THYLAKOID TARGETING SEQUENCE

Abstract: 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: THYLAKOID TARGETING SEQUENCE This invention relates to targeting proteins into the thylakoid lumen of chloroplasts in plants. The 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 synthesiεed 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 εuperoxide dismutase, into the chloroplast stroma. However, neither this protein nor a stromal protein, ferredoxin, was targeted into the thylakoid lumen by the plastocyanin pre-sequence (Smeekenε 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. We have now fused the pre-sequence of another lumenal protein, the 33kDa protein of the photosynthetic oxygen- evolving complex (33K) in front of mouεe dihydrofolate reductaεe (DHFR), a cytoplasmic protein which has been used as a passenger protein in mitochondrial protein transport studies (Eilers and Schatz, Nature 322, 228-232, 1986). In contrast to the previous attempts to target foreign proteins into the thylakoid lumen, the DHFR was efficiently targeted to the thylakoid lumen. This finding has general applicability. Accordingly, 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: (i) transforming a plant cell with a vector according to the invention, the said passenger protein being the desired protein; and (ii) culturing the transformed plant cell under conditions which allow expression of the protein. The invention additionally provides a method of producing a transgenic plant capable of producing a desired protein, which method comprises: (i) transforming a plant cell with a vector according to the invention, the said passenger protein being the desired protein; and (ii) regenerating plants from the transformed cells. 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. Preferably the pre-sequence is the pre-sequence of the 33kDa protein of wheat. For convenience here, 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-terminuε of the passenger protein. Alternatively, there may be an intervening linking sequence. 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. Preferably 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. TABLE 1 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 Val Ser GGC GCG ACG GCG Gly Ala Thr Ala The amino acid sequence of wheat 33K and the natural amino-terminal residues of the wheat 33kDa protein, and the corresponding DNA sequence, is shown in Table 2. * denotes the end of wheat 33K. TABLE 2 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. For example 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: - Ser by Thr and vice versa, - Glu by Asp and vice versa, and - Gin by Asn and vice versa. As far as extensions are concerned, 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-terminuε or to the carboxy-terminuε. Where 33K is modified, typically there iε a degree of homology of at least 70% between 33K and the modified pre-εequence. The degree of homology may be 85% or more or 95% or more. Any modified εequence, however, must be capable of targeting a pasεenger protein into the thylakoid lumen. The heterologous pasεenger protein may be any heterologous protein it is desired to target into the thylakoid lumen. By "heterologous" is meant that the passenger protein is not naturally linked to the pre-sequence. In particular, the passenger protein iε 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. Alternatively, the invention may be used to εynthesiεe large quantities of protein. A chimaeric gene may therefore contain the DNA sequence εhown 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 εhown in Table 1 may be provided. The DNA sequence encoding the pre-sequence iε ligated to a DNA sequence encoding the passenger protein. Optionally 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 polymeraεe and site-directed mutageneεis. A shorter DNA sequence therefore may be obtained by removing nucleotides from the 5'-terminuε or the 3'-terminus of the DNA sequence encoding 33K, for example using an exonuclease such as BAL 31. Whether 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. Such 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. Such regulatory elements include tranεcriptional 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 preεent 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 expresεed in plant cells. The promoter may be a plant promoter, for example the 35S cauliflower mosaic virus promoter or a nopaline synthase or octopine εynthaεe 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, εuch that the gene can be expreεεed. 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 compriεeε: (a) the chimaeric gene under the control of regulatory elements capable of enabling the gene to be expressed when integrated in the genome of a plant cell; (b) at least one DNA sequence which delineates the DNA to be integrated into the plant genome; and (c) a DNA sequence which enables this DNA to be transferred to the plant genome. Typically the DNA to be integrated into the plant cell genome is delineated by the T-DNA border εequences of a Ti-plasmid. If only one border sequence is present, it iε preferably the right border εequence. 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: (a) the chimaeric gene under the control of regulatory elements capable of enabling the gene to be expressed when integrated in the genome of a plant cell; and (b) at least one DNA sequence which delineates the DNA to be integrated into the plant genome. Agrobacterium tumefaciens, therefore, containing a hybrid plasmid vector or a binary vector i_n trans with a Ti-plasmid posεessing a vir region can be uεed to tranεform plant cells. Tiεεue explantε εuch aε εtemε or leaf discs may be inoculated with the bacterium. Alternatively, the bacterium may be co-cultured with regenerating plant protoplastε. 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 preεent or of a vector incorporating εuch a fragment. Direct introduction may be achieved uεing electroporation or polyethylene glycol. Plant cells from monocotyledonouε or dicotyledonouε 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 (rapeεeed) and poplars. Tisεue 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 expresεible in the cells. Seed from the regenerated plantε can be collected for future use. The following Example illustrates the invention. In the accompanying drawings: Figure 1 shows the structure of the wheat 33K-DHFR fusion protein. Sites of cleavage by the stromal and thylakoidal procesεing peptidaεes (SPP,TPP) are denoted by arrows. The precise site of cleavage by SPP is not known. Figure 2 showε the tranεport of wheat 33K-DHFR into iεolated pea chloroplasts. A: wheat 33K-DHFR (lane 1) was εyntheεiεed _in_ vitro, imported into pea chloroplaεtε, and samples were analysed without (lane 2) or with (lane 3) subsequent protease K treatment of the organelles. Lane 4 and 5, chloroplastε were proteaεe-treated after import and fractionated into stromal and thylakoid samples, respectively. Lane 6, as in lane 5 except that the thylakoidε were proteaεe K-treated. Lane 7, mature DHFR. B: wheat 33K-DHFR (lane 1) waε 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. Figure 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. Lanes 3,5, imported stromal and thylakoidal DHFR forms as in Figure 2A, lanes 4 and 5. Lane 6, mature DHFR. Symbols as in Figure 2. EXAMPLE 1.1 Materials Pea seedlings (Pisum εativum, var.. Feltham First) were grown, and chloroplastε isolated as described (Robinson and Ellis, Eur. J. Biochem. 152, 67-73, 1985). Radioactive materials were obtained from Amersham International, UK. 1.2 Construction of p33K-DHFR 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. 8_, 2251-2255, 1989) waε cut with EcoRl and Kpnl to remove the pre-εequence coding region together with some of the mature sequence. This fragment waε blunt-ended using SI nuclease. pDHFR2Z waε cut with BamHl, the endε were filled in with Klenow fragment, and the 33K fragment waε ligated in to generate p33KDHFR. The εtructure of p33KDHFR iε shown in Figure 1. The cloning procedure introduced a 3-reεidue linker between the 33K and DHFR polypeptide sections. Clones in which the pre-sequence region were in-frame with the DHFR sequence were transcribed using T7 polymerase and transcripts were translated in a wheatgerm system in the presence of [ 35 S] methionine (Melton e_t a_l, Nucleic Acids Res. 1^2, 7035-7056, 1984; Anderson e_t al ^ , Meth. Enzym. 101, 635-644, 1983). Mature DHFR for use as a marker was synthesiεed in the εame way from pDHFR2Z. 1.3 Import and proceεsing studies Chloroplast import asεays were carried out eεεentially 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 Triε-HCl, pH 8.0, and centrifuged at 10,000 x g for 10 min to generate stromal and thylakoid fractions. Thylakoidε were protease K-treated as above where appropriate. For procesεing εtudieε, 2 / /1 translation product was incubated with 20 l stromal or thylakoidal peptidase for 60 min at 27°C. Sa pleε were analysed by polyacrylamide gel electrophoreεiε followed by fluorography. 1.4 Results A chimaeric gene, p33KDHFR encoding the pre-sequence of wheat 33K protein (with 22 residues of mature protein) linked to DHFR, waε conεtructed aε εhown in Fig. 1. The encoded protein (wheat 33K-DHFR) was synthesiεed by _in vitro tranεcription/tranεlation 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 thylakoidε. The latter polypeptide iε about l-2kDa larger than mature DHFR, poεsibly indicating that the entire 33K pre-sequence has been removed, leaving a processed protein of 22 residues linked to DHFR. This protein is resiεtant to protease-digestion of the thylakoids. Control tests have confirmed that thiε protein is degraded if the vesicles are sonicated to allow access of the protease to the lumenal space contentε (not shown). We conclude that this polypeptide is located inside the vesicleε. The polypeptide iε released from the vesicleε by a very brief sonciation, indicating that it is essentially soluble in the thylakoid lumen (Figure 2B). The fusion protein was incubated with the stromal and thylakoidal procesεing peptidaεeε both of which have been extenεively 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.

Claims: CLAIMS 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 iε capable of targeting a passenger protein into the thylakoid lumen of a chloroplast, fused to (b) a heterologous passenger protein. 2. A chimaeric gene according to claim 1, wherein component (a) is the pre-εequence 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 paεεenger protein. 4. A chimaeric gene according to claim 3, wherein the linker sequence comprises up to the first 25 natural amino-terminal residueε of the mature wheat 33kDa protein. 5. A chimaeric gene according to any one of the preceding claims, wherein the DNA sequence encoding the said pre-sequence iε as follows: ATG GCA GCG TCT CTC CAA GCC GCG GCC ACG CTG ATG CCG GCC AAG ATC GGC GGC CGG GCC TCC TCC GCG CGA CCG TCG TCG CAC GTC GCC CGG GCG TTC GGC GTC GAC GCT GGC GCC AGG ATC ACC TGC TCC CTG CAG TCC GAC ATC AGG GAG GTC GCA AGC AAG TGC GCC GAC GCC GCC AAG ATG GCC GGC TTC GCC CTC GCC ACC TCT GCC CTC CTC GTC TCC GGC GCG ACG GCG. 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 expresεed in a plant cell tranεformed with the vector. 7. A vector according to claim 6, which further containε a region which enableε 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 iε 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 claimε 1 to 5. 11. A tranεgenic plant which haε been regenerated from plant cellε aε claimed in claim 9 or 10. 12. A transgenic plant which harbours in its cellε a chimaeric gene aε claimed in any one of claimε 1 to 5. 13. Seed obtained from a tranεgenic plant aε claimed in claim 11 or 12. 14. A method of producing a deεired protein in a plant cell, which method comprises: (i) transforming a plant cell with a vector aε claimed in any one of claimε 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 tranεgenic plant capable of producing a deεired 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 deεired protein; and (ii) regenerating plants from the transformed cellε

Inventors: ROBINSON COLIN, ROBINSON, COLIN
Applicants: AGRICULTURAL GENETICS CO, BIOTAL LTD, BP NUTRITION, CAMBRIDGE ADVANCED TECH, CIBA GEIGY PLC, ICI PLC, RHONE POULENC LTD, SCHERING AGROCHEMICALS LTD, SHELL RES LTD, TWYFORD SEEDS LTD, UNILEVER UK CENTRAL RESOURCES, 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 LTD., SHELL RESEARCH LTD., TWYFORD SEEDS LTD., UNILEVER U.K. CENTRAL RESOURCES LTD.
Application year: 1990
Main classification: C12N15
IPC codes: A01H5, C07K14, C12N5, C12N9, C12N15, C12P21, C12R1