WO1998045458A1 - Proteine seminale genetiquement manipulee contenant un pourcentage eleve d'acides amines essentiels - Google Patents

Proteine seminale genetiquement manipulee contenant un pourcentage eleve d'acides amines essentiels Download PDF

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WO1998045458A1
WO1998045458A1 PCT/US1998/006673 US9806673W WO9845458A1 WO 1998045458 A1 WO1998045458 A1 WO 1998045458A1 US 9806673 W US9806673 W US 9806673W WO 9845458 A1 WO9845458 A1 WO 9845458A1
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seq
protein
met
atg
lys
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PCT/US1998/006673
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English (en)
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Steven Gutteridge
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E.I. Du Pont De Nemours And Company
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Priority to AU68832/98A priority Critical patent/AU6883298A/en
Publication of WO1998045458A1 publication Critical patent/WO1998045458A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8253—Methionine or cysteine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C12N15/8254—Tryptophan or lysine

Definitions

  • This invention pertains to the development of seeds and seed storage proteins that are enhanced in the quantity of amino acids that are essential to humans and animals, and more particularly, the enhancement of the quantity of essential amino acids in the Brazil Nut 2S albumin seed storage protein.
  • Soybean meal is a good source of Lys and Tip but poor in sulfur-containing residues and thus must be supplemented with sulfur-rich corn meal to provide a suitably balanced diet.
  • a protein that has a substantial proportion of both the sulfur-amino acids and Lys in content that can be expressed to high levels in seeds of crop plants would have two advantages. First, the need to supplement meals with individual amino acids, or blend different meals would be obviated. Second, other meals that are left after extraction of other commodities and presently discarded for lack of nutrition, might become alternative sources of balanced dietary protein. With the molecular genetic tools now available, alteration of the amino acid composition of seed storage proteins to enhance their nutritional quality is possible. Such altered seed storage proteins can in turn enhance grain amino acid composition, thus adding value for the farmer.
  • the amino acid content of seeds is determined primarily by the storage proteins synthesized during seed development that serve as a major nutrient reserve following germination.
  • the quantity of this reserve varies from about 10% dry weight in cereals - to 40% in legumes.
  • storage proteins can account for 50% or more of the total protein. Although this abundance has meant that these proteins were some of the first to be isolated, it is only recently that their amino acid sequences have been determined.
  • a number of sulfur-rich plant seed storage proteins have been identified and their genes isolated.
  • Proteins with high lysine content are even less common, particularly ones with sufficiently high percentages of, for example, methionine and lysine so that the expression levels required to raise the level of those amino acids in seeds still expressing endogenous proteins are not beyond the limits of gene expression technology.
  • Modern protein engineering technology offers a route to create such proteins.
  • One solution is to design proteins completely de novo, such as taught by Falco et al. (World Patent Publication No. WO93/03160). This strategy is risky in that the fate of such a protein in the cell is difficult to predict.
  • the Brazil Nut 2S albumin represents a family of related proteins found in a variety of species (Youle and Huang (1981) Amer. J. Bot. 65:44-48). They are small proteins found in vivo as two subunits linked by disulfide bridges. The two subunits are derived from a single precursor peptide which is extensively processed (Crouch et al. (1983) J. Mol. App. Genet. 2, 273-283; Krebbers et al. (1988) Plant Physiol. 87, 859-866). Sequence analysis of 2S albumins from different species shows that while the sequences from different species are not always highly conserved, the number of cysteine residues and their arrangement in the sequence is, suggesting that the structure of 2S albumins is similar between species.
  • This invention pertains to a modified Brazil Nut 2S albumin seed storage protein wherein: (i) the amino acid sequence of the modified protein is at least 40% homologous to the wild type Brazil Nut 2S albumin seed storage protein; (ii) all cysteine residues of the modified protein are conserved relative to the wild type protein; (iii) at least 40% of proline residues are conserved relative to the wild type protein; (iv) at least 80% of leucine residues are conserved relative to the wild type protein; and (v) the modified protein comprises at least one non-conservative amino acid substitution not within the hypervariable loop, the substitution consisting of replacement of a non- essential amino acid with an essential amino acid.
  • a preferred embodiment of the instant invention is a modified Brazil Nut 2S albumin seed storage protein wherein: (i) the amino acid sequence of the modified protein is at least 82% homologous to the wild type Brazil Nut 2S albumin seed storage protein; (ii) all cysteine, proline, leucine and methionine residues of the modified protein are conserved relative to the wild type protein; (iii) all arginine residues of the wild type protein are substituted with lysine residues; and (iv) the modified protein comprises at least three non-conservative amino acid substitutions not within the hypervariable loop, said substitutions comprising substituting two glutamic acid residues with lysine residues and substituting one glutamine residue with a lysine residue.
  • Another embodiment of the instant invention is an isolated nucleic acid fragment encoding the modified Brazil Nut 2S albumin seed storage protein described above, and a chimeric gene wherein the nucleic acid fragment operably linked to suitable regulatory sequences.
  • a further embodiment of the instant invention is a transformed plant comprising in its genome the chimeric gene described above. Preferred plants include soybean, rapeseed, sunflower, cotton, corn, tobacco, alfalfa, wheat, barley, oats, sorghum, rice and forage grasses.
  • Yet another embodiment of the instant invention are seeds derived from the transformed plant described above wherein the seeds comprise the chimeric gene.
  • Still another embodiment of the instant invention is a method for increasing the essential amino acid content of seeds, the method comprising: (a) preparing a nucleic acid fragment encoding a modified Brazil Nut 2S albumin seed storage protein wherein (i) the amino acid sequence of the modified protein is at least 40% homologous to the wild type Brazil Nut 2S albumin seed storage protein; (ii) all cysteine residues of the modified protein are conserved relative to the wild type protein; (iii) at least 40% of proline residues are conserved relative to the wild type protein; (iv) at least 80% of leucine residues are conserved relative to the wild type protein; and (v) the modified protein comprises at least one non-conservative amino acid substitution not within the hypervariable loop, the substitution consisting of replacement of a non-essential amino acid with an essential amino acid; (b) preparing a chimeric gene comprising the nucleic acid fragment of step (a) operably linked to suitable regulatory sequences; (c) transforming a plant with the chimeric
  • Figure 1 is the nucleotide sequence and deduced amino acid sequence of the wild type Brazil Nut 2S albumin gene in plasmid pBNwt that was used as the starting point for the genetic modifications described herein. Relevant restriction enzyme cleavage sites are indicated.
  • Figure 2 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BNCNSS.
  • Figure 3 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN11.
  • Figure 4 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN15.
  • Figure 5 is the nucleotide sequence and deduced amino acid sequence of the - modified Brazil Nut 2S albumin gene BN17.
  • Figure 6 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN18.
  • Figure 7 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN19.
  • Figure 8 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN153KW.
  • Figure 9 is a composite of the amino acid sequences encoded by the wild type Brazil Nut 2S albumin gene (wt) and modified Brazil Nut 2S albumin genes exemplified herein. Sulfur-containing amino acid residues are indicated in bold.
  • Figure 10 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene AT2S1BN15.
  • Figure 11 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene AT2S1BN19.
  • Figure 12 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene AT2S1BN153W.
  • SEQ ID NO:l is the nucleotide sequence and deduced amino acid sequence of the wild type Brazil Nut 2S albumin gene in plasmid pBNwt that was used as the starting point for the genetic modifications described herein.
  • SEQ ID NO:2 is the amino acid sequence of the wild type Brazil Nut 2S albumin protein.
  • SEQ ID Nos:3-6 are four synthetic oligonucleotides used in the construction of the modified Brazil Nut 2S albumin gene BNCNSS.
  • SEQ ID NO:7 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BNCNSS.
  • SEQ ID Nos:8 and 9 are two synthetic oligonucleotide used in the construction of the modified Brazil Nut 2S albumin gene BN11.
  • SEQ ID NO: 10 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN11.
  • SEQ ID NOs:l 1-14 are four synthetic oligonucleotides used in the construction of the modified Brazil Nut 2S albumin gene BN15.
  • SEQ ID NO: 15 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN15.
  • SEQ ID Nos:16 and 17 are two synthetic oligonucleotides used in the construction of the modified Brazil Nut 2S albumin gene BN17.
  • SEQ ID NO: 18 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN17.
  • SEQ ID Nos:19 and 20 are two synthetic oligonucleotides used in the construction of the modified Brazil Nut 2S albumin gene BN18.
  • SEQ ID NO:21 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN18.
  • SEQ ID Nos:22 and 23 are two synthetic oligonucleotides used in the construction of the modified Brazil Nut 2S albumin gene BN19.
  • SEQ ID NO:24 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN19.
  • SEQ ID Nos:25-28 are four synthetic oligonucleotides used in the construction of the modified Brazil Nut 2S albumin gene BN153KW.
  • SEQ ID NO:29 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene BN153KW.
  • SEQ ID Nos:30 and 31 are two synthetic oligonucleotides used in the construction of the Brazil Nut 2S albumin genes comprising the Arabidopsis 2S albumin precursor sequence.
  • SEQ ID NO:32 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene AT2S1BN15.
  • SEQ ID NO:33 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene AT2S1BN19.
  • SEQ ID Nos:34 and 35 are two synthetic oligonucleotides used in the construction of the modified Brazil Nut 2S albumin gene AT2S1BN153W.
  • SEQ ID NO:36 is the nucleotide sequence and deduced amino acid sequence of the modified Brazil Nut 2S albumin gene AT2S1BN153W.
  • the present invention demonstrates that the 2S albumin from Brazil Nut is able to accommodate much more radical changes than had been demonstrated previously and that non-conservative replacements with the intent of enriching the protein with essential amino acids outside of the region between the 6th and 7th cysteines can be tolerated without influencing the ability of the protein to be expressed in the seeds of transgenic plants.
  • Such altered Brazil Nut 2S albumins modified such that they are composed of more than two essential amino acids can accumulate to sufficiently high levels to influence the nutritional value of the seed protein.
  • the present invention describes nucleic acid fragments that encode a modified high sulfur 2S albumin seed storage protein. This novel protein is analogous to the protein isolated from Brazil Nuts that is rich in methionine and cysteine amino acids, but has been altered to include other essential amino acids using site specific - replacement techniques.
  • the structure of the wild type Brazil Nut 2S albumin protein ( Figure 1 ; SEQ ID NO:2) is characterized by the presence of eight cysteine residues that form four disulfide bonds. Twenty percent of the sequence is methionine, distributed throughout the sequence both as isolated residues and in denser regions of adjacent occupancy. Between the 6th and 7th cysteine residues is a region that has been termed the "hypervariable loop," so designated because there are examples of engineered versions of the Arabidopsis homolog of the Brazil Nut 2S albumin where substantial amounts or even all of this segment, except for four amino acids immediately adjacent to the amino end of the region and 5 amino acids adjacent to the carboxyl end of the hypervariable loop have been replaced with other non-related sequences.
  • the Brazil Nut protein is also distinct from other 2S albumins because it is rich in arginine, glutamine and some glutamic acid residues.
  • more radical alterations might be achievable, since protein folding might depend mainly on the correct formation of disulfide bonds rather than the identity of other residues.
  • Genes have been constructed that encode a protein that has all of the native argininyl residues replaced by lysyl residues and then further supplemented with additional lysyl residues by alterations at positions not expected to tolerate such changes. These genes have been expressed in a microorganism and in a transgenic plant to alter the nutritional quality of the seed proteins.
  • the increase in methionine and lysine in the seed must be determined by a) the level of expression of the engineered gene in the transformed plant, which depends in part on the seed specific expression signals that are used, b) the percentage of methionine and lysine in the coding region of the engineered gene, c) the stability of the expressed protein in the seed of the transformed plant, which depends in part on its correct processing, intracellular targeting, folding into a structure to allow accumulation in the seed, and ability to withstand desiccation, and d) the compatibility of the new protein with the natural variants of the transformed plant.
  • Transfer of the gene constructs of the invention (linked to suitable regulatory sequences) into a living cell will result in the production of the encoded protein. Additionally, transfer of the gene constructs of the invention into plants, particularly Brassica, or other suitable crop plants such as corn, soybean or oil seed rape, with suitable regulatory sequences to direct expression of the protein in seeds may result in increased level of sulfur-containing and basic amino acids, particularly methionine and lysine, respectively, thus improving the nutritional quality of seed protein for animals.
  • essential amino acids refers to those amino acids which must be obtained by animals and humans from dietary sources.
  • the essential amino acids are arginine (Arg), histidine (His), isoleucine (He), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), threonine (Thr), tryptophan (Trp) and valine (Val).
  • nucleic acid refers to a polynucleotide of high molecular weight which can be single-stranded or double-stranded, composed of monomers (nucleotides) containing a sugar, phosphate and a base which is either a purine or pyrimidine.
  • a "nucleic acid fragment” is a fraction of a given nucleic acid molecule.
  • deoxyribonucleic acid (DNA) is the genetic material while ribonucleic acid (RNA) is involved in the transfer of information contained within DNA into proteins.
  • a “genome” is the entire body of genetic material contained in each cell of an organism.
  • nucleotide sequence refers to a polymer of DNA or RNA which can be single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases capable of incorporation into DNA or RNA polymers.
  • homologous to refers to the complementarity between the nucleotide sequence of two nucleic acid molecules or between the amino acid sequences of two protein molecules. Estimates of such homology are provided by either DNA-DNA or DNA-R A hybridization under conditions of stringency as is well understood by those skilled in the art (as described in Hames and Higgins (eds.) Nucleic Acid Hybridisation, IRL Press, Oxford, U.K.); or by the comparison of sequence similarity between two nucleic acids or proteins.
  • substantially similar refers to nucleotide and amino acid sequences that represent equivalents of the instant inventive sequences.
  • altered nucleotide sequences which simply reflect the degeneracy of the genetic code but nonetheless encode amino acid sequences that are identical to the inventive amino acid sequences are substantially similar to the inventive sequences.
  • amino acid sequences that are substantially similar to the instant sequences are those wherein overall amino acid identity is 95% or greater to the instant sequences. Modifications to the instant invention that result in equivalent nucleotide or amino acid sequences is well within the routine skill in the art.
  • nucleotide sequences encompassed by this invention can also be defined by their ability to hybridize, under stringent conditions (0.1X SSC, 0.1% SDS, 65°C), with the nucleotide sequences that are within the literal scope of the instant claims.
  • gene refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding) and following (3 ! non-coding) the coding region.
  • “Native” gene refers to the gene as found in nature with its own regulatory sequences.
  • Chimeric gene refers to a gene comprising heterogeneous regulatory and coding sequences.
  • Endogenous gene refers to the native gene normally found in its natural location in the genome.
  • a “foreign” gene refers to a gene not normally found in the host organism but that is introduced by gene transfer.
  • coding sequence refers to a DNA sequence that codes for a specific protein and excludes the non-coding sequences. It may constitute an "uninterrupted coding sequence", i.e., lacking an intron, such as in a cDNA or it may include one or more introns bounded by appropriate splice junctions.
  • An "intron” is a sequence of RNA which is contained in the primary transcript but which is removed through cleavage and re-ligation of the RNA within the cell to create the mature mRNA that can be translated into a protein.
  • initiation codon and “termination codon” refer to units of three adjacent nucleotides in a coding sequence that specify initiation and chain termination, respectively, of protein synthesis (mRNA translation).
  • RNA transcript refers to the product resulting from RNA polymerase- catalyzed transcription of a DNA sequence.
  • primary transcript When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.
  • Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into protein by the cell.
  • cDNA refers to a single- or a double-stranded DNA that is complementary to and derived from mRNA.
  • regulatory sequences means nucleotide sequences located upstream (5'), within, and/or downstream (3') to a coding sequence, which control the transcription and/or expression of the coding sequences, potentially in conjunction with the protein biosynthetic apparatus of the cell. These nucleotide sequences include a promoter sequence, a translation leader sequence, a transcription termination sequence, and a polyadenylation sequence.
  • promoter refers to a DNA sequence in a gene, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • a promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to - physiological or developmental conditions. It may also contain enhancer elements.
  • enhancer means a DNA sequence which can stimulate promoter activity. It may be an innate element of the promoter or a heterologous element inserted to enhance the level and/or tissue-specificity of a promoter.
  • Constutive promoters refers to those promoters that direct gene expression in substantially all tissues and at substantially all times.
  • “Organ-specific” or “development-specific” promoters as referred to herein are those that direct gene expression almost exclusively in specific organs, such as leaves or seeds, or at specific development stages in an organ, such as in early or late embryogenesis, respectively.
  • expression means the production of the protein product encoded by a gene.
  • 3' non-coding sequences refers to the DNA sequence portion of a gene that contains a transcription termination signal, polyadenylation signal, and any other regulatory signal capable of affecting mRNA processing or gene expression.
  • the polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3' end of the mRNA precursor.
  • the term "5' non-coding sequences” refers to the DNA sequence portion of a gene that contains a promoter sequence and a translation leader sequence.
  • translation leader sequence refers to that DNA sequence portion of a gene between the promoter and coding sequence that is transcribed into RNA and is present in the fully processed mRNA upstream (5') of the translation start codon.
  • the translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency.
  • mature protein refers to a post-translationally processed polypeptide without its signal peptide.
  • Precursor protein refers to the primary product of translation of an mRNA.
  • Signal peptide refers to the amino terminal extension of a polypeptide, which is translated in conjunction with the polypeptide forming a precursor peptide and which is required for its entrance into the secretory pathway.
  • signal sequence refers to a nucleotide sequence that encodes the signal peptide.
  • Intracellular localization sequence refers to a nucleotide sequence that encodes an intracellular targeting signal.
  • An “intracellular targeting signal” is an amino acid sequence which is translated in conjunction with a protein and directs it to a particular sub-cellular compartment.
  • Endoplasmic reticulum (ER) stop transit signal refers to a carboxy-terminal extension of a polypeptide, which is translated in conjunction with the polypeptide and causes a protein that enters the secretory pathway to be retained in the ER.
  • ER stop transit sequence refers to a nucleotide sequence that encodes the ER targeting signal.
  • Other intracellular targeting sequences encode targeting signals active in seeds and/or leaves and vacuolar targeting signals.
  • transgenic refers to the transfer of a nucleic acid fragment into the genome of a host cell, resulting in genetically stable inheritance.
  • Host cells containing the transformed nucleic acid fragments are referred to as "transgenic” cells, and organisms comprising transgenic cells are referred to as "transgenic organisms".
  • transgenic organisms Examples of methods of transformation of plants and plant cells include Agrobacterium-mediatcd transformation (De Blaere et al. (1987) Meth. Enzymol. 143, 277) and particle bombardment technology (Klein et al. (1987) Nature (London) 327, 70-73; U.S. Patent No. 4,945,050).
  • Whole plants may be regenerated from transgenic cells by methods well known to the skilled artisan (see, for example, Fromm et al. (1990) Bio/Technology 8, 833).
  • cassette means a nucleic acid fragment prepared by the annealing of two synthetic and complementary oligonucleotides. Based upon published sequences (Altenbach et al, (1987) Plant Molecular
  • oligonucleotides were synthesized to allow construction of modified forms of the wild type gene, either through mismatch site-specific procedures or as double-stranded DNA cassettes.
  • the first mutations were introduced into the wild type 2S gene using oligonucleotides that were complementary to the sequence except in those positions coinciding with the places where base changes were desired.
  • the construct used to achieve these changes was an M13-based plasmid that allowed isolation of single stranded form of the wild type 2S albumin gene (pM13BNwt).
  • the oligonucleotides synthesized to form the cassettes were therefore routinely extended at the 5' ends so that once the complementary pairs of strands had been annealed to form the cassette, restriction digestion would result in fragments bearing ends of single stranded DNA with precise complementarity to the single stranded ends of a vector that had been treated with the same restriction enzyme; these could then be ligated more efficiently into the expression vector of choice.
  • restriction digestion would result in fragments bearing ends of single stranded DNA with precise complementarity to the single stranded ends of a vector that had been treated with the same restriction enzyme; these could then be ligated more efficiently into the expression vector of choice.
  • the altered DNA from each series of new constructs could be readily assessed for success. All new versions of the gene were sequenced fully to confirm that the desired mutations had been introduced.
  • this version of the gene was introduced into pET24a (Novagen, Inc., Madison, WI), a commercial plasmid allowing expression from the T7 promoter in suitable hosts. This provided plasmid pETBNCNSS.
  • the first construct that was made using cassettes, pETBNl 1, comprising the modified Brazil Nut 2S albumin gene BN11 ( Figure 3; SEQ ID NO:10), is characterized by the introduction twelve of Lys codons (compared to zero Lys codons in the wild type 2S gene).
  • the threonine (Thr) codon corresponding to position 33 of the wild type protein was replaced with serine (Ser) codon to introduce an Sphl site adjacent to the SacII site.
  • pETBN15 was the version of the enriched gene (BN15; Figure 4; SEQ ID NO:15) with all fifteen Arg codons replaced by Lys codons and from which all other variants were made.
  • BN17 Figure 5; SEQ ID NO: 18
  • Ser 107 changed to Lys and glycine (Gly) 105 to Lys by oligonucleotide replacement
  • BN18 was a version of BN17 wherein Ser 44 changed to Lys ( Figure 6; SEQ ID NO: 21) by oligonucleotide replacement (SEQ ID NOs:19 and 20).
  • a version of BN15 was further enhanced with Lys and Met residues by replacing amino acids that are not considered of this type, i.e., non-conservative changes.
  • replacement of the Ndel- Sphl fragment of BN15 with two oligonucleotides (SEQ ID NOs:22 and 23) introduced two further Lys residues in place of two glutamic acid (Glu) residues at positions 4 and 28, and introduced an extra Met replacing Glu 27.
  • Other variants such as BN153KW ( Figure 8; SEQ ID NO:29), explored - introduction of the essential amino acid Trp into the sequence of the Brazil Nut 2S albumin.
  • Figure 9 is a composite of the amino acid sequences encoded by each of the wild type and modified Brazil Nut 2S albumin genes described above.
  • nucleic acid fragments were constructed that recreated the precursor sequence of an Arabidopsis 2S protein.
  • An oligonucleotide cassette (comprising SEQ ID NOs:30 and 31) encoding the Arabidopsis precursor sequence was designed so that introduction of the cassette into the 5'-end of the wild type and modified 2S albumin genes described above resulted in an in-frame fusion of the precursor sequence with the sequence of the mature genes.
  • the resulting genes had an Ncol site at the ATG initiation codon of precursor sequence and an Ndel site at the first codon of the mature sequence.
  • These precursor genes were designated AT2SlBNwt (not shown), AT2S1BN15 ( Figure 10; SEQ ID NO:32), and AT2S1BN19 ( Figure 11; SEQ ID NO:33) to indicate they contained the wild type and BN15, and BN19 variants of the 2S albumin gene, respectively.
  • Another precursor gene, AT2S1BN153W ( Figure 12; SEQ ID NO:36), was prepared by direct replacement of the Nhel to Hindlll fragment of pAT2SlBN15 with a cassette which directed the incorporation of an increased number of tryptophanyl residues in the gene product.
  • the nucleic acid fragment coding for the sulfur rich seed 2S protein may be attached to suitable regulatory sequences and used to overproduce the protein in microbes such as bacteria or yeast, or in transgenic plants such as Brassica, cereals or legumes.
  • suitable regulatory sequences such as bacteria or yeast, or in transgenic plants such as Brassica, cereals or legumes.
  • Such a DNA construction may include either the wild type 2S gene or an engineered gene.
  • One skilled in the art can isolate the coding sequences from the fragment of the invention by using and/or creating restriction endonuclease sites. Expression of enriched 2S protein in E. coli
  • the commercial expression vector pET24a was used to express the modified Brazil Nut 2S coding sequences in E. coli.
  • This vector employs the bacteriophage T7 RNA polymerase/T7 promoter system (Studier et al., (1990) Methods in Enzymology 185, 60-89) for gene transcription.
  • the variants of all the 2S albumin genes including the wild type construct were ligated into pET24a using the Ndel-Hindlll sites. These constructs were used to transform competent E. coli cells (strain BL21) which were grown to mid-log phase in LB before induction with IPTG. The protein expressed in transformed E.
  • the nucleic acid fragments of the invention can be used to produce large quantities of the 2S protein enriched in essential amino acids especially methionine and lysine via fermentation in E. coli or other microorganisms.
  • the nucleic acid fragment of the invention can be operably linked to a suitable regulatory sequence comprising a promoter sequence, a translation leader sequence and a 3' non-coding sequence.
  • the chimeric gene can then be introduced into a microorganism via transformation and the transformed organism grown under conditions resulting in high expression of the engineered gene.
  • the cells containing the protein rich in essential amino acids can be collected and the enriched protein extracted. Because high level production is not toxic to the cells, higher levels could be achieved using other strains.
  • a preferred class of hosts for the expression of the coding sequence of modified Brazil Nut 2S albumin proteins are eukaryotic hosts, particularly the cells of higher plants. Particularly preferred among the higher plants and the seeds derived from them are soybean, rapeseed (Brassica napus, B. campestris), sunflower (Helianthus annus), cotton (Gossypium hirsutum), corn, tobacco (Nicotiana tabacum), alfalfa (Medicago sativa), wheat (Triticum sp.), barley (Hordeum vulgar e), oats (Avena sativa, L), sorghum (Sorghum bicolor), rice (Oryza sativa), and forage grasses. Expression in plants will use regulatory sequences functional in such plants.
  • promoters for expression in all plant organs, and especially for expression in leaves include those directing the 19S and 35S transcripts in Cauliflower mosaic virus (Odell et al. (1985) Nature 313, 810-812; Hull et al.
  • promoters that are specific for expression in one or more organs of the plant.
  • examples include the light- inducible promoters of the small subunit of ribulose 1,5-bisphosphate carboxylase, if the expression is desired in photosynthetic organs, or promoters active specifically in seeds.
  • Preferred promoters are those that allow expression of the protein specifically in seeds. This may be especially useful, since seeds are the primary source of vegetable protein and also since seed-specific expression will avoid any potential deleterious effect in non-seed organs.
  • seed-specific promoters include, but are not limited to, the promoters of seed storage proteins, which represent more than 50% of total seed protein in many plants.
  • the seed storage proteins are strictly regulated, being expressed almost exclusively in seeds in a highly organ-specific and stage-specific manner (Higgins et al.( 1984) Ann. Rev. Plant Physiol. 35, 191 -221 ; Goldberg et al.(1989) Cell 56, 149-160; Thompson et al. (1989) BioEssays 10, 108-113). Moreover, different seed storage proteins may be expressed at different stages of seed development.
  • rapeseed napin (Radke et al. (1988) Theor. Appl. Genet. 75, 685-694) as well as genes from monocotyledonous plants such as for maize 15 kD zein (Hoffman et al. (1987) EMBOJ. 6, 3213-3221; Schemthaner et al. (1988) EMBOJ. 7, 1249-1253; Williamson et al. (1988) Plant Physiol. 88, 1002-1007), barley ⁇ -hordein (Marris et al. (1988) Plant Mol. Biol. 10, 359-366) and wheat glutenin (Colot et al. (1987) EMBO J.
  • promoters of seed-specific genes operably linked to heterologous coding sequences in chimeric gene constructs also maintain their temporal and spatial expression pattern in transgenic plants.
  • Such examples include Arabidopsis thaliana 2S seed storage protein gene promoter to express enkephalin peptides in Arabidopsis and B. napus seeds (Vandekerckhove et al. (1989) Bio/Technology 7,
  • nucleic acid fragment of the invention will be the heterologous promoters from several extensively-characterized soybean seed storage protein genes such as those for the Kunitz trypsin inhibitor (Jofuku et al. (1989) Plant Cell 1, 1079-1093; Perez-Grau et al. (1989) Plant Cell 1, 1095-1109), glycinin (Nielson et al. (1989) Plant Cell 1, 313-328), ⁇ -conglycinin (Harada et al. (1989) Plant Cell 1, 415-425).
  • Kunitz trypsin inhibitor Kunitz trypsin inhibitor
  • Plant Cell 1, 1079-1093 Perez-Grau et al. (1989) Plant Cell 1, 1095-1109
  • glycinin Naelson et al. (1989) Plant Cell 1, 313-328
  • ⁇ -conglycinin Harada et al. (1989) Plant Cell 1, 415-425.
  • Promoters of genes for ⁇ '- and ⁇ -subunits of soybean ⁇ -conglycinin storage protein will be particularly useful in expressing the modified Brazil Nut 2S albumin 2S albumin mRNA in the cotyledons at mid- to late-stages of soybean seed development (Beachy et al. (1985) EMBOJ. 4, 3047-3053; Barker et al. (1988) Proc. Natl. Acad. Sci. USA 85, 458-462; Chen et al. (1988) EMBO J. 7, 297-302; Chen et al. (1989) Dev. Genet. 10, 112-122; Naito et al. (1988) Plant Mol. Biol.
  • heterologous promoters from several extensively characterized com seed storage protein genes such as those from the 10 kD zein (Kirihara et al. (1988) Gene 71, 359-370), the 27 kD zein (Prat et al. (1987) Gene 52, 51-49; Gallardo et al. (1988) Plant Sci. 54, 211-281), and the 19 kD zein (Marks et al. (1985) J. Biol. Chem. 260, 16451-16459). The relative transcriptional activities of these promoters in com have been reported (Kodrzyck et al.
  • enhancers or enhancer-like elements into promoter constructs will also provide increased levels of primary transcription for modified Brazil Nut 2S albumin proteins to accomplish the invention.
  • the DNA sequence element isolated from the gene for the ⁇ '-subunit of ⁇ -conglycinin that can confer 40-fold seed-specific enhancement to a constitutive promoter (Chen et al. (1988) EMBO J. 7, 297-302; Chen et al. (1989) Dev. Genet. 10, 112-122).
  • One skilled in the art can readily isolate this element and insert it within the promoter region of any gene in order to obtain seed-specific enhanced expression with the promoter in transgenic plants. Insertion of such an element in any seed-specific gene that is expressed at different times than the ⁇ -conglycinin gene will result in expression in transgenic plants for a longer period during seed development.
  • the invention can also be accomplished by a variety of other methods to obtain the desired end.
  • the invention is based on modifying plants to produce increased levels of 2S enriched protein by having significantly larger numbers of copies of the modified gene either through enhanced promotion or multiple copies on each message.
  • Any 3' non-coding region capable of providing a transcription termination signal, a polyadenylation signal and other regulatory sequences that may be required for the proper expression of the modified Brazil Nut 2S albumin protein coding region can be used to accomplish the invention.
  • DNA sequences coding for intracellular localization sequences may be added to the modified Brazil Nut 2S albumin protein coding sequence if required for the proper expression of the proteins to accomplish the invention.
  • the signal sequence from the ⁇ subunit of phaseolin from the bean Phaseolus vulgar is, or the signal sequence from the a' subunit of ⁇ -conglycinin from soybean (Doyle et al. (1986) J. Biol. Chem. 261, 9228-9238), can be employed. Hoffman et al. ((1987) EMBO J.
  • a short amino acid domain that serves as a vacuolar targeting sequence has been identified from bean phytohemagglutinin which accumulates in protein storage vacuoles of cotyledons (Tague et al. (1990) Plant Cell 2, 533-546). In another report a carboxyl-terminal amino acid sequence necessary for directing barley lectin to vacuoles in transgenic tobacco was described (Bednarek et al. (1990) Plant Cell 2, 1145-1155). Construction of chimeric genes for expression of Brazil Nut 2S in plants
  • Three specific gene expression cassettes were used for construction of chimeric genes for expression of 2S in plants to explore expression of altered forms of the gene in a plant host. Specifically those variants of the 2S gene with conservative replacements as exemplified by Arg to Lys and also an example of non-conservative changes as in BN19.
  • the expression cassettes contained the regulatory regions from two highly expressed seed storage protein genes:
  • the precursor sequence of one of the 2S albumin genes from Arabidopsis thaliana was introduced in-frame at the 5'-end of the 2S native gene and selected variants to give AT2SlBNwt, AT2S1BN15, AT2S1BN19 andAT2SlBN153W.
  • the precursor versions of these genes were then ligated between the ⁇ -conglycinin promoter and the 3'- phaseolin termination region (Slightom et al., (1991,) Plant Mol. Biol. Man. B16, 1-55) in plasmid pCW109.
  • the vector pCW109 was made by inserting into the Hindlll site of the cloning vector pUC18 a 555 bp 5' non-coding region (containing the promoter region) of the ⁇ -conglycinin gene followed by the multiple cloning sequence containing the restriction endonuclease sites for Nco I, Sma I, Kpn I and Xba I, then 1174 bp of the common bean phaseolin 3' untranslated region into the Hindlll site (described above).
  • This plasmid allows the precursor, mature and flanking regulatory regions to be isolated as one large Hindlll fragment after amplification and isolation from E. coli (Odell et al., - (1994) Plant Physiol.
  • the starting Brazil nut 2S albumin sequence (SEQ ID No:l) used herein is supplemented with an initiation codon to facilitate expression in prokaryotic cells.
  • An Ndel-EcoRI fragment encompassing the nature sequence was ligated into a derivative of pET3a (Novagen Inc., Madison, WI) that has the Ncol site replaced by a short multiple cloning site containing Nde I and EcoRI.
  • This plasmid, pET3am was digested with Ndel and EcoRI to accept the BNwt gene fragment giving the plasmid pBNwt; see Figure 3.
  • This - plasmid which also carries the gene for ⁇ -lactamase, was used to transform competent E.coli (JM 83) cells that were grown in SOC medium (Hanahan, D. (1983) J. Mol. Biol. 166, 557) before selecting for plasmid-bearing organisms with ampicillin.
  • the cells were streaked onto agarose-LB plates that contained ampicillin (50 ug/mL) and grown overnight at 37°. A single colony was picked and inoculated into 50 mL of LB medium also containing ampicillin. The culture was shaken at 37° until the cells had reached an OD 6 oo of about 3.0.
  • the cells were harvested by centrifugation and the DNA isolated and purified using the procedures described by the suppliers of the Promega WizardTM kit. The purified DNA was verified by restriction site digestion and electrophoretic separation of the fragments on 1% agarose gels.
  • the 2S gene in the plasmid was also sequenced in both directions using primers that annealed to the vector sequence close to the T7 promoter outside the 5' end of the coding region and the 3' end at the T7 terminator.
  • the Muta-GeneTM kit provides a means of strongly selecting against the non-mutagenised strand of double-stranded DNA. This was achieved by transforming E. coli CJ236 competent cells with pM13BNwt, a host that has a double mutation in the dut and ur ⁇ g genes. Some of the thymines in the DNA are stabily replaced by uracil in this double mutant. The transformants were grown on LB plates containing chloramphenicol. One of the colonies was grown overnight in chloramphenicol-containing medium and the single-stranded DNA containing uracil was then isolated as a phagemid as described by the suppliers of the kit.
  • the four oligonucleotides used to introduce mutations into the 2S gene were first phosphorylated with T4 polynucleotide kinase at 37° for 60 min and the reaction stopped by heating at 65° for 10 min.
  • the oligonucleotides were simultaneously annealed to the uracil enriched single-stranded DNA of pM 13 BNwt at 70° followed by slow cooling to room temperature over 40 min.
  • the resulting partial duplex was then stored on ice before the double-stranded DNA was generated using T4 polymerase to extend the oligonucleotides in the presence of all four dNTPs and T4 DNA ligase to ligate the ends of the extended DNA.
  • the resulting double-stranded - DNA was used to transform E. coli MV1190 cells that have active uracil-N-glycosylase that inactivates the uracil-containing strand so that only the mutant strand replicates.
  • the transformed cells generated the double stranded form of the Ml 3 derivative; this was assessed by restriction analysis to ensure that the four unique restriction sites SacII, StuI, Nhe I and Cla I engineered into the oligonucleotides had been incorporated as a result of the manipulations, and that the internal Ncol site of the wild type gene was eliminated.
  • the modified gene was excised from the Ml 3 construct using ⁇ coRI and Xbal and ligated back into p ⁇ T3am.
  • the resulting plasmid, pBNCNSS contains a Brazil Nut 2S albumin gene with seven Arg codons replaced by Lys codon at positions corresponding to amino acids 37, 58, 63, 82, 83, 86, and 101 of the wild type protein, accompanied by a Met to phenylalanine (Phe) at position 104 ( Figure 2; SEQ ID NO:7).
  • EXAMPLE 3 Cassette Mutagenesis of the Brazil Nut 2S Albumin Gene
  • the modified 2S gene from pBNCSS was isolated by restriction enzyme digestion with Ndel and Hindlll, and ligated into pET24a to give pETBNCNSS.
  • the series of mutations that replaced a further four of the Arg residues with Lys were localized in the N-terminal half of the gene and were accomplished by annealing synthetic complementary oligonucleotides that coded for the altered sequence from the Ndel site to SacII site (SEQ ID NOs:8 and 9).
  • the individual 131 base oligonucleotides were first purified by electrophoreisis on 8% polyacrylamide gel.
  • the band was excised from the gel, eluted, and washed prior to annealing at 90° for 3 min.
  • the annealing solution was then cooled slowly to 30° and placed on ice for 3 min.
  • the oligonucleotides were designed with extended ends beyond the Nde I and Sac II sites so that, following annealing, the double-stranded cassette could be digested with these two enzymes to produce a high percentage of clean 'restriction' ends.
  • the resulting efficiencies and consistency of ligation into the Nde I/Sac Il-digested pETBNCSS vector with this cassette approach was evident from the number of transformants carrying the synthetic oligonucleotide insert.
  • the isolated vector was validated with respect to the correct insertion of the cassette by restriction analysis to show the presence of the new Sph I site introduced with the insert and by sequencing the region of the gene coding for the N-terminal segment of the protein.
  • the resulting construct, pETBNl 1, comprising the BN11 gene contained twelve Lys codons ( Figure 3; SEQ ID NO: 10).
  • the resulting ligated double cassette was isolated from an 8% polyacrylamide gel, washed and digested with Sphl and Hindlll before ligating into pETBNl 1 that had previously digested with the Sphl and Hindlll and isolated from a 1% agarose gel.
  • Transformants of competent E. coli carrying the altered vector were isolated and the DNA purified.
  • the DNA was validated by restriction analysis before sequencing the appropriate region of the gene. In this case, the introduction of a Styl site and removal of SacII was diagnostic of successful construction.
  • the derivative of pETBNl 1, now with all fifteen Arg replaced by Lys, was designated pETBN15 and encoded the modified BN15 gene ( Figure 4, SEQ ID NO: 15).
  • the cassette was first digested with the two restriction enzymes to provide clean Nhe I and BamHI ends and the fragment purified by gel electrophoresis.
  • the purified fragment was ligated into pETBNl 5 that had been cut with the same enzymes.
  • the resulting plasmid was termed pETBNl 7 and encoded the modified Brazil Nut 2S albumin gene designated BN17 ( Figure 5; SEQ ID NO:18).
  • the serine (Ser) residue at position 44 was replaced by
  • the 5'-end of the Brazil Nut 2S albumin gene was extended in-frame to include the 37 amino acid precursor sequence of the Arabidopsis 2S albumin protein that should contain all the information for the correct processing and targeting of the protein in the plant.
  • Two 137 base oligonucleotides were synthesized (SEQ ID NOs:30 and 31) with recessed Ncol and Ndel sites. The fragments were purified and isolated as described above before annealing together to form the double stranded cassette.
  • the cassette was also purified and isolated from an 8% polyacrylamide gel before restriction digestion with Ncol and Nde I to produce the clean 5'- and 3'-ends.
  • the pETBNl 5 vector was cut with Ndel and Hind III, and the fragment containing the BN15 gene was purified from the remaining vector using polyacrylamide gels and subsequent elution.
  • the Ndel sites of the cassette and the BN15 gene were then ligated together to produce the extended gene sequence with Ncol and Hindlll sites at the 5'- and 3'- ends, respectively.
  • the extended gene was then ligated into pET24d (Novagen Inc., Madison, WI) previously linearized by Ncol and Hindlll digestion.
  • the resulting vector was designated pAT2SlBN15 and contains the AT2S1BN15 gene ( Figure 10; SEQ ID NO:32).
  • Smaller segments of the vector could also be manipulated to enhance the amino acid content of the protein products.
  • the Nhel to Hindlll fragment of pAT2SlBN15 was replaced with a cassette composed of the oligonucleotides depicted in SEQ ID Nos: 22 and 23. This replaced Glu 89, Ser 93 and Phe 104 with Trp giving P AT2S1BN153W ( Figure 12; SEQ ID NO:36).
  • the wild type Brazil Nut 2S albumin gene and the modified genes BN15 and BN19 were positioned between the promoter that is normally responsible for controlling conglycinin expression and the 3' region normally found downstream of the phaseolin gene.
  • the vector that contained these control elements (pCW109) was cut with Ncol and Smal.
  • the plasmids containing the wild type and modified Brazil Nut 2S albumin genes (pAT2SlBNwt, pAT2SlBN15 and pAT2SlBN19) were first cut with EcoRI and blunt-ended with mung bean nuclease.
  • the DNA was precipitated from a solution containing 0.01% SDS and 0.1 M NaCl using two volumes of cold, dry ethanol.
  • the gene encoding the 2S precursor-containing protein was excised from the resulting linearised DNA using Ncol.
  • the Ncol (5 '-) blunt-ended (3') fragments from pAT2SlBN15 and pAT2SlBN19 were then ligated into pCW109 that was previously linearised by digestion with Ncol-Sma I.
  • the resulting constructs were designated pCW109BN15 and pCW109BN19.
  • the equivalent version that included the BNwt gene was a little more involved since this gene still has an internal Ncol site.
  • a vector suitable for plant transformation requires the presence of the right and left border sequences that Agrobacterium utilizes to introduce foreign DNA into the nuclear genome of plants, and also encompasses a selection cassette that allows for antibiotic selection of those plants that show successful integration of the foreign DNA into their genome. Ideally a second selection cassette should also be available for selecting those bacteria transformed with the binary vector for manipulation or amplification.
  • the vector chosen to achieve all these desired features was pzs96.
  • pzs96 has - genes encoding the N-phosphotransferase that imparts kanamycin resistance on transformed plants and the ⁇ -lactamase that imparts ampicillin resistance for selection in bacteria.
  • Each of the three pCW109AT2SlBN plasmids were digested with Hindlll.
  • the liberated Hindlll fragments were purified and then mixed separately with an equimolar amount of Hindlll-linearized pzs96 before ligation. After ligation, the DNA was used to transform competent E. coli; transformants were selected on media containing ampicillin. Correct construction was assessed by restriction digest analysis and DNA sequencing. In this way only those binary vectors with the Brazil Nut 2S albumin genes in the correct orientation were retained for use in transforming plants.
  • a suspension of Agrobacterium that had previously been grown overnight in LB containing kanamycin (25 ug/mL), rifampicin (50 ug/mL) and carbenicillin (100 ug/mL) was dispersed in 1 liter of infiltration medium to give an ODOOO of 0.8.
  • the bacterial suspension was poured into a tray that was placed into the bottom of the vacuum cabinet.
  • the pots were suspended inverted in the vacuum cabinet and the shoots of the plants submerged in the solution.
  • the door was closed and the vacuum of a rotary vane oil pump applied for 5 min reaching a final vacuum of 1.5-2.0 Torr.
  • the plants (TI generation) were removed and allowed to grow normally and set seed (4 weeks).
  • T2 seeds from this TI generation were harvested and selected for kanamycin resistance. This selection entailed sterilization of T2 seeds in 50% commercial bleach with 0.02% Tween-20 for 8-10 minutes and then washing in sterile water 3-5 times before sowing. Sterilized seeds were germinated in Petri plates with sterile media containing 1/2 strength Murashige-Skoog salts (Gibco #11117-066) plus 0.7% agar, 1% sucrose and 50 ug/mL kanamycin. Kanamycin was prepared as a 50 mg/mL stock in water, sterilized by passage through a 0.2 um filter, and added to the media after it had been autoclaved and cooled to 60°.
  • the plastic was then removed and plants were grown to maturity using standard practices in growth chambers at 20-25° with fluorescent and incandescent illumination of 100-300 umol/m 2 /sec photosynthetically active radiation and a photoperiod ranging from 12 h to continuous illumination.
  • the T2 plants in soil were allowed to self-fertilize to produce the T3 seeds which were harvested and used for analysis.
  • EXAMPLE 9 Expression of the Brazil Nut 2S Albumin Gene in Transformed Plants
  • the seeds from different lines of T2 generation transgenics harboring the AT2S1BN15, AT2S1BN19 and AT2SlBNwt genes were harvested for analysis.
  • the seeds (10 mg) from the mature plants were first ground to a fine powder in liquid nitrogen and then defatted at room temp with three washes of r ⁇ -hexane.
  • the resulting defatted flour was allowed to dry before extraction with a weak acidic buffer (0.1M citrate, pH 5.0) to solubilize the 2S proteins; the precipitate of other proteins removed by centrifugation.
  • a weak acidic buffer 0.1M citrate, pH 5.0
  • the acid extract was filtered using MicroconTM 0.2 um filtration units (Amicon Inc., Beverly, MA) and samples of the extracts from the transgenics were subjected to amino acid analysis and compared with the 2S albumin extracted from untransformed Arabidopsis (see Table 1).
  • T3 The seeds (T3) from those lines in the T2 generation that showed the most increased Met and Lys content of the 2S fractions were sown to provide a T4 set of seeds for analysis.
  • the seeds were treated as for the previous generation to obtain the 2S protein for analysis and the results shown in Table 1.
  • Table 1 shows the percent by weight of Met and Lys in the 2S fraction of untransformed and transgenic Arabidopsis seeds of the T3 and T4 generations.
  • the percent by weight of Arg was included to indicate that concomitant decrease was observed with Lys increase, as expected.
  • the percent by weight of valine (Val) is also reported, a residue not present in the Brazil Nut 2S albumin, thus providing an intemal reference for comparison of the various 2S extractions.
  • the vectors pETBNwt, pETBN15, pETBNl ⁇ , pETBN17, pETBN18 and pETBNl 9 were used to transform E. coli (BL21) cells and grown in LB medium with kanamycin (30 ug/mL) selection in 50 mL shake cultures at 37° ovemight on an incubated shaker (300 rpm). The next day, the cells were harvested to make glycerol stocks for long term storage and 1 mL was used to inoculate a fresh 50 mL batch of medium with the same selection. When the cells had reached an ODgoo of 0.9, protein expression was induced with 1 mM IPTG. The cells were harvested after overnight incubation on the shaker at 37°.
  • the protein content of the cells was analyzed by incubating a fraction of the cell paste that had been washed with 0.1M tris Cl buffer (pH 8.0) at 100° in a gel SDS loading buffer. Samples of the lysed cell extract were then run on a 18% SDS polyacrylamide gel. After the gel had been run it was allowed to wash in 0.1 M CAPS buffer, pH 10, to remove the Tris-Glycine gel running buffer. The proteins were transferred to PVDF membranes using electrophoretic transblotting procedures and visualized by coomassie blue staining. Those bands with the mobility of the 2S storage protein were identified as the recombinant product by N-terminal sequence and Western analyses.
  • GGC TTA AGG ATG ATG ATG ATG ATG AGG ATG CAA CAG GAG GAG ATG CAA CCC 241 Gly Leu Arg Met Met Met Met Arg Met Gin Gin Glu Glu Met Gin Pro 65 70 75
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO: 4: GAGCTGCAAA TGCGAAGGCC TAAAGATGAT GATG 34
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO: 5: GGGAGCAGAT GAAAAAGATG ATGAAGCTAG CCGAGAATA 39
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO: 6: GTCCCATGAA ATGCCCCTTC GGTGGATCGA TTGCCGGG 38
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO: 13: GCCGCAAGCT TGAATTCGGA TCCTCAGAAC CCGGCAATCG ATCCACCGAA GGGGCATTTC 60 ATGGGACTGA GGTTGCATTT GGAAGGGATA TTCTCGGCTA GCTTCATCAT CTTTTTCATC 120 TGCTCCCCCT TGGGTTGCAT C 141
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO: 19: CCGTACCAGA GCATGCCGAA GAAGGGAATG GAGCCGCACA TGAAAGAGTG CTGCGAGCAG 60 CTGGAGGGGA TGGACGAGAG CTGCAAATGC GAAGGCCTAA AGATG 105
  • MOLECULE TYPE other nucleic acid
  • SEQUENCE DESCRIPTION SEQ ID NO:25: GCGTACGACA TATGCAGGAG AAGTGTAAAG AGCAGATGCA GAAACAGAAG ATGCTTAAGC 60 ACTGCAAGAT GTACATGAAA CAGCAGATGG AGGAGAGCCC GTACCAGAGC ATGCCGAAGA 120 AGGG 124

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Abstract

La présente invention concerne la mise au point de semences et de protéines séminales qui contiennent une quantité accrue d'acides aminés essentiels pour l'homme et les animaux. Plus particulièrement, L'invention concerne la manipulation génétique de la protéine séminale d'albumine Brazil Nut 2S pour lui conférer un pourcentage plus élevé de résidus d'acides aminés essentiels. L'expression d'un gène codant cette protéine séminale obtenue par manipulation génétique chez des plantes transgéniques se traduit par une accumulation accrue d'acides aminés essentiels dans les sememces de ces plantes.
PCT/US1998/006673 1997-04-08 1998-04-06 Proteine seminale genetiquement manipulee contenant un pourcentage eleve d'acides amines essentiels WO1998045458A1 (fr)

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AU68832/98A AU6883298A (en) 1997-04-08 1998-04-06 An engineered seed protein having a higher percentage of essential amino acids

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