CA2064903A1 - Thylakoid targeting sequence - Google Patents
Thylakoid targeting sequenceInfo
- Publication number
- CA2064903A1 CA2064903A1 CA 2064903 CA2064903A CA2064903A1 CA 2064903 A1 CA2064903 A1 CA 2064903A1 CA 2064903 CA2064903 CA 2064903 CA 2064903 A CA2064903 A CA 2064903A CA 2064903 A1 CA2064903 A1 CA 2064903A1
- Authority
- CA
- Canada
- Prior art keywords
- protein
- sequence
- vector
- plant cell
- plant
- Prior art date
- Legal status (The legal status 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 status listed.)
- Abandoned
Links
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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Pharmacology & Pharmacy (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
ABSTRACT
THYLAKOID TARGETING SEQUENCE
A chimaeric gene expressible in plant cells encodes (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 heterologous passenger protein can therefore be delivered into the thylakoid lumen of chloroplasts in plant cells.
THYLAKOID TARGETING SEQUENCE
A chimaeric gene expressible in plant cells encodes (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 heterologous passenger protein can therefore be delivered into the thylakoid lumen of chloroplasts in plant cells.
Description
2~9~3 THYLAKOID TARGETING SEQUENC~
~ hi~ invention relates to targeting proteins into the thylakoid lumen of chloroplasts in plants.
The chloroplast genome ie too small to carry ~11 the genetic information needed for the large number of different proteins which function in that organelle. An esti~at_d 80 of chloroplast proteins are translated from cytopla~mic poly(A)~ RNA and are thu~ encoded ~n the nuclear genome.
The6e proteins are ~ynthe6i6ed as pr~curcor molecules that are po6t-translationally imported into chloropla6ts. The imported precursor6 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, 6troma, thylakoid membrane ~ thylakoid lumen.
Nuclear-encoded thylakoid lumen proteins are synthesised as larger precursors and imported into the chloroplast by a two-~tep proce~s. Precur~or~ are initially tran~ported into the strom~ and proces~cd to intermediate forms by a stromal proee661ng peptldaso, after which thc intermediates ~re tran6erred acro~ the thylakoid membrane and proce6sed to the mature ~ize by ~ thylDko1dal peptidase.
Analysis of the pre-~equence6 of lumenal protein~ has ~hown that they consist of two domain~, wh$ch are belleved to direct "envelope transfer" of the precursor and then "thylakoid transfer" of the stromal intermediate.
20~ ~Q'~
The t~rgeting propertie~ of the two do~ains have been tested in ~tudie~ using chimaeric proteins. Pl~stocyanin is normally located in the thylakoid lumen. The pre-~equence of Silene pl~ttocyanin was found to direct the tran~port of a foreign protein, yeast ~uperoxide dismut~se, into the chloropla~t 6troma. ~owever, neither ~his protein nor a etromal prctein, ferredoxin, W~5 t~rgeted into the thyl~koid lumen by the plastocy~nin pre-~equence (Smeeken~ et al, Cell 46, 365-375, l9B6; Smeekens et 1, Plant. Mol. Biol. 9, 377-38B, l9B7). Thi6 6ugsested th~t the mature ~equences of lumen~l proteins m~y contain ~ome information essenti~l for transport across the thylakoidal membr~ne.
We have now fused the pre-sequence of another lumenal protein, the 33kDa protein of the photosynthetic oxygen-evolving complex (33X) in front of mouse dihydrofol~te reductase (DHFR), ~ cytopla6mic protein whlch has been used a~ a pas~en~er protein in mitochondrlal protein tran6port studies (Eilers and Schatz, Nature 322, 22B-232, 19~6). In contrast to the previou6 ~ttempt~ to target foroign proteins into the thyl~koid lumen, the DHFR w~ efflciently t~rgeted to the thyl~kold lumen. Thl~ findlng h~ general ~pplicability.
~ ccordlngly, the present lnvent~on provide~ A
chimaerlc gene encoding (~) the pre-~equence of thc 33kDa proteln of the photosynthetic oxygen-evolving complex of photosynthetic org~nism or ~ modlfied vertion of the ~aid pre-sequence which i5 capable of targeting a pas~enger 2 ~
protein into the thylakoid lumen of a chloropla6t, fused to (b) a heterQlsgous passenger protein.
The invention also provides a vector which ~ncorporates ~uch a chimaeric gene under the control of regulatory elements capable of enabling the gene to be expressed ~n ~ plant cell transformed with the vector. The vector may further contain ~ region which enables the chimaeric gene and associ~ted regulatory ele~ents to be transferred to ~nd 6tably ~ntegrated in ~ plant cell genome.
The vector is generally a plasmid.
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 obta$ned which harbour~ in itC cells ~ 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:
(i) transforming a pl~nt cell with ~ vector according to the invention, the 4aid passenger proteln being the desired protein; and (li) culturing the tr~n~formed plant cell under condittons which allow expression of the protein.
The invent$on additionally provider a method of producing a transgenic plant capable of producing a desired protein, which method comprises:
~ i) transforminq a plant cell with a vector 2 ~ Q ~
according to the invention, the ~aid passenger protein being the desired protein; ~nd (ii) regenerating plants from the transformed cells.
The desired proteins can be isolated from the transformed plant cellE obtained by the fir~t method ~nd from the plants obtained by the ~econd method.
The chimaeric gene encodes the pre-sequence of the 33kDa protein of the photosynthetic oxygen-evolving complex of a photosynthetic organism, or a modified ver&ion of this pre-sequence which is al60 capable of targeting a p~ssenger protein into the thylakoid lumen, linked to a heterologous passenger protein. The pre-sequence may therefore by the pre-sequence of the 33kDa protein of the photosynthetic oxygen-evolving complex of a pl~nt, which may be a monocGtyledonous or dictotyledonous plant. Preferably the pre-sequence is the pre-6eguence of the 33kDa protein of wheat. For convenience here, the pre-~equence of the 33kDa protein will be design~ted 33R and both 33R and modified versions of it will be termed as a pre-sequence.
The pre-~equence may be fused directly to the amino-terminu~ of the pas~enger protein. Alternatively, there may be ~n intervening linking sequence. ~he linking sequence m~y be composed of up to 40 amino ncid residues, for ex~mple up to 30, up to 20 or up to 10 re6idue~ Any such linker must not interfere with the ability of the pre-sequence to t~rget the pa~senger protein into the thylakoid lumen. The portion of the gene encoding the - 5 - 20$~f3~
pre-6equence ~nd any intervening linking ~equences ~ay be prepared by st~ndard synthetic techniques.
Preferably the linking ~equence comprises the natural amino-terminal re~idues of the ~ature wheat 33kDa protein.
A pre-sequence, together with a number of the natural amino-terminal residue~ of the mature 33kDa prDtein, may be fused in front of the passenger protein. The fir~t 10 to 25, for example 20 to 25 ~nd ~n particular 12 or 22, natural ~mino-terminal resldue~ of the mature 33kDa protein may be pre6ent. A 6hort non-natural linker sequence, generally resulting from the DNA manlpulations necessary to construct A chimaeric qene, may separate the natural residue6 of the ~ature 33kDa protein from the amino terminus of the p~ssenger prote~n. For example, up to 4 non-natural further linker residue6 may be present.
A suita~le pre-cequence iB therefore wheat 33K. Thl6 pre-sequence and it~ corre~ponding DNA sequence lc chown ln Table l.
TABLE l ATG GCA GCG TCT CTC CAA GCC GCG GCC ACG CTG ATG CCG GCC AAG
Met Ala Ala Ser Leu Gln Ala Ala Ala Thr Leu Met Pro Ala Lys ATC GGC GGC CGG GCC TCC TCC GCG CGA CCG TCG TCG CAC GTC GCC
Ile Gly Gly Arg Ala Ser Ser Ala Arq Pro Ser Ser H16 V~l Al~
CGG GCG TTC GGC GTC GAC GCT GGC GCC AGG ATC ACC TGC TCC CTG
Arg Ala Phe Gly Val Asp Ala Gly Al~ Arq Ile Thr Cy~ Ser Leu CAG TCC GAC ATC AGG GAG GTC GCA AGC AAG TGC GCC GAC GCC GCC
Gln Ser Asp Ile Arg Glu Val Ala Ser Lys Cys Ala Asp Ala Ala 2 ~ 3 AAG ATG GCC GGC TTC GCC CTC GCC ACC TCT GCC CTC CTC GTC TCC
Lys Met Ala Gly Phe Ala Leu Ala Thr Ser Ala Leu Leu Vdl Ser GGC GCG ACG GCG
Gly Ala Thr Ala The amino ~cid ~e~uence of ~hea~ 33K ~nd the natural amino-terminal residues of the wheot 33kDa protein, ~nd the corresponding D~A ~equence, i~ ~hown in Table 2. ~ denotes the end of wheat 33R.
ATG GCA GCG TCT CTC CAA GCC GCG GCC ACG CTG ATG CCG GCC AAG
Met Ala Al~ S~r Leu Gln Ala Ala Ala Thr Leu Mct Pro Ala Ly8 ATC GGC GGC CGC GCC TCC TCC GCG CGA CCG TCG TCG CAC GTC GCC
Ile Gly Gly Arg Ala Ser Ser Ala Arg Pro Ser Ser Hi~ Val Ala CGG GCG TTC GGC GTC GAC GCT GGC GCC AGG ATC ACC TGC TCC CTG
Arg Alo Phe Gly Vol Asp Alo Gly Alo Arg Ile Thr Cy6 Ser Leu CAG TCC GAC ATC AGG GAG GTC GCA AGC AAG TGC GCC GAC GCC GCC
Gln Ser A6p Ile Arg Glu Vol Alo Ser Lys Cy8 Alo Asp Alo Al~
AAG ATG GCC GGC TTC GCC CTC GCC ACC TCT GCC C-C CTC GTC TCC
Ly5 Met Alo Gly Phe Ala Leu Ala Thr Ser Al~ Leu Leu V~l Ser GGC GCG ACG GCG GAG GGG GCG CCC AAG AGG CTG ACC TTC GAC GAG
Gly Alo Thr Al~Glu Gly Al~ Pro Ly6 Arg Leu Thr Ph~ A~p Glu ATC CAG AGC AAG ACC TAC ATG GAG GTG AAG GGT ACC GGC ACC GCG
Il~ Gln Ser Ly6 Thr Tyr Met Glu Val Ly6 Gly Thr Gly Thr Al~
~ modif~ed vercion of 33K moy ~ncorporate one or more amino acid ~ubstitutions, ~n~ertions ~nd/or deletion6 and/or An exten6ion at either or both ends. ror example one or more amino acid re~idues of 33K may be 6ubstituted or deleted, or one or more addit$onal residue may be inserted, 2 f~ L~
provided the physicochemical character of the pre-sequence is preserved, i.e. in terms of charge density, hydrophilicity/hydrophobicity and, ~ize and configuration.
Candidate substitutions include:
- Ser by Thr and vice versa, - Glu by Asp and vice ver~a, ~nd - Gln by Asn and vice versa.
As far as exten6ions are concerned, the sequence ~ay be extended by up to 10 amino acid residues at either end.
Up to, for ex~mple, 5 amino acid residues may be added therefore to the amlno-terminus or to the carboxy-terminus.
Where 33~ i~ modified, typlcally thcre ~ a degree of homology of at least 70% between 33X and the modified pre-sequence. The degree of homology mAy be 85~ or more or 95% or more. Any modified GeqUenCe, howover, must be capable of t~rgeting a pa6senger protein into the thylakoid lumen.
The heterologous p~s6enger protein may be ~ny heterologou6 proteln lt i6 deslred to target into the thylakold lumen. By "heterologou~" is moant that the passenger protein is not naturally linked to the pre-sequence. In part~cular, the passenger proteln i~ not the wheat 33kD~ protoin of the oxygen-evolving complox. The photo6ynthotic prop~rties of plants may bo alte~ed by targeting p~rticular proteins into the thylakoid lumen. A
passenger protein may therefore be ~ protein which could improve the photosynthetic c~pacity of a plant, for ex~mple - 8 - ~ 3 ~ utant ver~ion of a protein ~lready present in the lumen or a herbicide-resist~nt ~nalogue isolated from other pl~nts or bacteria. Alternatively, the invention may be used to ynthesi~e large quantities of protein.
A chimaeric gene may therefore contain the DNA
sequence 6hown in ~able 1. ~h$6 ~equence may be extended ~t the 3~-end ~8 ~bove, for example ba~ed on the DNA ~equence 6hown in Ta`ole 2. A modified version of the DNA sequence ~hown $n T~ble 1 may be provided. The DNA ~eguence encoding the pre-sequence i~ lig~ted to a DNA sequence encoding the passenger protein. Optionally an intervening DNA 6equence encoding linker ~mino acid re~idues is provided.
A modified pre-sequence may be obtained by lntroducing corre6ponding ch~nges into the DNA 6equence encoding 33K. This m~y be achieved by ~ny appropri~te technique, including restriction of the DNA 6equence for 33K
with ~n endonucle~6e, in6ertion of linkerc, u~e of ~n exonuclease ~nd/or a polymer~se ~nd ~ite-directed mut~genesis. A 6hortcr DNA cequence therefore m~y be obtained by removing nucleotldes rom the 5'-terminu~ or the 3'-terminus of the DNA sequence encoding 33K, for example using ~n exonuclea~e such ~6 ~A~ 31.
Whether ~ modified DNA ~equence encodes ~
pre-sequence c~p~ble of t~rqeting ~ p~ssenger protein into the thylakoid lumen may be readily ~scert~ined. Transcripts of ~ chimaeric gene encoding the modified pre-sequence ~nd a passenger protein ~re tr~nslated ~nd a chloroplast import 2 ~ 3 g assay effected as described in the Example.
Plant cells can be transformed with a chimaeric gene directly or by way of a vector incorporating the gene. Such ~ vector incorporates the chimaeric gene under the control of regulatory element~ cap~ble of enabling the gene to be expressed in a plant cell transformed with the vector. Such regulatory element~ include transoriptional control sequences, for example a6 above, and translational initiation and/or termination ~equences. The vector typically contains too a region wh$ch enable6 the chimaeric gene and associated regulatory control elements to be transferred to and ~tably integrated in the plant cell genome.
The chimaeric gene i5 therefore typically provided with transcriptional regulatory sequences and/or~ if not present at the 3'-end of the coding 6egucnce of the gene, a ~top codon. A DNA fr~gment m~y therefore also ~ncorporate a promoter, Shine-Dalgarno ~equencc and/or terminator ~equence which are capable of enabling the gene to be expre~ed in plant cells. The promoter may be a plant promoter, for example the 35S c~uli10wer mosaic virus promoter or a nopaline 6yntha~e or octopine synthase promoter.
Transformed cell~ ~re 6electcd by growth in an ~ppropriate medium. Plant tl~sue can therefore be obtained comprising a plant cell which harbours the chimaeric gene, for ex~mple in the plant cell genome, the gene being expressible in the plant cell. Plants can then be 2~5~ 3 regenerated which include the chimaeric gene in their cells, for example integrated in the plant cell genome, such that the gene can be expre sed. ~he re~enerated pl~nts can be reproduced ~nd, for example, Eeed obtained.
A preferred way of tr~n~forming a plant cell is to use Agrobacterium tumefaciens containing a vector comprisin~
the chimaeric gene. A hybrid pla~mid vector may therefore be employed which compri6es:
(~) the chimaeric gene under the control of regulatory element~ capable of enabling the gene to be expressed when integrated in the genome of ~ pl~nt cell;
(b) at least one DNA cequence which delineates the DNA to be integrated into the plant genome; ~nd (c) a DNA cequence which enables thifi DNA to be transferred to the plant genome.
Typically the DNA to be integrated into the pl~nt cell genome is deline~ted by the T-DNA border sequences of a Ti-plasmid. If only one border ~equence ls present, lt is preferably the right border cequence. She DNA ~equence which enable~ the DNA to be trancferred to the plant cell genome i6 generally the virulonco (vlr) rcgion of a Ti-plasmld.
The chimaeric gene ~nd ltfi tran~crlptionAl nnd trDnslational control elements can therefore be provided between the T-DNA border6 of a Ti-pla~mid. The plasmid may be a disarmed Ti-plasmid from whlch ehe genes for tumorigenicity have been deleted. The chimaeric gene and its transcriptional and control elements can, however, be provided between ~-DNA borders in a binary vector in trans with a Ti-plasmid with a vir re~ion. Such ~ binary vector therefcre compri~es:
(a) the chimaeric gene under the control of regulatory element6 c~p~ble of enabling the gene to be expressed when integrated in the genome of a plant cell; and (b) at least one DNA cequence which delineates the DNA to be integrated ~nto the plant genome.
Agrob~cterium tumefaciens, therefore, containing a hybrid plasmid vector or a binary vector in trans with a Ti-plasmid posse661ng a vir region c~n be used to tr~ns~orm plant cells. ~issue explant~ such a6 ~tems or leaf discs may be inocul~ted with the bacterium. Alternatively, the bacterlum may be co-cultured wlth rogenerating plant protoplast6. Plant protoplast6 mHy also be transformed by direct introduction of DNA fragments which encode the chimaer$ca gene and ln which the appropriate tran~criptional and translational control element6 ~re present or of a vector incorporat~ng cuch a fragment. Direct introduction may be ~chicved using electroporation or polyethylone glycol.
Plant cell6 from monocotylodonou~ or dicotylodonous pl~nt~ can be transform~d occording to the present invention. Monocotyledonou6 6pecies include barley, wheat, maize and rice. Dicotyledonou~ 6pecies lnclude ~obacco, tomato, sunflower, petunia, cotton, 6ugarbeet, potato, 2 ~ J3 ~
lettuce, melon, 60ybean, canola (r~peseed) ~nd poplars.
~issue cultures of transformed pl~nt cells are propagated to regenerate differentiated tr~nsfosmed whole plant~. The tr~nsformed pl~nt cell~ may be cultured on ~ ~uitable medium, prefer~bly a ~electable growth medium. Pl~nts ~y be regenerated from the resulting callus. Transgenic plants ~re thereby obt~ined who~e cells harbour the chim~eric gene, for example integrated in their genome, the gene being expressible in the cell~. Seed from.the regener~ted pl~nt6 c~n be collected for future use.
~ he following Example illustrates the invention. In the accompanying dr~wings:
Figure 1 shows the structure of the whe~t 33K-DHFR
fusion protein. Sites of cleavage by the 6tromal ~nd thyl~koidal processing peptid~se6 (SPP,~PP) are denoted by arrows. The preci~e rite of cleavage by SPP i~ not known.
Figure 2 chows the transport of wheat 33K-DHFR into isolated pea chloroplasts. A: wheat 33K-DHFR (l~ne 1) was synthesi~ed in vitro, imported into pea chloropl~st~, ~nd ~amples were analy6ed w~thout (lane 2) or with (l~ne 3) subsequent protease X tre~tment of the organolle6. Lane 4 nnd 5, chloropl~st6 were protea6~-trcated ~fter lmport and fractionated into stromal ~nd thyl~koid tample6, respectively. Lane 6, as in lane 5 oxcept that the thylakoids were protease ~-treated. Lane 7, mature DHFR.
~: wheat 33K-DHFR tlane 1) was imported and samples analy6ed without protease treatment (lane 2) and after protease 2 ~
treatment of the thylakoids (lane 3). Lanes 4,5 ~ thylakoid ~amples were sonicated for 5 sec and then centrifuged to generate soluble and ~embrane fractions, respectively.
S-DHFR, T-DHFR, ~tromal and thylakoidal DHFR for~, respectively.
Figure 3 shows the proce~sing of whe~t 33~-DHFR by the ~tromal and thylakoidal peptida~e~. Wheat 33R-DHFR
(lane 1) wac in~ub~ted with partially purified ~tromal ~lane 2) or thylakoidal ~lane 4) proce6~n~ peptidase. Lane~ 3,5, imported stromal and thylakoidal DHFR form~ as in Figure 2A, lanes 4 and 5. Lane 6, mature DHFR. Symbol~ as ln Figure 2.
EXAMPLE
1.1 Materlals Pea ~eedlings (Pisum 6~tivum, var. Feltham Fir~t) were grown, and chloroplasts i601ated at described (Robin~on and Ellis, Eur. J. Biochem. 152, 67-73, 1985). Radioactive materials were obt~ined from Amer6h~m international, UK.
1.2 Construction of p33K-DH~R
A mousa DHFR cDNA clone, pDHFR2Z, wa6 kindly provided by Dr J.V~ Cullimore ~Universlty of Warwick, GB). Th~
vector contained the coding region from pDS5/2 (Stuober et al, EMB0 J. 3, 3143-314B, 1984) exci~ed u6ing ~mH1/HindI~I
and lig~ted into pGem2Z ~Promega Biotech) which had been digested with BamH1 and SmaI. A cDNA clone encoding wheat pre-33K, p33K-2 ~Kirwin et al, EMB0 J. 8, 2251-2255, 1989) ~ 14 -was cut with EcoR1 and ~pnI to remove the pre-sequence coding region together with some of the mature sequence.
This fragment was blunt-ended using S1 nuclease. pD~FR2Z
was cut with B~m~1, the ends were ~illed in with ~lenow fragment, and the 33K fr~gment was ligated in to generate p33KDHFR~
The structure of p33KDHFR $s shown ~n Figure 1. The cloning procedure introduced ~ 3-recldue l$nker between the 33K ~nd DHFR polypeptide section~. Clones in which the pre-sequence region were ln-fr~me with the DHFR sequence were transcribed using T7 polymera6e and transcripts were tr~nslated in ~ whe~tgerm sy6tem ~n the pre6ence of l~ 5 S ]
methionlne (Melton et al, Nucleic Acids Res. 12, 7035-7056, l9B4; Anderson et al, Meth. Enzym. 101, 635-644, l9B3).
M~ture DHFR for use as a marker w~s synthesised in the same way from pDHFR2Z.
1.3 Import and processing studie6 Chloroplast import as6ays were carried out essentially ~5 de6cribed ~Robincon and Ellis, 1985). After incub~tion, non-lmported protelns were dlgest¢d uslng prote~se K (lSO~g ml~~, 4S min, 4C), and the chloropl~sts were washed once, ly6¢d ln 20mM Trlc-HCl, pH B.0, ~nd centrifuged ~t 10,000 x g for 10 mln to gener~te stromal and thyl~kold fractions. Thylakoids were protease R-treated a5 above where ~pproprlate. For processing studles, 2~1 translation product was incubated with 20~1 stromal or thylakoidal peptidase for 60 min at 27C. Samples were 2 ~ 9 ~3 3 analysed by polyacrylamide gel electrophoresis followed by fluorography.
1.4 Results A chimaeric gene, p33~DHFR encoding the pre-seguence of wheat 33K protein (with 22 residue6 of mature protein) linked to DHFR, wac con~tructed as shown $n Fiq. 1. The encoded protein (wheat 33x-DHFR) was 6ynthesi~ed by ln vitro tranccription/translation and incubated with ~solated pea chloroplastc. Figure 2 shows that the fusion protein was lmported and converted to two forms; the larger of which is located in the stroma and the smaller associated with the thylakoids. The latter polypeptide is about 1-2kDa larger than mature DHFR, possibly indicating that the entire 33R
pre-~equence has been removed, leaving a proce6sed protein of 22 residues linked to DHFR. This protein is resistant to protease-digestion of the thylakoids. Control tests have confirmed that this protein iE degraded if the ~esicle~ are sonicated to allow acce66 of the protease to the lumenal space content6 (not ~hown). We conclude that this polypeptide 18 located in~ide the ve6icles. The polypeptide ls rele~6ed from the vesicles by ~ very brief 60nclation, indicating that lt ls es6entially ~oluble in the thylakoid lumen (Flgur~ 2B).
The fu~ion proteln wa6 incubated with th~ ~tromal and thylakoidal proce6~ing peptldase~ both of whlch have been extensively purified (Roblnson and Ellis, Eur. J. Biochem.
142, 337-342, l9B4; Kirwin et al, J. ~iol. Chem. 262, ,7 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 Elight but reproducible. The thylakoidal peptidase processes the fusion protein to a polypeptide of identical mobility to imported, thylakoidal DHFR.
E. coli harbouring p33K-2 a~d 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.
~ hi~ invention relates to targeting proteins into the thylakoid lumen of chloroplasts in plants.
The chloroplast genome ie too small to carry ~11 the genetic information needed for the large number of different proteins which function in that organelle. An esti~at_d 80 of chloroplast proteins are translated from cytopla~mic poly(A)~ RNA and are thu~ encoded ~n the nuclear genome.
The6e proteins are ~ynthe6i6ed as pr~curcor molecules that are po6t-translationally imported into chloropla6ts. The imported precursor6 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, 6troma, thylakoid membrane ~ thylakoid lumen.
Nuclear-encoded thylakoid lumen proteins are synthesised as larger precursors and imported into the chloroplast by a two-~tep proce~s. Precur~or~ are initially tran~ported into the strom~ and proces~cd to intermediate forms by a stromal proee661ng peptldaso, after which thc intermediates ~re tran6erred acro~ the thylakoid membrane and proce6sed to the mature ~ize by ~ thylDko1dal peptidase.
Analysis of the pre-~equence6 of lumenal protein~ has ~hown that they consist of two domain~, wh$ch are belleved to direct "envelope transfer" of the precursor and then "thylakoid transfer" of the stromal intermediate.
20~ ~Q'~
The t~rgeting propertie~ of the two do~ains have been tested in ~tudie~ using chimaeric proteins. Pl~stocyanin is normally located in the thylakoid lumen. The pre-~equence of Silene pl~ttocyanin was found to direct the tran~port of a foreign protein, yeast ~uperoxide dismut~se, into the chloropla~t 6troma. ~owever, neither ~his protein nor a etromal prctein, ferredoxin, W~5 t~rgeted into the thyl~koid lumen by the plastocy~nin pre-~equence (Smeeken~ et al, Cell 46, 365-375, l9B6; Smeekens et 1, Plant. Mol. Biol. 9, 377-38B, l9B7). Thi6 6ugsested th~t the mature ~equences of lumen~l proteins m~y contain ~ome information essenti~l for transport across the thylakoidal membr~ne.
We have now fused the pre-sequence of another lumenal protein, the 33kDa protein of the photosynthetic oxygen-evolving complex (33X) in front of mouse dihydrofol~te reductase (DHFR), ~ cytopla6mic protein whlch has been used a~ a pas~en~er protein in mitochondrlal protein tran6port studies (Eilers and Schatz, Nature 322, 22B-232, 19~6). In contrast to the previou6 ~ttempt~ to target foroign proteins into the thyl~koid lumen, the DHFR w~ efflciently t~rgeted to the thyl~kold lumen. Thl~ findlng h~ general ~pplicability.
~ ccordlngly, the present lnvent~on provide~ A
chimaerlc gene encoding (~) the pre-~equence of thc 33kDa proteln of the photosynthetic oxygen-evolving complex of photosynthetic org~nism or ~ modlfied vertion of the ~aid pre-sequence which i5 capable of targeting a pas~enger 2 ~
protein into the thylakoid lumen of a chloropla6t, fused to (b) a heterQlsgous passenger protein.
The invention also provides a vector which ~ncorporates ~uch a chimaeric gene under the control of regulatory elements capable of enabling the gene to be expressed ~n ~ plant cell transformed with the vector. The vector may further contain ~ region which enables the chimaeric gene and associ~ted regulatory ele~ents to be transferred to ~nd 6tably ~ntegrated in ~ plant cell genome.
The vector is generally a plasmid.
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 obta$ned which harbour~ in itC cells ~ chimaeric gene.
Seed may be obtained from the transgenic plants.
The invention further provides a method of producing a desired protein in a plant cell, which method comprises:
(i) transforming a pl~nt cell with ~ vector according to the invention, the 4aid passenger proteln being the desired protein; and (li) culturing the tr~n~formed plant cell under condittons which allow expression of the protein.
The invent$on additionally provider a method of producing a transgenic plant capable of producing a desired protein, which method comprises:
~ i) transforminq a plant cell with a vector 2 ~ Q ~
according to the invention, the ~aid passenger protein being the desired protein; ~nd (ii) regenerating plants from the transformed cells.
The desired proteins can be isolated from the transformed plant cellE obtained by the fir~t method ~nd from the plants obtained by the ~econd 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 ver&ion of this pre-sequence which is al60 capable of targeting a p~ssenger 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 pl~nt, which may be a monocGtyledonous or dictotyledonous plant. Preferably the pre-sequence is the pre-6eguence of the 33kDa protein of wheat. For convenience here, the pre-~equence of the 33kDa protein will be design~ted 33R and both 33R and modified versions of it will be termed as a pre-sequence.
The pre-~equence may be fused directly to the amino-terminu~ of the pas~enger protein. Alternatively, there may be ~n intervening linking sequence. ~he linking sequence m~y be composed of up to 40 amino ncid residues, for ex~mple up to 30, up to 20 or up to 10 re6idue~ Any such linker must not interfere with the ability of the pre-sequence to t~rget the pa~senger protein into the thylakoid lumen. The portion of the gene encoding the - 5 - 20$~f3~
pre-6equence ~nd any intervening linking ~equences ~ay be prepared by st~ndard synthetic techniques.
Preferably the linking ~equence comprises the natural amino-terminal re~idues of the ~ature wheat 33kDa protein.
A pre-sequence, together with a number of the natural amino-terminal residue~ of the mature 33kDa prDtein, may be fused in front of the passenger protein. The fir~t 10 to 25, for example 20 to 25 ~nd ~n particular 12 or 22, natural ~mino-terminal resldue~ of the mature 33kDa protein may be pre6ent. A 6hort non-natural linker sequence, generally resulting from the DNA manlpulations necessary to construct A chimaeric qene, may separate the natural residue6 of the ~ature 33kDa protein from the amino terminus of the p~ssenger prote~n. For example, up to 4 non-natural further linker residue6 may be present.
A suita~le pre-cequence iB therefore wheat 33K. Thl6 pre-sequence and it~ corre~ponding DNA sequence lc chown ln Table l.
TABLE l ATG GCA GCG TCT CTC CAA GCC GCG GCC ACG CTG ATG CCG GCC AAG
Met Ala Ala Ser Leu Gln Ala Ala Ala Thr Leu Met Pro Ala Lys ATC GGC GGC CGG GCC TCC TCC GCG CGA CCG TCG TCG CAC GTC GCC
Ile Gly Gly Arg Ala Ser Ser Ala Arq Pro Ser Ser H16 V~l Al~
CGG GCG TTC GGC GTC GAC GCT GGC GCC AGG ATC ACC TGC TCC CTG
Arg Ala Phe Gly Val Asp Ala Gly Al~ Arq Ile Thr Cy~ Ser Leu CAG TCC GAC ATC AGG GAG GTC GCA AGC AAG TGC GCC GAC GCC GCC
Gln Ser Asp Ile Arg Glu Val Ala Ser Lys Cys Ala Asp Ala Ala 2 ~ 3 AAG ATG GCC GGC TTC GCC CTC GCC ACC TCT GCC CTC CTC GTC TCC
Lys Met Ala Gly Phe Ala Leu Ala Thr Ser Ala Leu Leu Vdl Ser GGC GCG ACG GCG
Gly Ala Thr Ala The amino ~cid ~e~uence of ~hea~ 33K ~nd the natural amino-terminal residues of the wheot 33kDa protein, ~nd the corresponding D~A ~equence, i~ ~hown in Table 2. ~ denotes the end of wheat 33R.
ATG GCA GCG TCT CTC CAA GCC GCG GCC ACG CTG ATG CCG GCC AAG
Met Ala Al~ S~r Leu Gln Ala Ala Ala Thr Leu Mct Pro Ala Ly8 ATC GGC GGC CGC GCC TCC TCC GCG CGA CCG TCG TCG CAC GTC GCC
Ile Gly Gly Arg Ala Ser Ser Ala Arg Pro Ser Ser Hi~ Val Ala CGG GCG TTC GGC GTC GAC GCT GGC GCC AGG ATC ACC TGC TCC CTG
Arg Alo Phe Gly Vol Asp Alo Gly Alo Arg Ile Thr Cy6 Ser Leu CAG TCC GAC ATC AGG GAG GTC GCA AGC AAG TGC GCC GAC GCC GCC
Gln Ser A6p Ile Arg Glu Vol Alo Ser Lys Cy8 Alo Asp Alo Al~
AAG ATG GCC GGC TTC GCC CTC GCC ACC TCT GCC C-C CTC GTC TCC
Ly5 Met Alo Gly Phe Ala Leu Ala Thr Ser Al~ Leu Leu V~l Ser GGC GCG ACG GCG GAG GGG GCG CCC AAG AGG CTG ACC TTC GAC GAG
Gly Alo Thr Al~Glu Gly Al~ Pro Ly6 Arg Leu Thr Ph~ A~p Glu ATC CAG AGC AAG ACC TAC ATG GAG GTG AAG GGT ACC GGC ACC GCG
Il~ Gln Ser Ly6 Thr Tyr Met Glu Val Ly6 Gly Thr Gly Thr Al~
~ modif~ed vercion of 33K moy ~ncorporate one or more amino acid ~ubstitutions, ~n~ertions ~nd/or deletion6 and/or An exten6ion at either or both ends. ror example one or more amino acid re~idues of 33K may be 6ubstituted or deleted, or one or more addit$onal residue may be inserted, 2 f~ L~
provided the physicochemical character of the pre-sequence is preserved, i.e. in terms of charge density, hydrophilicity/hydrophobicity and, ~ize and configuration.
Candidate substitutions include:
- Ser by Thr and vice versa, - Glu by Asp and vice ver~a, ~nd - Gln by Asn and vice versa.
As far as exten6ions are concerned, the sequence ~ay be extended by up to 10 amino acid residues at either end.
Up to, for ex~mple, 5 amino acid residues may be added therefore to the amlno-terminus or to the carboxy-terminus.
Where 33~ i~ modified, typlcally thcre ~ a degree of homology of at least 70% between 33X and the modified pre-sequence. The degree of homology mAy be 85~ or more or 95% or more. Any modified GeqUenCe, howover, must be capable of t~rgeting a pa6senger protein into the thylakoid lumen.
The heterologous p~s6enger protein may be ~ny heterologou6 proteln lt i6 deslred to target into the thylakold lumen. By "heterologou~" is moant that the passenger protein is not naturally linked to the pre-sequence. In part~cular, the passenger proteln i~ not the wheat 33kD~ protoin of the oxygen-evolving complox. The photo6ynthotic prop~rties of plants may bo alte~ed by targeting p~rticular proteins into the thylakoid lumen. A
passenger protein may therefore be ~ protein which could improve the photosynthetic c~pacity of a plant, for ex~mple - 8 - ~ 3 ~ utant ver~ion of a protein ~lready present in the lumen or a herbicide-resist~nt ~nalogue isolated from other pl~nts or bacteria. Alternatively, the invention may be used to ynthesi~e large quantities of protein.
A chimaeric gene may therefore contain the DNA
sequence 6hown in ~able 1. ~h$6 ~equence may be extended ~t the 3~-end ~8 ~bove, for example ba~ed on the DNA ~equence 6hown in Ta`ole 2. A modified version of the DNA sequence ~hown $n T~ble 1 may be provided. The DNA ~eguence encoding the pre-sequence i~ lig~ted to a DNA sequence encoding the passenger protein. Optionally an intervening DNA 6equence encoding linker ~mino acid re~idues is provided.
A modified pre-sequence may be obtained by lntroducing corre6ponding ch~nges into the DNA 6equence encoding 33K. This m~y be achieved by ~ny appropri~te technique, including restriction of the DNA 6equence for 33K
with ~n endonucle~6e, in6ertion of linkerc, u~e of ~n exonuclease ~nd/or a polymer~se ~nd ~ite-directed mut~genesis. A 6hortcr DNA cequence therefore m~y be obtained by removing nucleotldes rom the 5'-terminu~ or the 3'-terminus of the DNA sequence encoding 33K, for example using ~n exonuclea~e such ~6 ~A~ 31.
Whether ~ modified DNA ~equence encodes ~
pre-sequence c~p~ble of t~rqeting ~ p~ssenger protein into the thylakoid lumen may be readily ~scert~ined. Transcripts of ~ chimaeric gene encoding the modified pre-sequence ~nd a passenger protein ~re tr~nslated ~nd a chloroplast import 2 ~ 3 g assay effected as described in the Example.
Plant cells can be transformed with a chimaeric gene directly or by way of a vector incorporating the gene. Such ~ vector incorporates the chimaeric gene under the control of regulatory element~ cap~ble of enabling the gene to be expressed in a plant cell transformed with the vector. Such regulatory element~ include transoriptional control sequences, for example a6 above, and translational initiation and/or termination ~equences. The vector typically contains too a region wh$ch enable6 the chimaeric gene and associated regulatory control elements to be transferred to and ~tably integrated in the plant cell genome.
The chimaeric gene i5 therefore typically provided with transcriptional regulatory sequences and/or~ if not present at the 3'-end of the coding 6egucnce of the gene, a ~top codon. A DNA fr~gment m~y therefore also ~ncorporate a promoter, Shine-Dalgarno ~equencc and/or terminator ~equence which are capable of enabling the gene to be expre~ed in plant cells. The promoter may be a plant promoter, for example the 35S c~uli10wer mosaic virus promoter or a nopaline 6yntha~e or octopine synthase promoter.
Transformed cell~ ~re 6electcd by growth in an ~ppropriate medium. Plant tl~sue can therefore be obtained comprising a plant cell which harbours the chimaeric gene, for ex~mple in the plant cell genome, the gene being expressible in the plant cell. Plants can then be 2~5~ 3 regenerated which include the chimaeric gene in their cells, for example integrated in the plant cell genome, such that the gene can be expre sed. ~he re~enerated pl~nts can be reproduced ~nd, for example, Eeed obtained.
A preferred way of tr~n~forming a plant cell is to use Agrobacterium tumefaciens containing a vector comprisin~
the chimaeric gene. A hybrid pla~mid vector may therefore be employed which compri6es:
(~) the chimaeric gene under the control of regulatory element~ capable of enabling the gene to be expressed when integrated in the genome of ~ pl~nt cell;
(b) at least one DNA cequence which delineates the DNA to be integrated into the plant genome; ~nd (c) a DNA cequence which enables thifi DNA to be transferred to the plant genome.
Typically the DNA to be integrated into the pl~nt cell genome is deline~ted by the T-DNA border sequences of a Ti-plasmid. If only one border ~equence ls present, lt is preferably the right border cequence. She DNA ~equence which enable~ the DNA to be trancferred to the plant cell genome i6 generally the virulonco (vlr) rcgion of a Ti-plasmld.
The chimaeric gene ~nd ltfi tran~crlptionAl nnd trDnslational control elements can therefore be provided between the T-DNA border6 of a Ti-pla~mid. The plasmid may be a disarmed Ti-plasmid from whlch ehe genes for tumorigenicity have been deleted. The chimaeric gene and its transcriptional and control elements can, however, be provided between ~-DNA borders in a binary vector in trans with a Ti-plasmid with a vir re~ion. Such ~ binary vector therefcre compri~es:
(a) the chimaeric gene under the control of regulatory element6 c~p~ble of enabling the gene to be expressed when integrated in the genome of a plant cell; and (b) at least one DNA cequence which delineates the DNA to be integrated ~nto the plant genome.
Agrob~cterium tumefaciens, therefore, containing a hybrid plasmid vector or a binary vector in trans with a Ti-plasmid posse661ng a vir region c~n be used to tr~ns~orm plant cells. ~issue explant~ such a6 ~tems or leaf discs may be inocul~ted with the bacterium. Alternatively, the bacterlum may be co-cultured wlth rogenerating plant protoplast6. Plant protoplast6 mHy also be transformed by direct introduction of DNA fragments which encode the chimaer$ca gene and ln which the appropriate tran~criptional and translational control element6 ~re present or of a vector incorporat~ng cuch a fragment. Direct introduction may be ~chicved using electroporation or polyethylone glycol.
Plant cell6 from monocotylodonou~ or dicotylodonous pl~nt~ can be transform~d occording to the present invention. Monocotyledonou6 6pecies include barley, wheat, maize and rice. Dicotyledonou~ 6pecies lnclude ~obacco, tomato, sunflower, petunia, cotton, 6ugarbeet, potato, 2 ~ J3 ~
lettuce, melon, 60ybean, canola (r~peseed) ~nd poplars.
~issue cultures of transformed pl~nt cells are propagated to regenerate differentiated tr~nsfosmed whole plant~. The tr~nsformed pl~nt cell~ may be cultured on ~ ~uitable medium, prefer~bly a ~electable growth medium. Pl~nts ~y be regenerated from the resulting callus. Transgenic plants ~re thereby obt~ined who~e cells harbour the chim~eric gene, for example integrated in their genome, the gene being expressible in the cell~. Seed from.the regener~ted pl~nt6 c~n be collected for future use.
~ he following Example illustrates the invention. In the accompanying dr~wings:
Figure 1 shows the structure of the whe~t 33K-DHFR
fusion protein. Sites of cleavage by the 6tromal ~nd thyl~koidal processing peptid~se6 (SPP,~PP) are denoted by arrows. The preci~e rite of cleavage by SPP i~ not known.
Figure 2 chows the transport of wheat 33K-DHFR into isolated pea chloroplasts. A: wheat 33K-DHFR (l~ne 1) was synthesi~ed in vitro, imported into pea chloropl~st~, ~nd ~amples were analy6ed w~thout (lane 2) or with (l~ne 3) subsequent protease X tre~tment of the organolle6. Lane 4 nnd 5, chloropl~st6 were protea6~-trcated ~fter lmport and fractionated into stromal ~nd thyl~koid tample6, respectively. Lane 6, as in lane 5 oxcept that the thylakoids were protease ~-treated. Lane 7, mature DHFR.
~: wheat 33K-DHFR tlane 1) was imported and samples analy6ed without protease treatment (lane 2) and after protease 2 ~
treatment of the thylakoids (lane 3). Lanes 4,5 ~ thylakoid ~amples were sonicated for 5 sec and then centrifuged to generate soluble and ~embrane fractions, respectively.
S-DHFR, T-DHFR, ~tromal and thylakoidal DHFR for~, respectively.
Figure 3 shows the proce~sing of whe~t 33~-DHFR by the ~tromal and thylakoidal peptida~e~. Wheat 33R-DHFR
(lane 1) wac in~ub~ted with partially purified ~tromal ~lane 2) or thylakoidal ~lane 4) proce6~n~ peptidase. Lane~ 3,5, imported stromal and thylakoidal DHFR form~ as in Figure 2A, lanes 4 and 5. Lane 6, mature DHFR. Symbol~ as ln Figure 2.
EXAMPLE
1.1 Materlals Pea ~eedlings (Pisum 6~tivum, var. Feltham Fir~t) were grown, and chloroplasts i601ated at described (Robin~on and Ellis, Eur. J. Biochem. 152, 67-73, 1985). Radioactive materials were obt~ined from Amer6h~m international, UK.
1.2 Construction of p33K-DH~R
A mousa DHFR cDNA clone, pDHFR2Z, wa6 kindly provided by Dr J.V~ Cullimore ~Universlty of Warwick, GB). Th~
vector contained the coding region from pDS5/2 (Stuober et al, EMB0 J. 3, 3143-314B, 1984) exci~ed u6ing ~mH1/HindI~I
and lig~ted into pGem2Z ~Promega Biotech) which had been digested with BamH1 and SmaI. A cDNA clone encoding wheat pre-33K, p33K-2 ~Kirwin et al, EMB0 J. 8, 2251-2255, 1989) ~ 14 -was cut with EcoR1 and ~pnI to remove the pre-sequence coding region together with some of the mature sequence.
This fragment was blunt-ended using S1 nuclease. pD~FR2Z
was cut with B~m~1, the ends were ~illed in with ~lenow fragment, and the 33K fr~gment was ligated in to generate p33KDHFR~
The structure of p33KDHFR $s shown ~n Figure 1. The cloning procedure introduced ~ 3-recldue l$nker between the 33K ~nd DHFR polypeptide section~. Clones in which the pre-sequence region were ln-fr~me with the DHFR sequence were transcribed using T7 polymera6e and transcripts were tr~nslated in ~ whe~tgerm sy6tem ~n the pre6ence of l~ 5 S ]
methionlne (Melton et al, Nucleic Acids Res. 12, 7035-7056, l9B4; Anderson et al, Meth. Enzym. 101, 635-644, l9B3).
M~ture DHFR for use as a marker w~s synthesised in the same way from pDHFR2Z.
1.3 Import and processing studie6 Chloroplast import as6ays were carried out essentially ~5 de6cribed ~Robincon and Ellis, 1985). After incub~tion, non-lmported protelns were dlgest¢d uslng prote~se K (lSO~g ml~~, 4S min, 4C), and the chloropl~sts were washed once, ly6¢d ln 20mM Trlc-HCl, pH B.0, ~nd centrifuged ~t 10,000 x g for 10 mln to gener~te stromal and thyl~kold fractions. Thylakoids were protease R-treated a5 above where ~pproprlate. For processing studles, 2~1 translation product was incubated with 20~1 stromal or thylakoidal peptidase for 60 min at 27C. Samples were 2 ~ 9 ~3 3 analysed by polyacrylamide gel electrophoresis followed by fluorography.
1.4 Results A chimaeric gene, p33~DHFR encoding the pre-seguence of wheat 33K protein (with 22 residue6 of mature protein) linked to DHFR, wac con~tructed as shown $n Fiq. 1. The encoded protein (wheat 33x-DHFR) was 6ynthesi~ed by ln vitro tranccription/translation and incubated with ~solated pea chloroplastc. Figure 2 shows that the fusion protein was lmported and converted to two forms; the larger of which is located in the stroma and the smaller associated with the thylakoids. The latter polypeptide is about 1-2kDa larger than mature DHFR, possibly indicating that the entire 33R
pre-~equence has been removed, leaving a proce6sed protein of 22 residues linked to DHFR. This protein is resistant to protease-digestion of the thylakoids. Control tests have confirmed that this protein iE degraded if the ~esicle~ are sonicated to allow acce66 of the protease to the lumenal space content6 (not ~hown). We conclude that this polypeptide 18 located in~ide the ve6icles. The polypeptide ls rele~6ed from the vesicles by ~ very brief 60nclation, indicating that lt ls es6entially ~oluble in the thylakoid lumen (Flgur~ 2B).
The fu~ion proteln wa6 incubated with th~ ~tromal and thylakoidal proce6~ing peptldase~ both of whlch have been extensively purified (Roblnson and Ellis, Eur. J. Biochem.
142, 337-342, l9B4; Kirwin et al, J. ~iol. Chem. 262, ,7 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 Elight but reproducible. The thylakoidal peptidase processes the fusion protein to a polypeptide of identical mobility to imported, thylakoidal DHFR.
E. coli harbouring p33K-2 a~d 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.
Claims (15)
1. 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 n chloroplast, fused to (b) a heterologous passenger protein.
2. A chimaeric gene according to claim 1, wherein component (a) is the pre-sequence of the 33kDa protein of the photosynthetic oxygen-evolving complex of wheat or a said modified version thereof.
3. A chimaeric gene according to claim 1 or 2, which further encodes an intervening linker sequence between the said pre-sequence or modified version thereof and the passenger protein.
4. A chimaeric gene according to claim 3, wherein the linker sequence comprises up to the first 25 natural amino-terminal residues of the mature wheat 33kDa protein.
5. A chimaeric gene accordinq to any one of the preceding claims, wherein the DNA sequence encoding the said pre-sequence is as follows:
.
.
6. A vector which incorporates a chimeric gene as claimed in any one of the preceding claims under the control of regulatory elements capable of enabling the gene to be expressed in a plant cell transformed with the vector.
7. A vector according to claim 6, which further contains a region which enables the chimaeric gene and associated regulatory elements to be transferred to and stably integrated in a plant cell genome.
8. A vector according to claim 6 or 7, which is a plasmid.
9. A plant cell which has been transformed with a vector as claimed in any one of claims 6 to 8.
10. A plant cell which harbours a chimaeric gene as claimed in any one of claims 1 to 5.
11. A transgenic plant which has been regenerated from plant cells as claimed in claim 9 or 10.
12. A transgenic plant which harbours in its cells a chimaeric gene at claimed in any one of claims 1 to 5.
13. Seed obtained from a transgenic plant as claimed in claim 11 or 12.
14. A method of producing a desired protein in a plant cell, which method comprises:
(i) transforming a plant cell with a vector as claimed in any one of claims 6 to 8, the said passenger protein being the desired protein; and (ii) culturing the transformed plant cell under conditions which allow expression of the protein.
(i) transforming a plant cell with a vector as claimed in any one of claims 6 to 8, the said passenger protein being the desired protein; and (ii) culturing the transformed plant cell under conditions which allow expression of the protein.
15. A method of producing a transgenic plant capable of producing a desired protein, which method comprises:
(i) transforming a plant cell with a vector as claimed in any one of claims 6 to 8, the said passenger protein being the desired protein; and (ii) regenerating plants from the transformed cells.
(i) transforming a plant cell with a vector as claimed in any one of claims 6 to 8, the said passenger protein being the desired protein; and (ii) regenerating plants from the transformed cells.
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 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2064903A1 true CA2064903A1 (en) | 1991-02-15 |
Family
ID=10661583
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2064903 Abandoned CA2064903A1 (en) | 1989-08-14 | 1990-08-14 | Thylakoid targeting sequence |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0487618A1 (en) |
| JP (1) | JPH07501681A (en) |
| CA (1) | CA2064903A1 (en) |
| GB (1) | GB8918496D0 (en) |
| WO (1) | WO1991002800A1 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ATE360072T1 (en) | 1998-07-10 | 2007-05-15 | Calgene Llc | EXPRESSION OF EUKAROTIC PEPTIDES IN PLANT PLASTIDS |
| US6512162B2 (en) | 1998-07-10 | 2003-01-28 | Calgene Llc | Expression of eukaryotic peptides in plant plastids |
| KR100362320B1 (en) * | 1998-08-19 | 2004-03-20 | 학교법인 명지학원 | New gene expression method for targeting chloroplasts |
-
1989
- 1989-08-14 GB GB8918496A patent/GB8918496D0/en active Pending
-
1990
- 1990-08-14 EP EP19900913200 patent/EP0487618A1/en not_active Withdrawn
- 1990-08-14 JP JP2512183A patent/JPH07501681A/en active Pending
- 1990-08-14 CA CA 2064903 patent/CA2064903A1/en not_active Abandoned
- 1990-08-14 WO PCT/GB1990/001281 patent/WO1991002800A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0487618A1 (en) | 1992-06-03 |
| JPH07501681A (en) | 1995-02-23 |
| GB8918496D0 (en) | 1989-09-20 |
| WO1991002800A1 (en) | 1991-03-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0131623B1 (en) | Chimeric genes suitable for expression in plant cells | |
| KR0154872B1 (en) | Agrobacterium mediated transformation of germinating plant seeds | |
| US8871468B2 (en) | Synthetic genes for plant gums and other hydroxyproline-rich glycoproteins | |
| US5618699A (en) | Plant virus vector, plasmid, process for expression of foreign gene and process for obtaining foreign gene product | |
| US8936937B2 (en) | System for expression of genes in plants from a virus-based expression vector | |
| JPH08505289A (en) | Cross reference for applications related to virus amplification of recombinant messenger RNA in transgenic plants | |
| KR102444024B1 (en) | Recombinant vectors for the preparation of antigens for diagnosis of African swine fever and uses thereof | |
| US6232099B1 (en) | Method of producing a chimeric protein | |
| US6472211B1 (en) | Method of increasing transcription with nucleic acids encoding PABF | |
| BG61275B1 (en) | Production of plants resistant to attacks of sclerotiniasp.by introducing a gene coding for an oxalate oxidase | |
| US6548642B1 (en) | Synthetic genes for plant gums | |
| CA2064903A1 (en) | Thylakoid targeting sequence | |
| Kuroda et al. | Nucleotide sequence and synthesis of infectious RNA from cloned cDNA of broad bean wilt virus 2 RNA 2 | |
| CN1301308A (en) | Disease resistant transgenic plants | |
| WO1995005471A2 (en) | Method for stable transformation of plants | |
| DE10155862A1 (en) | Production of recombinant antibodies by fusion with elastin-like peptides | |
| CN111073868B (en) | Plant flavone methyltransferase protein and coding gene and application thereof | |
| CN114480346B (en) | DNA hydrolase and preparation method thereof | |
| KR100797912B1 (en) | -1 Promoters Originated from Chlorella Virus HS-1 and Use Thereof | |
| JP3512217B2 (en) | Endo-type xyloglucan transferase gene | |
| JP2022089006A (en) | Production of active human furin fragment in plant | |
| EP1162267A2 (en) | Plant virus vector, plasmid, process for expression of foreign gene and process for obtaining foreign gene product | |
| EP0622461A2 (en) | Preparation of yellow mosaic disease-resistant barley | |
| US5428144A (en) | Maize dwarf mosaic virus cDNA | |
| AU2003200734B2 (en) | Transgenic Lemnaceae |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 19960214 |