisin BPN” or “BPN” is a B.V. amyloliqu efaciens refers to a protease enzyme naturally produced, and Or a sequence homologous thereto.   "Pro-BPN '" is a chaperone polypeptide that assists in folding and activating BPN' Has a "pro" portion of about 78 amino acids that functions as a tide and Refers to a form of BPN 'having a sequence or a sequence homologous thereto.   "Codon optimization" converts native codons to optimal codons in the host plant Refers to a change in the coding sequence of a gene in order to replace it with a codon.   DNA sequence if it matches the sequence of a segment or region of that gene For example, "derived" from the rice or barley α-amylase gene. gene The segment of the gene, which may be derived, is the promoter region of the gene, the 5 'untranslated region. And the 3 ′ untranslated region.II . Transformed plant cells   The plants used in the process of the invention are of monocotyledonous origin, in particular Gramineae. Is a member of the taxonomic family known as. This family is edible The species includes all members of the herbaceous family, known as cereals. The grain is Wheat (Triticum sps.), Rice (Oryza sps.), Barley (Hordeum sps.), Barley (Avena sps.), Rye (Secale sps.), Corn (Zea sps) .) And millet (Pennisettum sps.). In the present invention In the above, preferred family members are rice and barley.   Plant cells or tissues from members of the family can be obtained using a variety of standard techniques (eg, For example, electroporation, protoplast fusion or microparticulation The expression construct (ie, the gene of interest is inserted) Transformed plasmid DNA). The expression construct comprises a transcription regulatory region ( Promoter). The transcription is performed on sugar (eg, in culture medium or in plant tissue ( The presence or absence of small molecules, such as, for example, a reduction or deficiency of sucrose in germinated seeds) Is specifically up-regulated by its absence. In the present invention, Cycle bombardment is a preferred transformation procedure.   The construct also comprises a mature heterologous protein in a form suitable for secretion from plant cells. And a gene encoding Genes encoding recombinant heterologous proteins Placed under the control of a specifically regulated promoter. Metabolically regulated Promoters inhibit mRNA synthesis or transcription by small or hormonal molecules. Or up-regulated promoters, such as rice RA my3D and RAmy3E promoters. These are caused by sugar deficiency in cell culture. It is regulated. In germinated seeds from regenerated transgenic plants For protein production of yeast, preferred promoters are dibe Ramy 1A promoter upregulated by rephosphoric acid. Departure The present constructs may also be used to facilitate efficient translation of AAT, as described below. Additional regulatory DNA sequences (eg, preferred codons, termination sequences) are utilized.   A.Plant expression vector   An expression vector for use in the present invention comprises a chimeric gene (or expression cassette). including. It is designed for operation in plants, upstream from the expression cassette and And downstream companion sequences. Companion sequences can be plasmid or And from the bacteria to the desired plant host in the vector. Provides the necessary features for the vector to be moved. A suitable transformation vector is No. PCT WO 95/14099, published May 25, 1995, which is hereby incorporated by reference. Which are incorporated herein by reference). Inducible promoter, sig A sequence encoding a null peptide, a sequence encoding a mature heterologous protein, and Suitable components of an expression vector containing a suitable termination sequence are discussed below. One An exemplary vector is the p3D (AAT) v1.0 vector illustrated in FIGS. 3A and 3B. A1.promoter   Transcriptional regulatory or promoter regions may be selected under selected culture conditions (eg, culture media). Sugar deficiency in nutrients or water uptake in budding seeds, followed by gibberellic acid production ) Is selected to be regulated in a manner that allows induction. The right promoter And their selected methods are described in detail in PCT application WO 95/14099, incorporated above. Have been. Examples of such promoters are the cereal α-amylase gene and And a promoter that transcribes the sucrose synthase gene, and Small molecules, sugar deficiency, or plant hormones (eg, gibberellic acid or abscisic acid) Abscissic acid). Representative professional The motor was rice α-amylase RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RA promoters from the my3C, RAmy3D, and RAmy3E genes, and pM / C, gKAmy1 41, gKAmy155, Amy32b, and HV18 barley α-amylase gene-derived pro Including motor. These promoters are, for example, ADVANCES IN PLANT BIOTEC HNOLOG YRyu, D.D.Y. et al., Eds., Elsevier, Amsterdam, 1994, p. Which is incorporated herein by reference). Other suitable promoters are rice Synthase and sucrose-6-phosphate from barley and barley Includes the setase (SPS) promoter.   Other suitable promoters are available in a manner to allow induction under seed maturation conditions. And regulated promoters. Examples of such promoters are: Conserved cotyledon proteins: rice glutelin, oryzin, and prolami , Barley hordein, wheat gliadin and glutelin, Maize zein and glutelin, oat glutelin, and sugar corn Koshkafilin (kafirin), millet penisetin (pennisetin), and rye giseca Includes promoters related to phosphorus (secalin).   A preferred promoter for expression in budding seeds is rice α-amylase RA my1A promoter (upregulated by siberellic acid). Fine A preferred promoter for expression in vesicle culture is rice α-amylase RAmy3D Promoter and α-amylase RAmy3E promoter (due to sugar deletion in culture) Strongly up-regulated). These promoters are also Active during bud. Preferred promoters for expression in mature seeds are , A barley endosperm-specific B1 hordein promoter (Brandt, A. et al., (1985) ) Carlsberg Res. Commun. 50: 333-345).   A chimeric gene further comprises an inducible singleton between the promoter and the coding sequence. 5 ′ untranslated region (5 ′ UTR) of a leaf plant gene (for example, the rice α-amylase gene described above) Or 5 'UTR from one of the barley alpha amylase genes). One good The 5'UTR is effective for RAmy1A gene to enhance the stability of gene transcription. The next 5'UTR. The 5 'UTR has the sequence provided by SEQ ID NO: 5 herein. Having. A2.Signal sequence   In addition to encoding the protein of interest, the chimeric gene Signal sequence (or signal peptide) that enables processing and transposition Code properly. Suitable signal sequences are described in PCT application WO 95/1409 referenced above. Described in 9. One preferred signal sequence is identified as SEQ ID NO: 1; It is derived from the Amy3D promoter. Another preferred signal sequence is as SEQ ID NO: 4 Identified and derived from RAmy1A promoter. Plant signal sequence is mature protein Arranged in frame with a heterologous nucleic acid encoding A fusion protein having a corresponding N-terminal region, and a signal peptide C-terminal amino acid, construction immediately adjacent to the N-terminal amino acid of mature heterologous protein Form an object. The expressed fusion protein is subsequently secreted and the signal peptide Is precisely processed by signal peptidase at the junction of Produce mature heterologous proteins.   In another embodiment of the present invention, the coding sequence in the fusion protein gene (less At least in the signal sequence coding region) is a plant cell as described below. (Eg, codon optimization for optimal expression in rice cells). Figure The upper row in 1 is the RAmy3D signal sequence identified herein as SEQ ID NO: 3. 1 shows one codon-optimized coding sequence. A3.Naturally occurring heterologous protein coding sequence   (i)α-antitrypsin: Mature human AAT is identified herein as SEQ ID NO: 7. Consists of 394 amino acids with a defined sequence. Protein is asparagine Have N-glycosylation sites at positions 46, 83, and 247. Corresponding natural DNA code The nucleotide sequence is identified herein as SEQ ID NO: 8.   (ii)Antithrombin III: Mature human ATIII is referred to herein as SEQ ID NO: 9. Consists of 432 amino acids having the sequence identified. This protein has four It has N-glycosylation sites at sparagine residues 96, 135, 155, and 192. Correspondence The natural DNA coding sequence to be identified is identified herein as SEQ ID NO: 10.   (iii)Human serum albumin: The mature HSA as found in human serum has the sequence Consists of 585 amino acids having the sequence identified herein as No. 11. this The protein has no N-linked glucosylation sites. Corresponding natural DNA coding sequence Is identified herein as SEQ ID NO: 12.   (iv)Subtilisin BPN ': B. amyloliquefaciens BPN 'is a 352 amino acid having the sequence identified herein as SEQ ID NO: 13 Consists of acids. The corresponding natural DNA coding sequence is referred to herein as SEQ ID NO: 14. Identified. The pro-BPN 'polypeptide has been identified herein as SEQ ID NO: 15. It contains a "pro" portion of 77 amino acids. The remainder of the polypeptide (forming the mature active BPN ' Is the amino acid sequence of 275 identified herein by SEQ ID NO: 16 . Native BPN 'as produced in Bacillus is not glycosylated. A4. Codon-optimized coding sequence   According to one aspect of the invention, a predominantly or exclusively found in a plant cell host. Alter the native coding sequence of a heterologous gene by including the most frequent codons By varying, a several-fold enhanced expression level can be achieved in plant cell culture. Has been discovered.   This method has been demonstrated for the expression of heterologous genes in rice plant cells, It is recognized that it is generally applicable to any monocotyledonous plant. First steps and Thus, a representative set of known gene coding sequences from rice is assembled. Next The sequence was analyzed for codon frequency at each amino acid, and Frequent codons are selected for each amino acid. This approach Is different from the codon matching method reported earlier, Frequent codons are selected for at least some amino acids. Rice plant The optimal codons selected in this manner for and barley are shown in Table 1.   As described above, the sequence encoding the fusion protein in the chimeric gene is The final (C-terminal) codon in the sequence is the N-terminal amino acid in the mature form of the heterologous protein. It is constructed to directly follow the acid codon. In accordance with the present invention, an exemplary fusion tan The protein gene is identified herein as follows:   SEQ ID NO: 18, which is from the RAmy3D signal sequence / mature α1-antitrypsin Corresponding to the codon-optimized coding sequence of the fusion protein;   SEQ ID NO: 19, which contains the codon optimized RAmy3D signal sequence and the native mature Corresponds to a fusion protein coding sequence consisting of a Tithrombin III sequence;   SEQ ID NO: 20, which is a codon optimized RAmy3D signal sequence and native mature human Corresponds to a fusion protein coding sequence consisting of a serum albumin sequence;   SEQ ID NO: 21, which is the fusion protein RAmy3D signal sequence / prosbutyridine It corresponds to the codon-optimized coding sequence of BPN '. In this case, prosubtilisin is "Ripe" protein is considered secreted prosubtilisin, where active mature subtilis Can autocatalyze to tilisin.   In a preferred embodiment, the sequence encoding BPN 'is a native BPN' And is further modified to remove strong N-glycosylation sites. You. Table 2 removes all strong N-glycosylation sites in subtilisin BPN ' Shows preferred codon substitutions. SEQ ID NO: 17 comprises the substitutions provided in Table 2. Corresponds to mature BPN 'amino acid sequence. ^1.Improved thermostable; Bryan et al., Proteins: Structure, Function, and Genet ics1: 326 (1986). A5.Transcription and translation terminators   Chimeric genes can also be derived from inducible monocot genes (eg, For example, as described above, one rice α-amylase gene or barley α-amylase gene Offspring) from the 3 'untranslated region (3'UTR). One preferred 3 'UTR is the RAmy1A gene (This sequence is provided by SEQ ID NO: 6). This array is Non-coding sequence 5 'to polyadenylation site, polyadenylation site, and transcription Including termination sequence. Transcription termination region is important for mRNA stability to enhance expression In particular it can be chosen. Polyadenylation tail (Alber and Kawasaki, 1982, Mol. An d Appl. (Genet. 1: 419-434) also states that to optimize high levels of transcription, And each are generally added to the expression cassette for proper transcription termination . The polyadenylation sequence is the Agrobacterium octopine synthetase signal (Gielen EMBO J. et al. 3: 835-846 (1984)), or a homologous nopaline synthase (Depicker et al.). , Mol. Appl. Genet. 1: 561-573 (1982)).   The ultimate expression of the heterologous protein is determined by eukaryotic cells (in this case, grass family families). As such, any site in the cloned gene may be Whether to include sequences that are processed as introns by the pricing mechanism It is desired to determine In that case, site-specific abrupt “intron” region Mutagenesis prevents deletion of some of the genetic messages as a pseudo-intron code (Reed and Maniatis, Cell41: 95-105 (1985)).   FIG. 2 shows a tandem cell suspension culture constructed according to the present invention. One preferred chimeric gene element specifically contemplated for use in protein expression Is shown. This gene is a promoter derived from the RAmy3D gene in the 5 'to 3' direction. -(Can be induced with sugar deficiency in cell culture), 5 'UTR from RAmy1A gene Encodes the RAmy3D signal sequence (confers enhanced stability to gene transcripts) Region (as identified above), the coding region of the heterologous protein to be produced, And the 3'UTR region from the RAmy1A gene. III.Plant transformation   For plant transformation, a chimeric gene was engineered for use in plants. In an expression vector. Vectors transfer DNA from bacteria to the desired plant host Plasmids that provide the necessary properties to the vector to enable it to Or contains appropriate elements of viral origin. A suitable transformation vector is The related PCT application WO 95/14099 (May 25, 1995, incorporated herein by reference). Published). Appropriate construction of the expression vector containing the above chimeric gene The components are discussed below. One exemplary vector is p3Dv1. Described in Example 1. 0 vector. A.Transformation vector   The vector containing the chimeric gene of the present invention can also be used for plant cells. Selectable markers (eg, np for selection in media containing kanamycin) In a medium containing the tII kanamycin resistance gene or phosphinothricin (PPT) Phosphinothricin acetyltransferase gene for selection I can do it.   Vectors also contain an origin of replication, and antibiotic and herbicide resistance genes (eg, HPH (Hagio et al., Plant Cell Reports14: 329 (1995); van der Elzer, Plant Mo l. Biol.Five: 299-302 (1985)). Sequences that allow for their selection and propagation in two hosts may be included. representative Typical second hosts include bacteria and yeast. In one embodiment Thus, the second host has an origin of replication of type colE1 and a selectable marker of Escherichia coli which is a gene encoding a sylin resistance gene. like this Sequences are well known in the art and are also commercially available (Eg, Clontech, Palo Alto, CA; Stratagene, La Jolla, CA)   The vector of the present invention also contains a region homologous to the Agrobacterium tumefaciens vector. , A T-DNA border region from Agrobacterium tumefaciens, and a chimeric gene or Can be modified into an intermediate plant transformation plasmid containing the expression cassette (described above). You. Furthermore, the vectors of the present invention have been neutralized for Agrobacterium tumefaciens. It may contain a plant tumor inducible plasmid.   Vector described in Example 1 and having a promoter from the RAmy3D gene -Cell culture, where the RAmy3D promoter is induced by sugar deficiency in the cell culture medium Suitable for use in a method of producing a mature protein in a plant. Other Promos Data may also be selected for the other uses described above. For example, mature tags in germinating seeds For protein expression, the coding sequence was induced by gibberellic acid during seed germination. Can be placed under the control of the rice α-amylase RAmy1A promoter. B.Transformation of plant cells   Plant cells (eg, plant protoplast cells) directly or by vector Various methods of transformation have been described (eg, in the PCT application cited above). WO 95/14099). Used for plant transformation as described in references Promoters that direct the expression of a selectable marker (eg, nptII) are useful in plant hosts. Should work effectively. One such promoter is native Ti Nos promoter derived from plasmid (Herrera-Estella et al., Nature,303: 209-213 ( 1983)). In addition, the 35S and 19S promoters of cauliflower mosaic virus (Odell et al., Nature313: 810-812 (1985)) and the 2 'promoter (Velten et al.). , EMBO J.Three: 2723-2730 (1984)).   In one preferred embodiment, for exposing scutulum tissue cells Germ cells and endosperm of mature seeds are removed. Cells can be bombarded or injected with DNA or Or vector transformation (for example, cells are susceptible to Agrobacterium infection). Agrobacteriu after blastocyst cells were bombarded with microparticles m infection (Bidney et al., Plant Mol. Biol.18: 301-313,1992)) obtain.   One preferred transformation is generally described in Sivamani, E. et al., Plant Cell Reports.1 Five : 465 (1996); Zhang, S. et al., Plant Cell Reports.Fifteen: 465 (1996); and Li, L. et al. , Plant Cell Reports12: 250 (1993). In short, Rice seeds are sterilized by standard methods and callus induction from seeds includes 2,4D Perform on MB medium. During the first incubation period, callus tissue is transformed into seed embryos. Formed around. End of incubation period (eg, 14 days at 28 ° C) By then the callus is about 0.25-0.5 cm in diameter. The callus mass is then seeded More detached, place on fresh NB medium and incubate again at 28 ° C for 14 days Work. After the second incubation period, the satellite callus is It occurs around the "mother" callus mass. These satellite culls Is slightly smaller, more dense, and more circular than the original tissue Guo is clear. It is these calli that are transferred to fresh medium. The "mother" callus is not transferred. The goal is to have the strongest and most The only choice is to select vigorous proliferating tissue.   Bombarded calli were selected from 14 day old subcultures. Size, form, color and And density are all important when selecting calli under optimal physiological conditions for transformation. is important. Calli should be between 0.8-1.1 mm in diameter. Callus is coarse It should appear as a spherical mass with an outer surface.   Transformation is performed by particle bombardment, as detailed in the references cited above. . After the transformation step, the cells typically allow expression of the selectable marker gene. Grown under different conditions. In a preferred embodiment, the selectable marker gene is HPH is there. To produce a uniformly and stably transformed cell line, the transformed cells are It is preferable to culture under multi-stage selection. IV.Cell culture production of mature heterologous proteins   Transgenic cells (typically callus cells) support plant cell growth Cells until they reach the desired cell density, and then Cultured under conditions supporting expression of the mature protein under the control of the promoter. Preferred culture conditions are described below and in Example 2. Mature secreted into the medium Purification of the protein is by standard techniques known to those skilled in the art.   Production of mature AAT: In a preferred embodiment, the culture medium is catalyzed by a metal. In order to reduce the degradation of AAT, a phosphate buffer (eg, 20 mM phosphate buffer p H6.8, described in Example 2). Or, or in addition, for example, EDTA Such a metal chelating reagent may be added to the medium.   Following the cell culture method described in Example 2, the cell culture medium was partially purified and As shown in 4, the fractions containing AAT were analyzed by Western blot. First two records ("Phosphate") in the presence and absence of elastase ("+ E" and A-A bands in both "-E") are shown. In this case, the presence of elastase Lower high molecular weight band corresponds to approximately 58-59 kdal AAT / elastase complex I do. Also, as shown in the figure, the expression was high in the absence of sucrose, Almost undetectable in the presence.   Check the degree of glycosylation (determined by apparent molecular weight on SDS-PAGE) For this purpose, the proteins produced in culture were fractionated by SDS-PAGE and shown in FIG. As described above, immunodetection was performed with a labeled antibody produced against the C-terminal portion of AAT. lane 4 contains human AAT and its movement position corresponds to about 52 kdal. Lane 3 , AAT produced in plants, having an apparent molecular weight of about 49-50 kdal, Degree of glycosylation indicates that it reaches 60-80% of the glycosylation found in human AAT (The molecular weight of unglycosylated AAT is 45 kdal).   Similar results are shown in the Western blot of FIG. Lanes 1-3 in this figure are To reduced amounts (15, 10, and 5 ng) of human AAT; lane 4 shows non-expressing plant cells In 10 μl of supernatant from the strain; lanes 5 and 6 show AAT expressing plant cell lines 11B and And lane 7 corresponds to 250 ng of trypsin. This corresponds to 10 μl of the supernatant derived from the cell line 27F to which the enzyme was added. Of the upward mobility of lane 7 Changes indicate an association between trypsin and AAT produced in the plant.   The ability of plant-generated AAT to bind elastase is shown in FIG. Figure 7 shows 52k 30 minutes of dal human AAT (lanes 1-4) and AAT produced in 49-50 kdal plants 3 shows the molecular weight change over the binding interval.   Demonstrates that the mature protein is produced as a secreted form with the desired N-terminus To construct, as described above, and mature α₁Contains the coding sequence for antitrypsin The resulting chimeric gene was expressed and secreted into cell culture as described in Example 2. Next, the N-terminal region of the isolated protein was sequenced, and the N-terminal region shown in FIG. The sequence was obtained. This sequence, which is identical to SEQ ID NO: 22 herein, α₁It has the same N-terminal residue as antitrypsin.   Production of mature ATIII: In a preferred embodiment, the culture medium is MES buffer, pH 6.8 It contains. The produced ATIII protein shown in lanes 4 and 6 of FIG. Western blot analysis shows growth in the absence (but not the presence) of sucrose In the cell lines 42 and 46, a band corresponding to ATIII (lane 1) was shown.   Production of mature BPN ': One embodiment of the present invention wherein BPN 'is secreted as the pro-BPN' form of the enzyme In embodiments, the chaperone "pro" portion of the enzyme is responsible for folding of the enzyme. Promotes and is excised from the enzyme leaving the active mature BPN '. Another embodiment In one embodiment, the enzyme is converted to the active form that occurs in the presence of the free chaperone ( Eder et al., Biochem. (1993)32: 18-26; Eder et al., (1993) J. Am. Mol. Biol.223: 293- 304), the mature enzyme is co-expressed with the “pro” chaperone moiety and co-secreted You. In yet another embodiment of the present invention, BPN 'may have a pH in the range of 6-8. In some cases, it is secreted as an inactive form, followed by activation of the inactive form (eg, For example, after isolation of the enzyme, exposure to a "pro" By immobilization on a support).   In both of these embodiments, during the period of active expression and secretion of BPN ', The nutrient medium is maintained at a pH between 5 and 6, preferably at about 5.5, and is usually alkaline pH. Keep the BPN 'active at a pH below the optimal activity.   Optimizing codons for the most frequent codons in the host plant is shown in FIG. As shown in the figure, several fold increase in the level of heterologous protein expressed in cell culture Brought strength. The extent of the enhancement can be seen from Western blot analysis (two detailed The cell line is shown in FIG. 10 and further demonstrated in FIG. 11). Lanes in FIG. 2 (second from left) is from cells transformed with native proBPN 'coding sequence 5 shows a western blot of BPN 'obtained in the culture of Example 1. Two bands observed Corresponds to a low molecular weight protein with a molecular weight of approximately 35 kdal, which corresponds to the molecular weight of pro-BPN ' You. The upper band is probably associated with a glycosylated, somewhat higher molecular weight species. Hit.   The first lane in the figure shows the codon-optimized proBPN 'sequence identified by SEQ ID NO: 21. 2 shows BPN 'polypeptide produced in culture with transformed plant cells. ratio For comparison purposes, the same volume of culture medium, prepared for cell density, was And 2 were added. As shown, the codon-optimized sequence produced BPN 'enzyme amount is less than subtilisin BPN' produced in the native coding sequence. It was twice as expensive. In addition, corresponds to a dark band or mature peptide (molecular weight 27.5 kdal) A band was observed. However, the 35kD band directly above was cut into more active forms. Note that this is a more pronounced band, which may be a premature product .   FIG. 11 shows codon-optimized BPN '(AP106) vs. native in rice callus cell culture. Specific activity of the expressed BPN '(AP101) expression was determined by DelMar, E. et al. G. et al., (1979; Anal. Biochem. 99: 316-320) using the chromogenic peptide substrate suc-Ala-Ala-Pro-Phe-pNA Assay and compare. As shown in FIG. 11, a codon-optimized chimeric gene Some of the cell lines transformed in As demonstrated, it was observed in plant cells transformed with the native proBPN 'sequence. 2-5 times the highest level of BPN 'produced.   According to another aspect of the invention, the transformed plant cell culture comprises a cell culture having a mature pH. It is capable of expressing and secreting BPN 'at pH 5.5, which greatly inhibits autolysis of active BPN'. Was found. To assay for optimal pH conditions, the BPN ' For testing cultures from transformed cell lines, disclosed by DelMar (supra) et al. The assay (supra) was used. The transformed rice callus cells were disclosed in Example 2. Under similar conditions (but the pH of the medium was maintained at the selected pH between 5 and 8.0) ) In MES medium. At each pH, the total amount of BPN 'expression and secretion is Was determined by Western blot analysis. BPN 'activity was described by DelMar Can be tested by the assay described above (supra). V.Production of mature heterologous proteins in germinating seeds   In this embodiment, the monocotyledon cells transformed as described above, Used to grow, plant seeds are harvested, then emerged and matured Protein was isolated from germinated seeds.   Regeneration of plants from cultured protoplasts or callus tissue is performed using standard methods (eg, For example, Evans et al.HANDBOOK OF PLANT CELL CULTURESVolume 1: (MacMillan Publishi ng Co. New York, 1983); and Vasil I.R. (Ed)CELL CULTURE AND SOM ATIC CELL GENETICS OF PLANTS Acad. Press, Orlando, Volume I, 1984, and (As described by Vol. III, 1986), and in the PCT application cited above. Performed as described. A.Seed germination conditions   Transgenic seeds from regenerated plants are harvested and first infiltrated (Steeping) process (where the seeds soaked or sprayed with water are treated with seed water (The content is increased to between 35-45%). this The process initiates germination. Infiltration typically causes the seeds to flow freely Where possible, it is typically done in an infiltration tank equipped with a conical end . Adding compressed air to add oxygen to the infiltration process is an option. Warm The degree is controlled at about 22 ° C., depending on the seed.   After infiltration, the seeds contain air saturated with moisture, and have a controlled temperature and It is transferred to a germination compartment under air flow. Typical temperature is 12-25 ° C And germination is continued for 3-7 days.   The gene encoding the heterologous protein is produced by metabolic production such as sugar or plant hormones (Eg, 2-100 μM gibberellic acid). When operably linked, this metabolite can be added, removed or depleted to the infiltration medium. Thirst and / or added to air saturated with moisture used during germination You. Seeds absorb the aqueous medium and begin to germinate, expressing heterologous proteins. The media can then be recovered and the seeds can be placed in a controlled humidity and temperature aerated environment. By maintaining, malting can be initiated. In this way, Since the seed can begin to grow prior to expression, the expression product may be partially Less likely to be degraded or denatured.   More specifically, the temperature during the absorption or infiltration phase was kept in the range of about 15-25 ° C On the other hand, the temperature during germination is usually about 20 ° C. Absorption time is usually about 1 to 4 days Yes, while germination time is usually an additional 1-10 days, more usually 3-7 days . Normally, the malting time does not exceed about 10 days. During the malting period, Plant hormones (especially gibberellic acid) can be used during harvesting and shortened.   To achieve maximum production of recombinant protein over malting, The order is to remove the hull as described in PCT application WO 95/14099 cited above. And can be modified to accommodate seeds from which embryos have been removed. Of endosperm-derived sugar In the absence of RAmy3D promoter activity, and therefore expression of the heterologous protein, It is expected to rise 5 to 10 times. Alternatively, half of the seeds without embryos are Cl_TwoAnd 5 μM gibberellic acid incubate RAmy1A promoter activity. Sex increases by 50 times.   Production of mature HSA: Germination as outlined above and in more detail in Example 3 According to the conditions, the supernatant was analyzed by Western blot. Western blot The analysis shows HSA production in germinating rice seeds (seed samples 24, 72, and 120 hours after induction with gibberellin). HSA production induced Approximately 24 hours later, it was highest (FIG. 12, lanes 3 and 4). Biliru bin) (measuring the correct folding of HSA produced in plants) is described in Example 3. Assayed according to the indicated method. VI.Production of mature heterologous proteins in maturing seeds   In this embodiment, the monocotyledon cells transformed as described above Seeds from this plant and the resulting seeds It allows maturation (typically in the field) with production of the seed protein.   For example, it is intended that the heterologous protein as a whole provides benefits to the seed ( For example, enriching a seed with a selected protein), following seed maturation, The offspring and its heterologous protein are used directly, i.e., without protein isolation. Can be   Alternatively, the seed is enriched or purified by standard methods. Can be fractionated to obtain a heterologous protein. One general approach First, crush the seeds and then extract the desired protein or metabolite Seeds in a suitable extraction medium (eg, aqueous or organic solvent) I do. If desired, heterologous proteins can be further purified using standard purification methods. Can be fractionated and purified.   The following examples are provided by way of illustration, not limitation. Business Can be changed or modified to yield essentially similar results A variety of non-critical parameters are easily recognized.General method   In general, nomenclature and laboratory procedures for standard recombinant DNA techniques are: Sambrook et al.MOLECULAR CLONING-A LABORATORY MANUAL, Cold Spring Harbor La boratory, Cold Spring Harbor, New York 1989 and S.B. Gelvin and R.A . Schilperoot,PLANT MOLECULAR BIOLOGY, 1988. Other common References are provided throughout this document. The procedure herein is It is known in the art and is provided for the convenience of the reader.Example 1 The α ₁ antitrypsin sequences to optimize the codon Of transformation vector containing A.Insertion of hygromycin resistance gene   A 3 kb BamHI fragment containing 35S promoter-Hph-NOS was transferred to plasmid pMON410. (Monsanto, St. Louis, MO) Plasmid pUCH18 + was placed at the BglII site at position 63. B.Inserting a terminator   pOSg1ABK5 was cloned into pBluescript KS- (Stratagene, San Diego, CA). Lambda clone λOSg1A (Huang, N., et al. (1990) Nuc. Acids Res. 18: 7007) This is a 5 kb BamHI-KpnI fragment derived from the present invention. Digest plasmid pOSg1ABK5 with MspI And blunt-ended with T4 DNA polymerase, followed by digestion with SpeI. 350bp Digested with BamHI and blunted with T4 DNA polymerase And pUC19 (New England BioLabs, Beverly, MA) digested with XbaI. It was cloned to form pUC19 / terminator. C.RAmy3D Promoter insertion   1.1 kb from p1AS1.5 (Huang, N. et al. (1993) Plant Mol. Biol. 23: 737-747). The NheI-PstI fragment was transferred to the vector pGEM5zf- [Multiple Cloning Site (MCS) (Prome ga, Madison, WI): ApaI, AatII, SphI, NcoI, SstII, EcoRV, SpeI, NotI, PstI , SalI, NdeI, SacI, MluI, NsiI] and cloned into the SpeI and PstI sites, and pGEM 5zf- (3D / NheI-PstI) was formed. Then, pGEM5zf- (3D / NheI-PstI) was converted to PstI and Digest with SacI and the complementary sequence, 5 ′ GCTTG ACCTG TAACT CGGGC CAGGC GAGCT 3 '(SEQ ID NO: 23) and 5' CGCCT AGCCC GAGTT ACAGG TCAAG CAGCT3 '(SEQ ID NO: 24) Two kinase-untreated 30-mers having the following were ligated into it to form p3DProSig . NcoI digestion of p3DProSig, blunting with T4 DNA polymerase, and Ss The promoter fragment prepared by tI digestion is digested with EcoRI, and T4 DNA Subcloned into pUC19 / terminator digested with Merase and SstI digested. To form p3DProSigEND. D.Insertion of multiple cloning sites   After digestion of p3DProSigEND with SstI and SmaI, the untreated kinase Reotide (5 'AGCTC CATGG CCGTG GCTCG AGTCT AGACG CGTCC CC 3' (SEQ ID NO: 25) And 5 'GGGGA CGCGT CTAGA CTCGA GCCAC GGCCA TGG 3' (SEQ ID NO: 26)) The new synthetic linker fragment constructed in Step 3 to form p3DProSigENDlink. Done. E. FIG.p3DProSigENDlink Modification of lateral part   After digesting p3DProSigENDlink with SalI and blunting with T4 DNA polymerase , Digested with EcoRV. Next, a HindIII site is located proximal to the promoter sequence, The blunt fragment so that the EcoRI site is proximal to the terminator sequence. And pBluescript KS + (Stratagene) at the EcoRV site. Then, HindIII-EcoR Transfer the I fragment into the pUCH18 + polylinker to form the p3Dv1.0 expression vector did. F.RAmy1A Promoter insertion   From lambda clone λOSg2 (Huang et al., (1990) Plant Mol. Biol. 14: 655-668). 1.9 kb NheI-PstI fragment from subclone pOSG2CA2.3 of SpeI and P Cloned into the vector pGEM5zf- at the stI site to form pGEM5zf- (1A / NheI-PstI) Was. pGEM5zf- (1A / NheI-PstI) is digested with PstI and SacI and the two kinases The untreated 35-mer and the four kinase-treated 32-mers were ligated with their complementary sequences as follows: , P1AProSig: 5 ′ GCATG CAGGT GCTGA ACACC ATGGT GAACAAACAC 3 ′ Column number 27); 5 'TTCTT GTCCC TTTCG GTCCT CATCG TCCTC CT 3' (SEQ ID NO: 28); 5 'T GGCC TCTCC TCCAA CTTGA CAGCC GGGAG CT 3 '(SEQ ID NO: 29); 5' TTCAC CATGG TGT TC AGCAC CTGCA TGCTG CA 3 '(SEQ ID NO: 30); 5' CGATG AGGAC CGAA A GGGAC AAGAA GTGTT TG 3 ′ (SEQ ID NO: 31); 5 ′ CCCGG CTGTC AAGTT GGAGG AGAGG CCAAG GAGGA 3 '(SEQ ID NO: 32). HindIII-SacI 0.8kb promoter fragment Was subcloned from p1AProSig into HindIII-SacI digested p3Dv1.0 vector Thus, a p1Av1.0 expression vector was obtained. G. FIG.p3D-AAT Plasmid construction   According to the sequence disclosed as Genebank Accession No. Two PCR primers were used to amplify the fragment: N-terminal primer -5 'GAGGA TCCCC AGGGA GATGC TGCCC AGAA 3' (SEQ ID NO: 33) and C-terminal primer Mer 5 'CGCGC TCGAG TTATT TTTGG GTGGG ATTCA CCAC 3' (SEQ ID NO: 34). N-terminal Primers are blunt for in-frame insertion with the end of the p3D signal peptide Position and the C-terminal primer was used as shown in FIGS. 3A and 3B. Includes an XhoI site for cloning the fragment into the vector. Or, The sequence encoding mature AAT (SEQ ID NO: 8) or codon-optimized AAT is described above. An XhoI restriction site was incorporated 3 ′ to the termination codon for insertion into the expression vector, It can be chemically synthesized using techniques known in the art.                                 Example 2 Production of mature α-antitrypsin in cell culture   After selection of transgenic calli, callus cells were transferred to AA2 medium (Thompson, J. .A., Et al., Plant Science47: 123 (1986), liquid culture containing 3% sucrose, pH15.8) Suspended in it. The cells were then transferred to a 10 mL multi-well tissue culture plate. Transfer to a phosphate buffered medium (20 mM phosphate buffer, pH 6.8) Shaking at 120 rpm for 48 hours. After that, remove the supernatant and Western Blotting Before analysis, they were stored at -80 ° C.   The supernatant was purified using a Centricon-10 filter (Amicon cat. # 4207). Concentrated and washed with induction medium to remove substances that interfere with electrophoretic migration. Samples were concentrated approximately 10-fold and mature AAT was purified by SDS PAGE electrophoresis. Purifier The protein was extracted from the electrophoresis medium and its N-terminal sequence was determined. The sequence shown in FIG. 8 identified as SEQ ID NO: 22 in the description is shown.                                 Example 3 HSA induction in germinating seeds   GA_Three-Codon-optimized HSA gene driven by responsive RAmy1A promoter Seeds are harvested after selection of transgenic plants that test positive for the presence of Then, in a dark place at 25 ° C., water was absorbed while being rotationally shaken at 100 rpm for 24 hours. GA_ThreeThe final concentration 5 It was added to μM and incubated for a further 24-120 hours. 120 μl grinding buffer All the lysates are crushed twice and centrifuged at 23,000 xg at 4 ° C for 1 minute to obtain all soluble Sex protein was isolated. Carefully remove the clear supernatant from the pellet and remove Transferred to tube.   Bilirubin binding assay   Bilirubin binding to its high affinity site in mature HSA was determined by Jacobsen, J. et al. (1974; Clin. Chem. 20: 783) and Reed, R.G. (1975; Biochemistry 14: 4 Assay using the method described in 578-4583). In short, protein The concentration of free bilirubin in equilibrium with bound bilirubin is determined by peroxide Determined by the rate of oxidation of free bilirubin catalyzed by oxidase. Biril Bottle (Nutritional Biochemicals Corp.) stock solution with 5 mM water Prepared in 1 mM EDTA containing sodium oxide and concentration was 47,500 M at 440 nm^-1cm^-1 Determined using the molar absorbance of Alico containing between 5 and 30 nmol bilirubin The plate was added at 37 ° C. to an lcm cuvette containing 1 ml of PBS and about 30 nmol HSA. You. The absorption spectrum between 500 and 350 nm is recorded. Horseradish peroxida (Sigma) (0.05 mg / ml in PBS) and 0.05% Recoat is added and the change in absorbance at λmax is recorded for 3-5 minutes. Free and calculated as calculated from oxidation rates observed using different total bilirubin concentrations. Scatchard plots were made using the concentrations of From this, the association constant at the single binding site is determined.   Although the present invention has been described with respect to particular embodiments, without departing from the invention, , It is understood that many changes and modifications can be made.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme court ゛ (Reference) C12R 1:91) C12N 5/00 C (31) Priority claim number 60 / 038,169 (32) Priority date February 13, 1997 (Feb. 13, 1997) (33) Priority claim country United States (US) (31) Priority claim number 60/038, 170 (32) Priority date February 13, 1997 ( (Feb. 13, 1997) (33) Priority country United States (US) (81) Designated country EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU , MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS) , MW, SD, SZ, UG, Z ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU , CZ, DE, DK, EE, ES, FI, GB, GE, GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR , TT, UA, UG, US, UZ, VN, YU, ZW Transformed with the gene: (i) an N-terminal portion corresponding to the rice α-amylase signal sequence peptide, and (i iii) C.) Immediately adjacent to the C-terminal amino acid of said peptide, Protein portion corresponding to that the mature protein.

Claims (1)

[Claims] 1. Less than:   (I) mature glycosylated α 1 -antitrypsin (AAT) The same N-terminal amino acid sequence as mature AAT produced and the half-life of serum Has a glycosylation pattern that substantially increases the half-life of glycosylated AAT Mature glycosylated AAT;   (Ii) mature glycosylated antithrombin III (ATIII), which Mature glycosylated ATIII having the same N-terminal sequence as mature ATIII produced in   (Iii) mature human serum albumin (HSA) produced in humans Has the same N-terminal amino acid sequence as mature HSA and its bilirubin binding properties. Has the folding pattern of natural mature HSA as evidenced by Mature HSA; and   (Iv) Mature, active subtilisin BPN '(BPN'), which is odorant to Bacillus Active subtilis having the same N-terminal amino acid sequence as BPN 'produced BPN '; Mature heterologous proteins selected from the group consisting of A method comprising:   (A) (i) by addition or removal of small molecules or during seed maturation An inducible monocot transcriptional regulatory region, (ii) a first encoding a heterologous protein. A DNA sequence, and (iii) a second DNA sequence encoding a signal peptide. Obtaining monocotyledonous plant cells transformed with the chimeric gene The DNA sequence of the second and the second DNA sequence are in frame and the fusion protein And (i) the transcriptional regulatory region is operable on the second DNA sequence And (ii) the signal peptide is composed of the transformed cells. A step effective to facilitate secretion of the mature heterologous protein;   (B) culturing the transformed cells under conditions effective to induce the transcription regulatory region The fusion protein expression and the mature heterologous protein Promoting secretion of cytoplasm from the transformed cells; and   (C) a step of isolating the mature heterologous protein produced by the transformed cell About A method comprising: 2. 2. The method of claim 1, wherein said first DNA sequence encodes proBPN '. And the culturing step promotes the expression and secretion of pro-BPN 'from the transformed cells. Culturing said transformed cells at a pH between 5 and 6 to Conditions effective for the isolation step to allow automatic conversion of pro-BPN 'to active mature BPN' Incubating pro-BPN 'below. 3. 2. The method of claim 1, wherein said first DNA sequence encodes mature BPN '. And the method further comprises:   (I) induction by addition or removal of small molecules or during seed maturation A third DNA encoding a possible transcriptional regulatory region, (ii) the propeptide portion of BPN ' Sequence, and (iii) a fourth DNA sequence encoding a signal polypeptide. Transforming said cells with a second chimeric gene comprising: Is operably linked to the transcription control region and the third DNA sequence. Where the signal polypeptide is in the translation frame with the propolypeptide portion. And allows for secretion of the expressed propeptide moiety from the transformed cells. Steps that are effective to facilitate;   The culturing step includes the step of expressing BPN 'and the propeptide moiety from the transformed cells. And culturing the cells at a pH between 5 and 6 to promote secretion;   And the isolation step is effective to allow the conversion of BPN 'to active mature BPN' Incubating the BPN 'and the pro-moiety under Isolating mature BPN ', Method. 4. The signal peptide comprises an amino acid sequence identified by SEQ ID NO: 1. The method according to claim 1, which is a signal peptide having RAmy3D. 5. The second DNA sequence encodes a RAmy3D signal peptide (SEQ ID NO: 1) And has a codon optimized nucleotide sequence identified by SEQ ID NO: 3 The method of claim 1, wherein 6. The signal peptide comprises an amino acid sequence identified by SEQ ID NO: 4. The method according to claim 1, which is a signal peptide having RAmy1A. 7. The second DNA sequence, the first DNA sequence, or the second and the first DN Both A sequences are codon-optimized for enhanced expression in the plant The method of claim 1, wherein 8. The transcription regulatory region is RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C. , RAmy3D, and RAmy3E, pM / C, gKAmy141, gKAmy155, Amy32b, and HV18 Rice or barley α-amylase gene selected from the group consisting of genes The method according to claim 1, which is a promoter of 9. The chimeric gene comprises the transcription regulatory region and the second DNA coding sequence; In between, selected from the group consisting of RAmy1A, RAmy3B, RAmy3C, RAmy3D, HV18, and RAmy3E. 9. The method of claim 8, further comprising a selected 5 'untranslated region of an inducible monocot gene. The described method. 10. The chimeric gene, downstream of the sequence encoding the fusion protein, RAmy1A, RAmy1B, RAmy2A, RAmy3A, RAmy3B, RAmy3C, RAmy3D, and RAmy3E, pM / C, gKAmy141, gKAmy155, Amy32b, and the HV18 gene Of an inducible monocot gene derived from rice or barley α-amylase gene 9. The method of claim 8, further comprising a 3 'untranslated region. 11. The culturing step comprises converting the transformed plant cells into a sugar-free medium or a sugar-free medium. 2. The method of claim 1, comprising culturing in a poor medium. The regulatory region is derived from the RAmy3E or RAmy3D gene, and the 5 ′ untranslated region is RAmy3E. Having the sequence derived from the 1A gene and identified by SEQ ID NO: 5, and The method wherein the 3 ′ untranslated region is derived from the RAmy1A gene. 12. The transformed cells are mature seed aleurone cells, and the transcription regulatory region is Up-regulated by the addition of small molecules that promote germination of offspring, and The culturing step germinates the seed in either an embryo-less or embryoless form. The method of claim 1, comprising causing to occur. 13. The transcription control region is a rice α-amylase RAmy1A promoter or The wheat HV18 promoter, and wherein the small molecule is gibberellic acid. 12. The method according to 12. 14． A mature heterologous protein produced by the method of claim 1, Where the protein is:   (I) mature glycosylated α1-antitrypsin (AAT), which Has the same N-terminal amino acid sequence as mature AAT produced in Glycosylation pattern significantly increases the half-life of mature unglycosylated AAT over half-life Mature glycosylated AAT with   (Ii) mature glycosylated antithrombin III (ATIII), which Glycosylated A having the same N-terminal amino acid sequence as mature ATIII produced in TIII; and   (Iii) mature glycosylated subtilisin BPN '(BPN'), comprising Bacill Mature glycosylation with the same N-terminal amino acid sequence as BPN 'produced in us Subtilisin BPN '; Wherein the protein is produced in said monocotyledonous plant. Mature heterologous protein with a glycosylation pattern characteristic of the protein to be . 15. The monocotyledon plant cells are transformed rice, barley, corn , Wheat, oats, rye, sugar corn, or millet cells, The method of claim 1. 16. The monocot plant cell is a transformed rice or barley cell, The method of claim 1. 17． The mature heterologous protein may be produced according to the method of claim 1. A plant cell, wherein the culturing step comprises culturing the transformed plant cell without sugar. Culturing in a medium or in a sugar-deficient medium, wherein the transcription regulatory region is RAmy3E or Derived from the RAmy3D gene, the 5 'untranslated region is derived from the RAmy1A gene, and the sequence Has the sequence identified by No. 5 and the 3 'untranslated region is from the RAmy1A gene Is a plant cell. 18. The mature heterologous protein may be produced according to the method of claim 1. A seed, wherein the transformed cells are aleurone cells, and the transcription regulatory region is Upregulated by the addition of small molecules that promote seed germination, and The culturing step germinates the seed in a form having an embryo or a form having no embryo Seeds, including to do.