WO1991002800A1 - Sequence de ciblage de thylakoide - Google Patents

Sequence de ciblage de thylakoide Download PDF

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Publication number
WO1991002800A1
WO1991002800A1 PCT/GB1990/001281 GB9001281W WO9102800A1 WO 1991002800 A1 WO1991002800 A1 WO 1991002800A1 GB 9001281 W GB9001281 W GB 9001281W WO 9102800 A1 WO9102800 A1 WO 9102800A1
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WO
WIPO (PCT)
Prior art keywords
protein
gcc
sequence
plant
plant cell
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Application number
PCT/GB1990/001281
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English (en)
Inventor
Colin Robinson
Original Assignee
Advanced Technologies (Cambridge) Ltd.
Agricultural Genetics Company Ltd.
Biotal Ltd.
Bp Nutrition Ltd.
Ciba-Geigy Plc
Imperial Chemical Industries Plc
Rhone-Poulenc Ltd.
Schering Agrochemicals Limited
Nickerson International Seed Company Limited
Twyford Seeds Ltd.
Unilever U.K. Central Resources Ltd.
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Application filed by Advanced Technologies (Cambridge) Ltd., Agricultural Genetics Company Ltd., Biotal Ltd., Bp Nutrition Ltd., Ciba-Geigy Plc, Imperial Chemical Industries Plc, Rhone-Poulenc Ltd., Schering Agrochemicals Limited, Nickerson International Seed Company Limited, Twyford Seeds Ltd., Unilever U.K. Central Resources Ltd. filed Critical Advanced Technologies (Cambridge) Ltd.
Priority to JP2512183A priority Critical patent/JPH07501681A/ja
Publication of WO1991002800A1 publication Critical patent/WO1991002800A1/fr

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Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
    • 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)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8221—Transit peptides
    • 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
    • C12N15/8257—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 for the production of primary gene products, e.g. pharmaceutical products, interferon
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/08—Fusion polypeptide containing a localisation/targetting motif containing a chloroplast localisation signal

Definitions

  • This invention relates to targeting proteins into the thylakoid lumen of chloroplasts in plants.
  • chloroplast genome is too small to carry all the genetic information needed for the large number of different proteins which function in that organelle.
  • An estimated 80% of chloroplast proteins are translated from cytoplasmic poly(A) + RNA and are thus encoded in the nuclear genome. These proteins are synthesi ⁇ ed as precursor molecules that are post-translationally imported into chloroplasts. The imported precursors are processed within the chloroplasts by proteases to form mature proteins. In addition to being imported, these proteins are directed to one of several different chloroplast compartments: outer membrane, intermembrane space, inner membrane, stroma, thylakoid membrane or thylakoid lumen.
  • Nuclear-encoded thylakoid lumen proteins are synthesised as larger precursors and imported into the chloroplast by a two-step process. Precursors are initially transported into the stroma and processed to intermediate forms by a stromal processing peptidase, after which the intermediates are transferred across the thylakoid membrane and processed to the mature size by a thylakoidal peptidase. Analysis of the pre-sequences of lumenal proteins has shown that they consist of two domains, which are believed to direct "envelope transfer" of the precursor and then "thylakoid transfer" of the stromal intermediate. The targeting properties of the two domains have been tested in studies using chimaeric proteins.
  • Plastocyanin is normally located in the thylakoid lumen.
  • the pre-sequence of Silene plastocyanin was found to direct the transport of a foreign protein, yeast ⁇ uperoxide dismutase, into the chloroplast stroma.
  • a foreign protein yeast ⁇ uperoxide dismutase
  • ferredoxin was targeted into the thylakoid lumen by the plastocyanin pre-sequence (Smeeken ⁇ e_t al, Cell 46, 365-375, 1986; Smeekens e_t al , Plant. Hoi. Biol. 9_, 377-388, 1987).
  • This suggested that the mature sequences of lumenal proteins may contain some information essential for transport across the thylakoidal membrane.
  • the present invention provides a chimaeric gene encoding (a) the pre-sequence of the 33kDa protein of the photosynthetic oxygen-evolving complex of a photosynthetic organism or a modified version of the said pre-sequence which is capable of targeting a passenger protein into the thylakoid lumen of a chloroplast, fused to (b) a heterologous passenger protein.
  • the invention also provides a vector which incorporates such a chimaeric gene under the control of regulatory elements capable of enabling the gene to be expressed in a plant cell transformed with the vector.
  • the vector may further contain a region which enables the chimaeric gene and associated regulatory elements to be transferred to and stably integrated in a plant cell genome.
  • the vector is generally a plas id.
  • Plant cells can be transformed with such a vector.
  • the invention therefore further provides plant cells which harbour a chimaeric gene as above.
  • Transgenic plants may be regenerated from such plant cells.
  • a transgenic plant can be obtained which harbours in its cells a chimaeric gene. Seed may be obtained from the transgenic plants.
  • the invention further provides a method of producing a desired protein in a plant cell, which method comprises:
  • the invention additionally provides a method of producing a transgenic plant capable of producing a desired protein, which method comprises:
  • the desired proteins can be isolated from the transformed plant cells obtained by the first method and from the plants obtained by the second method.
  • the chimaeric gene encodes the pre-sequence of the 33kDa protein of the photosynthetic oxygen-evolving complex of a photosynthetic organism, or a modified version of this pre-sequence which is also capable of targeting a passenger protein into the thylakoid lumen, linked to a heterologous passenger protein.
  • the pre-sequence may therefore by the pre-sequence of the 33kDa protein of the photosynthetic oxygen-evolving complex of a plant, which may be a monocotyledonous or dictotyledonous plant.
  • the pre-sequence is the pre-sequence of the 33kDa protein of wheat.
  • the pre-sequence of the 33kDa protein will be designated 33K and both 33K and modified versions of it will be termed as a pre-sequence.
  • the pre-sequence may be fused directly to the amino-terminu ⁇ of the passenger protein.
  • the linking sequence may be composed of up to 40 amino acid residues, for example up to 30, up to 20 or up to 10 residues. Any such linker must not interfere with the ability of the pre-sequence to target the passenger protein into the thylakoid lumen.
  • the portion of the gene encoding the pre-sequence and any intervening linking sequences may be prepared by standard synthetic techniques.
  • the linking sequence comprises the natural amino-ter inal residues of the mature wheat 33kDa protein.
  • a pre-sequence, together with a number of the natural amino-terminal residues of the mature 33kDa protein, may be fused in front of the passenger protein.
  • the first 10 to 25, for example 20 to 25 and in particular 12 or 22, natural amino-terminal residues of the mature 33kDa protein may be present.
  • a short non-natural linker sequence generally resulting from the DNA manipulations necessary to construct a chimaeric gene, may separate the natural residues of the mature 33kDa protein from the amino terminus of the passenger protein. For example, up to 4 non-natural further linker residues may be present.
  • a suitable pre-sequence is therefore wheat 33K.
  • This pre-sequence and its corresponding DNA sequence is shown in Table 1.
  • GGC GCG ACG GCG Gly Ala Thr Ala
  • a modified version of 33K may incorporate one or more amino acid substitutions, insertions and/or deletions and/or an extension at either or both ends.
  • one or more amino acid residues of 33K may be substituted or deleted, or one or more additional residue may be inserted, provided the physicochemical character of the pre-sequence is preserved, i.e. in terms of charge density, hydrophilicity/hydrophobicity and, size and configuration.
  • Candidate substitutions include:
  • the sequence may be extended by up to 10 amino acid residues at either end. Up to, for example, 5 amino acid residues may be added therefore to the amino-terminu ⁇ or to the carboxy-terminu ⁇ .
  • 33K is modified, typically there i ⁇ a degree of homology of at least 70% between 33K and the modified pre- ⁇ equence. The degree of homology may be 85% or more or 95% or more. Any modified ⁇ equence, however, must be capable of targeting a pas ⁇ enger protein into the thylakoid lumen.
  • the heterologous pas ⁇ enger protein may be any heterologous protein it is desired to target into the thylakoid lumen.
  • heterologous is meant that the passenger protein is not naturally linked to the pre-sequence.
  • the passenger protein i ⁇ not the wheat 33kDa protein of the oxygen-evolving complex.
  • the photosynthetic properties of plants may be altered by targeting particular proteins into the thylakoid lumen.
  • a passenger protein may therefore be a protein which could improve the photosynthetic capacity of a plant, for example a mutant version of a protein already present in the lumen or a herbicide-resistant analogue isolated from other plants or bacteria.
  • the invention may be used to ⁇ ynthesi ⁇ e large quantities of protein.
  • a chimaeric gene may therefore contain the DNA sequence ⁇ hown in Table 1. This sequence may be extended at the 3'-end as above, for example based on the DNA sequence shown in Table 2. A modified version of the DNA sequence ⁇ hown in Table 1 may be provided.
  • an intervening DNA sequence encoding linker amino acid residues is provided.
  • a modified pre-sequence may be obtained by introducing corresponding changes into the DNA sequence encoding 33K. This may be achieved by any appropriate technique, including restriction of the DNA sequence for 33K with an endonuclease, insertion of linkers, use of an exonuclease and/or a polymera ⁇ e and site-directed mutagene ⁇ is.
  • a shorter DNA sequence therefore may be obtained by removing nucleotides from the 5'-terminu ⁇ or the 3'-terminus of the DNA sequence encoding 33K, for example using an exonuclease such as BAL 31.
  • a modified DNA sequence encodes a pre-sequence capable of targeting a passenger protein into the thylakoid lumen may be readily ascertained.
  • Transcripts of a chimaeric gene encoding the modified pre-sequence and a passenger protein are translated and a chloroplast import assay effected as described in the Example.
  • Plant cells can be transformed with a chimaeric gene directly or by way of a vector incorporating the gene.
  • a vector incorporates the chimaeric gene under the control of regulatory elements capable of enabling the gene to be expressed in a plant cell transformed with the vector.
  • regulatory elements include tran ⁇ criptional control sequences, for example as above, and translational initiation and/or termination sequences.
  • the vector typically contains too a region which enables the chimaeric gene and associated regulatory control elements to be transferred to and stably integrated in the plant cell genome.
  • the chimaeric gene is therefore typically provided with transcriptional regulatory sequences and/or, if not pre ⁇ ent at the 3'-end of the coding sequence of the gene, a stop codon.
  • a DNA fragment may therefore also incorporate a promoter, Shine-Dalgarno sequence and/or terminator sequence which are capable of enabling the gene to be expres ⁇ ed in plant cells.
  • the promoter may be a plant promoter, for example the 35S cauliflower mosaic virus promoter or a nopaline synthase or octopine ⁇ yntha ⁇ e promoter.
  • Transformed cells are selected by growth in an appropriate medium.
  • Plant tissue can therefore be obtained comprising a plant cell which harbours the chimaeric gene, for example in the plant cell genome, the gene being expressible in the plant cell.
  • Plants can then be regenerated which include the chimaeric gene in their cells, for example integrated in the plant cell genome, ⁇ uch that the gene can be expre ⁇ ed.
  • the regenerated plants can be reproduced and, for example, seed obtained.
  • a preferred way of transforming a plant cell is to use Agrobacterium tumefaciens containing a vector comprising the chimaeric gene.
  • a hybrid plasmid vector may therefore be employed which compri ⁇ e ⁇ :
  • the DNA to be integrated into the plant cell genome is delineated by the T-DNA border ⁇ equences of a Ti-plasmid. If only one border sequence is present, it i ⁇ preferably the right border ⁇ equence.
  • the DNA sequence which enables the DNA to be transferred to the plant cell genome is generally the virulence (vir) region of a Ti-plasmid.
  • the chimaeric gene and its transcriptional and translational control elements can therefore be provided between the T-DNA borders of a Ti-plasmid.
  • the plasmid may be a disarmed Ti-plasmid from which the genes for tumorigenicity have been deleted.
  • the chimaeric gene and its transcriptional and control elements can, however, be provided between T-DNA borders in a binary vector n trans with a Ti-plasmid with a vir region.
  • Such a binary vector therefore comprises:
  • Agrobacterium tumefaciens therefore, containing a hybrid plasmid vector or a binary vector i_n trans with a Ti-plasmid pos ⁇ essing a vir region can be u ⁇ ed to tran ⁇ form plant cells.
  • Ti ⁇ ue explant ⁇ ⁇ uch a ⁇ ⁇ tem ⁇ or leaf discs may be inoculated with the bacterium.
  • the bacterium may be co-cultured with regenerating plant protoplast ⁇ .
  • Plant protoplasts may also be transformed by direct introduction of DNA fragments which encode the chimaerica gene and in which the appropriate transcriptional and translational control elements are pre ⁇ ent or of a vector incorporating ⁇ uch a fragment. Direct introduction may be achieved u ⁇ ing electroporation or polyethylene glycol.
  • Plant cells from monocotyledonou ⁇ or dicotyledonou ⁇ plants can be transformed according to the present invention.
  • Monocotyledonous species include barley, wheat, maize and rice.
  • Dicotyledonous species include tobacco, tomato, sunflower, petunia, cotton, sugarbeet, potato, lettuce, melon, soybean, canola (rape ⁇ eed) and poplars.
  • Tis ⁇ ue cultures of transformed plant cells are propagated to regenerate differentiated transformed whole plants.
  • the transformed plant cells may be cultured on a suitable medium, preferably a selectable growth medium. Plants may be regenerated from the resulting callus. Transgenic plants are thereby obtained whose cells harbour the chimaeric gene, for example integrated in their genome, the gene being expres ⁇ ible in the cells. Seed from the regenerated plant ⁇ can be collected for future use.
  • FIG. 1 shows the structure of the wheat 33K-DHFR fusion protein. Sites of cleavage by the stromal and thylakoidal proces ⁇ ing peptida ⁇ es (SPP,TPP) are denoted by arrows. The precise site of cleavage by SPP is not known.
  • Figure 2 show ⁇ the tran ⁇ port of wheat 33K-DHFR into i ⁇ olated pea chloroplasts.
  • Lane 1 wheat 33K-DHFR (lane 1) wa ⁇ imported and samples analysed without protease treatment (lane 2) and after protease treatment of the thylakoids (lane 3).
  • Lanes 4,5 thylakoid samples were sonicated for 5 sec and then centrifuged to generate soluble and membrane fractions, respectively.
  • S-DHFR, T-DHFR, stromal and thylakoidal DHFR forms, respectively.
  • FIG 3 shows the processing of wheat 33K-DHFR by the stromal and thylakoidal peptidases.
  • Wheat 33K-DHFR (lane 1) was incubated with partially purified stromal (lane 2) or thylakoidal (lane 4) processing peptidase.
  • Pea seedlings (Pisum ⁇ ativum, var.. Feltham First) were grown, and chloroplast ⁇ isolated as described (Robinson and Ellis, Eur. J. Biochem. 152, 67-73, 1985). Radioactive materials were obtained from Amersham International, UK.
  • a mouse DHFR cDNA clone, pDHFR2Z was kindly provided by Dr J.V. Cullimore (University of Warwick, GB).
  • This vector contained the coding region from pDS5/2 (Stueber e_t al, EMBO J. 3_, 3143-3148, 1984) excised using BamHl/HindHI and ligated into pGem2z (Promega Biotech) which had been digested with Ba Hl and Smal.
  • a cDNA clone encoding wheat pre-33K, p33K-2 (Kirwin et al, EMBO J.
  • Chloroplast import as ⁇ ays were carried out e ⁇ entially as described (Robinson and Ellis, 1985). After incubation, non-imported proteins were digested using protease K (150//g ml "1 , 45 min, 4°C), and the chloroplasts were washed once, lysed in 20mM Tri ⁇ -HCl, pH 8.0, and centrifuged at 10,000 x g for 10 min to generate stromal and thylakoid fractions. Thylakoid ⁇ were protease K-treated as above where appropriate.
  • the encoded protein (wheat 33K-DHFR) was synthesi ⁇ ed by _in vitro tran ⁇ cription/tran ⁇ lation and incubated with isolated pea chloroplasts.
  • Figure 2 shows that the fusion protein was imported and converted to two forms, the larger of which is located in the stroma and the smaller associated with the thylakoid ⁇ .
  • the fusion protein was incubated with the stromal and thylakoidal proces ⁇ ing peptida ⁇ e ⁇ both of which have been exten ⁇ ively purified (Robinson and Ellis, Eur. J. Biochem. 142, 337-342, 1984; Kirwin et al, J. Biol. Che . 262, 16386-16390, 1987) .
  • Figure 3 shows that the stromal peptidase converts wheat 33K-DHFR to a form which is slightly larger (as judged by SDS-polyacrylamide gels) than the stromal polypeptide generated during import. The difference in mobilities is slight but reproducible.
  • the thylakoidal peptidase processes the fusion protein to a polypeptide of identical mobility to imported, thylakoidal DHFR.
  • E. co i harbouring p33K-2 and E. coli harbouring p33K-DHFR were deposited on 23 August 1989 at the National Collection of Industrial and Marine Bacteria, Aberdeen, GB under accession number NCIMB 40179 and NCIMB 40180 respectively.

Abstract

Un gène chimérique s'exprimant dans les cellules de plantes code pour (a) la pré-séquence de la protéine 33kDa du complexe photosynthétique évoluant dans l'oxygène d'un organisme photosynthétique ou une version modifiée de ladite pré-séquence qui est capable de cibler une protéine passagère dans le lumen thylakoïde d'un chloroplaste, soudée à (b) une protéine passagère hétérologue. La protéine passagère hétérologue peut donc être déchargée dans le lumen thylakoïde de chloroplastes de cellules de plantes.
PCT/GB1990/001281 1989-08-14 1990-08-14 Sequence de ciblage de thylakoide WO1991002800A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2512183A JPH07501681A (ja) 1989-08-14 1990-08-14 チラコイドへ運搬する配列

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB8918496.4 1989-08-14
GB8918496A GB8918496D0 (en) 1989-08-14 1989-08-14 Thylakoid targeting sequence

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WO1991002800A1 true WO1991002800A1 (fr) 1991-03-07

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EP (1) EP0487618A1 (fr)
JP (1) JPH07501681A (fr)
CA (1) CA2064903A1 (fr)
GB (1) GB8918496D0 (fr)
WO (1) WO1991002800A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000003012A2 (fr) * 1998-07-10 2000-01-20 Calgene Llc Expression de peptides eukaryotes dans les plastes de vegetaux
US6512162B2 (en) 1998-07-10 2003-01-28 Calgene Llc Expression of eukaryotic peptides in plant plastids
KR100362320B1 (ko) * 1998-08-19 2004-03-20 학교법인 명지학원 엽록체를타겟팅하는신규한유전자발현방법

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
CELL, Volume 56, 27 January 1989, Cell Press, Pub., K. KEEGSTRA: "Transport and Routing of Proteins into Chloroplasts", pages 247-253 see page 250, right-hand-column, last paragraph - page 251, left-hand-column *
EMBO Journal, Volume 7, No. 9, 1988 IRL Press Limited, (Oxford, GB), D. DE BOER et al.: "In Vivo Import of Plastocyanin and a Fusion Protein into Developmentally Different Plastids of Transgenic Plants", pages 2631-2635 see the whole document *
EMBO Journal, Volume 8, No. 11, November 1989, K. KO et al.: Targeting of Proteins to the Thylakoid Lumen by the Bipartite Transsit Peptide of the 33 kd Oxygenevolving Protein", pages 3187-3194 see the whole document *
EMBO Journal, Volume 8, No. 8, 1989, IRL Press, P.M. KIRWIN et al.: "ATP-Dependent Import of a Lumenal Protein by Isolated Thylakoid Vesicles", pages 2251-2255, see the whole Document *
FEBS Letters, Volume 253, No. 1,2, 14 August 1989, Elsevier Science B.V., J.W. MEADOWS et al.: "Targeting of a Foreign Protein into the Thylakoid Lumen of Pea Chloroplasts", pages 244-246 see the whole document *
Journal of Biological Chemistry, Volume 263, No. 29, 15 October 1988, The American Society for Biochemistry and Molecular Biology, Inc. (US), L. COMAI et al.: "Chlorplast Transport of a Ribulose Bisposphate Carboxylase Small Subunit-5-Enolpyru-vyl- 3-Phosphoshikimate Synthase Chimeric Protein Requires part of the Mature Small Subunit in Addition to the Transit Peptide", pages 15104-15109 see the whole document *
Plant Molecular Biology, Volume 9, 1987, Martinus Nijhoff, Pub., Dordrecht, NL; S. SMEEKENS et al.: "Import into Chlorplasts of a Yeast Mitochondrial Protein Directed by Ferredoxin and Plastocyanin Transit Peptides", pages 377-388 see the whole document *
Plant Physiology, Volume 89, No. 4, April 1989, Annual Meeting of the American Society of Plant Physiologists, (Toronto, Ontario, Canada), 30 July - 3 August 1989, K. KO et al.: "Targeting of Proteins to the Thylakoid Lumen", page 143 see page 143; Abstract 855 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000003012A2 (fr) * 1998-07-10 2000-01-20 Calgene Llc Expression de peptides eukaryotes dans les plastes de vegetaux
WO2000003012A3 (fr) * 1998-07-10 2000-06-08 Calgene Llc Expression de peptides eukaryotes dans les plastes de vegetaux
US6512162B2 (en) 1998-07-10 2003-01-28 Calgene Llc Expression of eukaryotic peptides in plant plastids
US6812379B2 (en) 1998-07-10 2004-11-02 Calgene Llc Expression of eukaryotic peptides in plant plastids
US7259293B2 (en) 1998-07-10 2007-08-21 Calgene Llc Expression of eukaryotic peptides in plant plastids
KR100362320B1 (ko) * 1998-08-19 2004-03-20 학교법인 명지학원 엽록체를타겟팅하는신규한유전자발현방법

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GB8918496D0 (en) 1989-09-20
CA2064903A1 (fr) 1991-02-15
EP0487618A1 (fr) 1992-06-03
JPH07501681A (ja) 1995-02-23

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