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WO1991004270A1 - Proteines modifiees a conservation des semences - Google Patents

Proteines modifiees a conservation des semences Download PDF

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Publication number
WO1991004270A1
WO1991004270A1 PCT/AU1990/000430 AU9000430W WO9104270A1 WO 1991004270 A1 WO1991004270 A1 WO 1991004270A1 AU 9000430 W AU9000430 W AU 9000430W WO 9104270 A1 WO9104270 A1 WO 9104270A1
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WO
WIPO (PCT)
Prior art keywords
protein
phaseolin
plant
dna
storage protein
Prior art date
Application number
PCT/AU1990/000430
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English (en)
Inventor
Commonwealth Scientific And Industrial Research Organisation
Peter Malcolm Colman
Michael Colin Lawrence
Joseph Noozhumurry Varghese
Timothy Couzens Hall
Mauricio Martin Bustos
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Commw Scient Ind Res Org
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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Publication of WO1991004270A1 publication Critical patent/WO1991004270A1/fr

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Classifications

    • 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

Definitions

  • This invention relates to plant seed storage proteins, and in particular to methods of modifying said proteins at specific sites.
  • WO/8903887 discloses a process for production of a mammalian peptide via expression of a modified seed storage protein gene in a transgenic plant.
  • the most commonly used vector is Agrobacterium tumefaciens Ti plasmid, which is successful in transferring genetic
  • seed storage proteins Most of the protein found in mature seeds belongs to a class called the seed storage proteins. This term is used to describe those proteins whose function is to provide, upon germination, a source of fixed carbon and nitrogen to sustain the early heterotrophic growth of the seedling. However, it is because of the importance of seeds to human nourishment that so much work has been directed at understanding seed proteins.
  • seed proteins are those from the agriculturally important legumes, soya bean (Glycine max) , garden pea (Pisum sativum) and French bean (Phaseolus vulgaris), although increasing attention is being given to food crops of the Third World, including cow pea (Vigna unguiculata), mung bean (V. radiata) and pigeon pea (Cajanus cajan).
  • legume storage proteins which are mostly of the salt-soluble globulin class, are a
  • vicilins 15 has been found to occur twice in vicilins 16 , suggesting a repeated structural motif in vicilins.
  • arcelin Another seed storage protein, arcelin, has been identified in certain wild forms of Phaseolus vulgaris (Romero A. et al (1986) Theor. Appl. Genet 72.123-128; Osborn et al. (1986) Theor. Appl. Genet. 71 847-855); these proteins are toxic to bruchid pests of beans, and transfer of the cloned arcelin gene to other bean plant confers insect resistance (European Patent Application No. 337750: The Plant Cell
  • phaseolin the major 7S storage protein from the French Bean Phaseolus vulgaris.
  • Specific protein engineering targets in this system include improved nutritional value, altered stability with respect to functionality in food systems, and general use as a high-level expression system.
  • Phaseolin a vicilin-like molecule
  • Phaseolin trimer M r about 150 000
  • the phaseolin trimer associates into a dodecamer 18 below pH 4.5.
  • phaseolin is a
  • tPA tissue plasminogen activator
  • PAI-1 plasminogen activator inhibitor-1
  • polypeptides of the trimeric seed storage protein phaseolin comprise two amino acids
  • structurally-similar units each made up of a ⁇ -barrel and an ⁇ -helical domain.
  • the ⁇ -barrel has the "jelly-roll" folding topology shown by viral coat proteins and the ⁇ -helical domain shows structural similarity to the helix-turn-helix motif found in certain DNA-binding proteins.
  • the tetramer of trimers referred to above turns out to be the form of the molecule in the crystals studied here.
  • the major barrier to modifying plants to produce seed storage proteins with commercially desirable properties is knowledge of the three-dimensional structure of the storage proteins.
  • phaseolin structure of a representative seed storage protein, phaseolin.
  • thermostability for heat-setting properties in snack foods (c) thermostability for heat-setting properties in snack foods.
  • the present invention provides a mutein which is a variant of a naturally-occurring legume seed storage protein, wherein said mutein has a modified primary structure relative to said legume storage protein, but retains the tertiary and quaternary structure of said legume storage protein.
  • tertiary and quaternary structure For the purposes of this specification, retention of the tertiary and quaternary structure is to be understood to mean that elements of that tertiary and quaternary structure which are not the subject of primary structure modifications are substantially unaffected by said modifications.
  • a plant 7S or 11S storage protein modified at a specific amino acid residue or a specific region of its amino acid sequence, wherein the tertiary and quarternary structure of the naturally occurring storage protein is retained.
  • the modification is selected from the group consisting of:
  • a DNA molecule whose sequence encodes a mutein, as defined above.
  • this DNA encodes a protein having the properties of a plant 7S or 11S storage protein, and also encodes one or more of the modifications set out above.
  • Plasmids, expression vectors, and microorganisms comprising said DNA are also within the scope of the invention.
  • transgenic plant or part thereof having a DNA sequence as definee above.
  • the plant part is a seed.
  • the modified DNA thus represents the initial embodiment of the changes; such DNA will be converted, via the processes of transcription and translation in the cell, to yield the modified seed storage protein.
  • Figure 1 (25 sheets) represents the atomic co-ordinates of phaseolin in orthogonal ⁇ units.
  • phaseolin trimer is coincident with the Y axis of the co-ordinate frame.
  • the coordinates of the C atoms have been deposited by the applicant in the
  • Figure 2 represents schematic diagrams showing the two observed patterns of interaction between the ⁇ + ⁇
  • Figure 3 represents a stereo pair showing a C ⁇ trace of the phaseolin trimer, viewed down the molecular three-fold towards the centre of the tetramer.
  • the N- and C-termini of each subunit are labelled.
  • the two possible ways of linking the two ⁇ + ⁇ units to form the subunit are
  • Figure 4 is a diagram showing the structure of native phaseolin (A) and the proposed high-methionine mutant phaseolin (B) in the neighbourhood of residues 84 to 88. The methionine sidechains of the mutant are seen to be
  • residues in the same ⁇ -sheet as residues 84 to 88 are not shown.
  • Figure 5 is a diagram showing the structure of native phaseolin (A) and the proposed high-methionine mutant phaseolin (B) in the neighbourhood of residues 261-265.
  • the methionine sidechains of the mutant are seen to be
  • residues in the same ⁇ -sheet as residues 261-265 are not shown.
  • the secondary structure assignments are based on the current unrefined model and thus that the precise start and end points of the strands and helices may be in error by one or two amino acids in some places.
  • the 411 residue protein has a five amino-acid insertion after residue 189 and a nine amino-acid insertion after residue 390.
  • the 412 residue protein has a further insertion of one amino acid after residue 100.
  • the chain trace of the electron density map (not shown) is that of the 397 amino-acid protein (termed the ⁇ -type polypeptide 11 ) and shows no break or weakening of electron density in the vicinity of residue 189.
  • the map shows weak evidence of glycan binding at both known Asn-X-Ser/Thr glycosylation triplets 24 .
  • the density is not yet sufficiently clear to provide information about the nature of heterogeneity of the glycans.
  • Figure 2 shows a schematic drawing of the polypeptide. It consists of two structurally-similar units of about 160 amino acids each. These are related by a
  • Each unit is itself an ⁇ + ⁇ two-domain structure, the first of some 110 residues being a classic viral capsid jelly-roll structure, and the second smaller domain being a cluster of three helices, including a
  • a fourth helix in the N-terminal unit which is not structurally associated with the three-helix cluster, forms part of the connection through to the C-terminal unit.
  • the helical domains comprise residues ca 156-181 and 340-371, each being a three-helix cluster. These helices bear a
  • the internal sequence repeat 16 observed in phaseolin, jack bean canavalin and pea vicilin is the basis for the structural repeat described here.
  • the sequence similarity in the internal repeat is low ( ⁇ 15% identity), but the amino-acid alignment corresponds to the structural repeat perfectly in many places and to within a few residues in the worst cases.
  • the domains are remarkably similar; a least-squares fit of the C ⁇ positions in corresponding structural elements of the pair of units yields an r.m.s. deviation of only 2.2 ⁇ .
  • the six structural ⁇ + ⁇ units which comprise the phaseolin trimer are arranged alternately up and down around the
  • trimer is
  • the jelly-roll ⁇ -barrel has a remarkable capacity to exist in different states of oligomerization 32,33 .
  • the B-I-D-G face of the two barrels associate with each around the pseudo-diad in what appears to be a typical example of aligned packing of ⁇ -sheets.
  • the two potential N-linked glycosylation sites are both within the C-terminal barrel on strands A and I respectively. The sugar attached to strand I could contact the neighbouring N-terminal barrel.
  • the helical domains of the subunits are closely associated with each other around the pseudo-diad relating the neighbouring subunits (see Figure 2b). As mentioned above, the
  • helix-turn-helix motifs formed by helices 2 and 3 of each domain are similar to those found in certain DNA-binding proteins 35 , typified by the Cro protein.
  • phaseolin to the helix-turn-helix motif formed by residues 16-36 of Cro yields an r.m.s. deviation of 1.9 ⁇ and 1.3 ⁇ respectively, within the spread determined for these motifs 35 .
  • the capacity of helix-turn-helix structures to bind DNA is associated with their condensation into dimers with diad symmetry coincident with that of double-stranded DNA, in particular with elements of the diad-related helices positioned 34 ⁇ apart and binding to consecutive major grooves of DNA. No such 34 ⁇ period is evident in the dimerisation of the phaseolin helical domains, nor indeed are the two domains identical.
  • phaseolin the second helix in the motif (i.e. the third in the helical domain) is exposed to the environment in both structural units on the polypeptide and within the trimer
  • phaseolin polypeptide translocation of the phaseolin polypeptide from its site of synthesis on membrane-bound polysomes through the endoplasmic reticulum into storage bodies 41,3 makes such an interaction unlikely. Nevertheless, DNA-binding studies could provide direct evidence for or against a role for storage proteins in the regulation of their own high level of synthesis.
  • the inter-trimer contact in the tetramer occurs at the tetrahedral diad axes. It consists of a symmetry-related pair of interactions, each involving the N-terminal ⁇ -barrel of one subunit with the C-terminal ⁇ -barrel of a subunit from the neighbouring trimer.
  • the N-terminal strands of polypeptides from neighbouring trimers are also in contact at the tetrahedral diad.
  • Sequence alignments imply structural similarity of 7S proteins from common bean, jack bean, soybean and pea seeds 12,15,16. Insertions and deletions in these alignments are, with minor modifications, compatible with the phaseolin structure (see Table 1). For the most part, the phaseolin sequence is shorter than the other sequences, the exception being a five residue insertion around position 350, which may affect the structure of the connection between helices 1 and 2 of the C-terminal structural unit.
  • a preliminary structure for jack bean canavalin shows pseudo 32 point group symmetry and a viral capsid domain in each of the two structural units per polypeptide.
  • legumin protein cucurbitin has point group symmetry 23, at least at low resolution (ca 20 ⁇ ) .
  • oligomeric form is believed to proceed via a 7S (trimer) intermediate, the final coalescing of trimers not occurring until cleavage of the polypeptides into the basic and acidic chains 44 . If the trimer form is indeed similar to the vicilin 7S molecules, with pseudo point group 32, then a rearrangement of the subunits within the trimer must accompany the formation of the 11S particle, in order to satisfy the resulting cubic arrangement of diads and triads.
  • phaseolin Transgenic tobacco plants containing normal or modified phaseolin genes were then monitored for the production and deposition of phaseolin. Whereas in both cases phaseolin expression was achieved, deposition of the modified protein in the storage bodies did not occur, suggesting prior
  • site directed mutagenesis is used to introduce desired mutations at defined sites in known fragments of wild type phaseolin cDNA.
  • Mutations are introduced by known methods, such as those described by Kunkel (46), into restriction fragments which include the segment of DNA encoding the locus for the desired mutation.
  • the resulting DNA is used to transfect a host, such as E.coli, and single stranded DNA prepared from plaques.
  • the presence of the desired mutation is confirmed by complete sequencing of the same restriction fragment, for example using the method of Sanger et al. (47).
  • the double stranded replicative form of DNAs of proven mutants is
  • phaseolin protein in transgenic tobacco seeds (Bustos et al., 1990; (49)).
  • the advantages of using the minigene instead of phaseolin genomic DNA fragments derive from the absence of intervening sequences. This facilitates the design of gene constructions and permutations of DNA fragments to combine different mutations.
  • a 381 bp EcoRI-PstI restriction fragment from clone ⁇ wt i- (Bustos et al., 1990) is subcloned into the phagemid vector pBSKS+ (Stratagene) and the resulting clone is used to produce a U-containing single stranded DNA template.
  • Three different synthetic single-stranded oligonucleotide DNA molecules (shown below as oligos I to III) are utilized to mutagenize the wt sequence by the method of Kunkel (46). The individual base substitutions are indicated in boldface.
  • oligo II 5' G GTC TTG ATG aaa cct 3' oligo III 5' gcc ATG ATG ATG ATG aaa cct 3' final gly ser ala Met Met Met Met Met lys pro asp 91
  • a 2.0 kbp Xbal-BamHI restriction fragment from clone ⁇ wt i- (Bustos et al., 1990) is subcloned into the phagemid vector pBSKS+ (Stratagene) and the resulting clone used to produce a U-containing single-stranded DNA template.
  • three synthetic oligonucleotides (oligos IV to VI, shown below) are used to mutagenize the wt sequence using the same method.
  • each oligo is designed to mutate two or three closely spaced nucleotides, and all oligos result in at least one new methionine codon.
  • the sequences at and around the mutated sites are verified using a sequencing primer that hybridizes near the site. After mutagenesis the Xbal-BamHI fragments are replaced into ⁇ wt i- or into the high-methionine mutants modified at the ILVLV site. A maximum of ten new methionine residues result from the combination of mutations at both sites.
  • phaseolin genes are subsequently subcloned into the binary vector pBIN19 and transferred into the genome of tobacco by A. tumefaciens-mediated
  • the structure of the high-methionine mutant phaseolin can be investigated by model-building as follows.
  • An interactive molecular-graphics computer program (QUANTA, Polygen Corporation) was used to model the replacement of residues 84 to 88 and 261 to 265 by methionine.
  • the energyrefinement computer-program XPLOR (Harvard University) was then used to refine the atomic coordinates of the mutant structure to ensure that favourable stereochemistry is achieved, keeping the remainder of the protein as close as possible to the native structure.
  • Leu 65 Leu 76, Phe 252, Leu 264, Ile 177, Leu 181.
  • Elimination of the loop of structure between beta strands E and F of the C-terminal structural unit may be accomplished by deleting residues numbered 278 to 287 and replacing them with a linker of two glycine residues. This increases the stability of phaseolin to enzymes with
  • the selection of sites for mutagenesis may be influenced by the accessibility of the target site to restriction endonucleases.
  • the criteria for assessing whether the desired mutation has been achieved will depend on the nature of the mutation attempted, but include;
  • the mutation is to substitute methionine for another amino acid
  • the presence of one or two met residues could be detected by cyanogen bromide cleavage; the presence of about 10 met residues could be readily assayed by amino acid analysis.
  • Retention of three dimensional structure can be assessed in advance by model building, and tested in the protein actually produced by success in expression in vivo, and by various physical analyses such as molecular weight, solubility, and circular dichroism spectral studies. Other suitable methods will be known to those skilled in the art.
  • sequence homologies indicate that similar results may be expected with related proteins, such as the homologous protein from pea, and to some extent with legumins as implied by the homology described in reference 16.

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Abstract

On prévoit des protéines modifiées à conservation des plantes, danbs lesquelles la modification est réalisée dans la structure primaire de la protéine, les structures tertiaire et quaternaire de la protéine étant retenues. Le site des modifications est sélectionné avec référence à la structure à trois dimensions de la protéine, laquelle vient d'être établie par les inventeurs. On revendique également des molécules d'ADN modifiées, des vecteurs, des plantes génovariées, et des parties et des produits de ceux-ci.
PCT/AU1990/000430 1989-09-20 1990-09-19 Proteines modifiees a conservation des semences WO1991004270A1 (fr)

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AUPJ6441 1989-09-20
AUPJ644189 1989-09-20

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994008018A1 (fr) * 1992-09-28 1994-04-14 Unilever Plc Modification de polypeptides
WO1994010315A3 (fr) * 1992-10-23 1994-09-15 Pioneer Hi Bred Int Procede permettant de renforcer la teneur d'une proteine seminale de stockage de semence en un acide amine selectionne
FR2757538A1 (fr) * 1996-12-19 1998-06-26 Mini Ricerca Scient Tecnolog Gene de glutenine a faible masse moleculaire, vecteur d'expression contenant ce gene, microorganisme modifie avec ce vecteur, plants transgeniques contenant ce gene, et proteine codee par ce gene
WO1998026064A3 (fr) * 1996-12-09 1998-09-03 Dekalb Genetics Corp Methodes d'accroissement du contenu nutritionnel de plantes
US6169232B1 (en) 1997-07-15 2001-01-02 Dow Agrosciences Llc Nucleotide sequences of genes encoding sink protein and uses thereof for improving the nutritional quality of feeds
US7064248B2 (en) 1990-01-22 2006-06-20 Dekalb Genetics Corp. Method of preparing fertile transgenic corn plants by microprojectile bombardment
US7615685B2 (en) 1990-01-22 2009-11-10 Dekalb Genetics Corporation Methods of producing human or animal food from stably transformed, fertile maize plants
US8785130B2 (en) 2005-07-07 2014-07-22 Bio-Id Diagnostic Inc. Use of markers including nucleotide sequence based codes to monitor methods of detection and identification of genetic material
US9150906B2 (en) 2006-06-28 2015-10-06 Bio-Id Diagnostic Inc. Determination of variants produced upon replication or transcription of nucleic acid sequences
EP4139333A4 (fr) * 2020-04-23 2024-10-23 Pioneer Hi-Bred International, Inc. Soja à protéine de graine modifiée

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BIOCHEM. PHYSIOL. PFLANZEN, 183, 1988, pp. 211-218, GERHARD SAALBACH et al., "Construction of Storage Protein Genes with Increased Number of Methionine Codons and their Use in Transformation Experiments". *
FED. PROC. AM. SOC. EXPTL. BIOL, Vol. 46, 6, p. 2023, C.D. DICKINSON et al., "Engineering of Soybean Seed Storage Proteins". *
NATURE, Volume 339, 29 June 1989, pp. 658-659, DAGMAR RINGE, "The Sheep in Wolf's Clothing". *
PLANT MOLECULAR BIOLOGY, Vol. 11, 1988, pp. 717-729, L.M. HOFFMAN et al., "A Modified Storage Protein is Synthesized Processed, and Degraded in the Seeds of Transgenic Plants". *
PROTEIN ENGINEERING, Volume 3, No. 8, 1980, pp. 725-731, CHANSHICK et al., "Improvement of Nutritional Value and Functional Properties of Soybean Glycinin by Protein Engineering". *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7064248B2 (en) 1990-01-22 2006-06-20 Dekalb Genetics Corp. Method of preparing fertile transgenic corn plants by microprojectile bombardment
US7615685B2 (en) 1990-01-22 2009-11-10 Dekalb Genetics Corporation Methods of producing human or animal food from stably transformed, fertile maize plants
WO1994008018A1 (fr) * 1992-09-28 1994-04-14 Unilever Plc Modification de polypeptides
WO1994010315A3 (fr) * 1992-10-23 1994-09-15 Pioneer Hi Bred Int Procede permettant de renforcer la teneur d'une proteine seminale de stockage de semence en un acide amine selectionne
US7547820B2 (en) 1993-08-25 2009-06-16 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
US6960709B1 (en) 1993-08-25 2005-11-01 Dekalb Genetics Corporation Method for altering the nutritional content of plant seed
WO1998026064A3 (fr) * 1996-12-09 1998-09-03 Dekalb Genetics Corp Methodes d'accroissement du contenu nutritionnel de plantes
FR2757538A1 (fr) * 1996-12-19 1998-06-26 Mini Ricerca Scient Tecnolog Gene de glutenine a faible masse moleculaire, vecteur d'expression contenant ce gene, microorganisme modifie avec ce vecteur, plants transgeniques contenant ce gene, et proteine codee par ce gene
US6169232B1 (en) 1997-07-15 2001-01-02 Dow Agrosciences Llc Nucleotide sequences of genes encoding sink protein and uses thereof for improving the nutritional quality of feeds
US8785130B2 (en) 2005-07-07 2014-07-22 Bio-Id Diagnostic Inc. Use of markers including nucleotide sequence based codes to monitor methods of detection and identification of genetic material
US9150906B2 (en) 2006-06-28 2015-10-06 Bio-Id Diagnostic Inc. Determination of variants produced upon replication or transcription of nucleic acid sequences
US10036053B2 (en) 2006-06-28 2018-07-31 Bio-ID Diagnostics Inc. Determination of variants produced upon replication or transcription of nucleic acid sequences
EP4139333A4 (fr) * 2020-04-23 2024-10-23 Pioneer Hi-Bred International, Inc. Soja à protéine de graine modifiée

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