Patent Grant
8053632
This application is the U.S. National Stage of International Application PCT/EP2003/013018 filed Nov. 20, 2003, which designates the U.S. and was published by the International Bureau in English on Jun. 3, 2004, and which claims the benefit of German Patent Application No. 102 54 166.3 filed Nov. 20, 2002; both of which are hereby incorporated herein in their entirety by reference.
The present invention relates to a method of controlling a cellular process of interest in a plant by an external signal like an externally applied polypeptide. The invention further relates to a transiently or stably genetically-modified plant adapted for said method and to a genetically-modified plant which has been controlled according to the method of the invention. Moreover, the present invention relates to a method of producing a product in a genetically-modified plant by controlling a cellular process of interest using an encrypted external signal. The process of the invention allows for the selective control of transgene expression in a transiently or stably genetically modified plant, whereby a cellular process of interest previously non-operable in the plant may be selectively switched on at any predetermined time.
One of the major problems in plant biotechnology is the achievement of reliable control over transgene expression. Tight control over gene expression in plants is essential if a downstream product of transgene expression is growth inhibitory or toxic, like for example, biodegradable plastics (Nawrath, Poirier & Somerville, 1994, Proc. Natl. Acad. Sci., 91, 12760-12764; John & Keller, 1996, Proc. Natl. Acad. Sci., 93, 12768-12773; U.S. Pat. Nos. 6,103,956; 5,650,555) or protein toxins (U.S. Pat. No. 6,140,075). Existing technologies for controlling gene expression in plants, are usually based on tissue-specific and inducible promoters and practically all of them suffer from a basal expression activity even when uninduced, i.e. they are “leaky”. Tissue-specific promoters (U.S. Pat. No. 5,955,361; WO09828431) represent a powerful tool but their use is restricted to very specific areas of applications, e.g. for producing sterile plants (WO9839462) or expressing genes of interest in seeds (WO00068388; U.S. Pat. No. 5,608,152). Inducible promoters can be divided into two categories according to their induction conditions: those induced by abiotic factors (temperature, light, chemical substances) and those that can be induced by biotic factors, for example, pathogen or pest attack. Examples of the first category are heat-inducible (U.S. Pat. No. 5,187,287) and cold-inducible (U.S. Pat. No. 5,847,102) promoters, a copper-inducible system (Mett et al., 1993, Proc. Natl. Acad. Sci., 90, 4567-4571), steroid-inducible systems (Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; U.S. Pat. No. 6,063,985), an ethanol-inducible system (Caddick et al., 1997, Nature Biotech., 16, 177-180; WO09321334), and a tetracycline-inducible system (Weinmann et al., 1994, Plant J., 5, 559-569). One of the latest developments in the area of chemically inducible systems for plants is a chimaeric promoter that can be switched on by glucocorticoid dexamethasone and switched off by tetracycline (Bohner et al., 1999, Plant J., 19, 87-95). For a review on chemically inducible systems see: Zuo & Chua, (2000, Current Opin. Biotechnol., 11, 146-151). Other examples of inducible promoters are promoters which control the expression of patogenesis-related (PR) genes in plants. These promoters can be induced by treatment of a plant with salicylic acid, an important component of plant signaling pathways in response to pathogen attack, or other chemical compounds (benzo-1,2,3-thiadiazole or isonicotinic acid) which are capable of triggering PR gene expression (U.S. Pat. No. 5,942,662).
There are reports of controllable transgene expression systems using viral RNA/RNA polymerase provided by viral infection (for example, see U.S. Pat. Nos. 6,093,554; 5,919,705). In these systems, a recombinant plant DNA sequence includes the nucleotide sequences from the viral genome recognized by viral RNA/RNA polymerase. The effectiveness of these systems is limited because of the low ability of viral polymerases to provide functions in trans, and their inability to control processes other than RNA amplification. Another way is to trigger a process of interest in a transgenic plant by using a genetically-modified virus which provides a heterologous nucleic acid encoding a switch for a biochemical process in a genetically-modified plant (WO02068664).
The systems described above are of significant interest as opportunities of obtaining desired patterns of transgene expression, but they do not allow tight control over the expression patterns, as the inducing agents (copper) or their analogs (brassinosteroids in case of steroid-controllable system) can be present in plant tissues at levels sufficient to cause residual expression. Additionally, the use of antibiotics and steroids as chemical inducers is not desirable or economically unfeasible for large-scale applications. When using promoters of PR genes or viral RNA/RNA polymerases as control means for transgenes, the requirements of tight control over transgene expression are also not fulfilled, as casual pathogen infection or stress can cause expression. Tissue- or organ-specific promoters are restricted to very narrow areas of application, since they confine expression to a specific organ or stage of plant development, but do not allow the transgene to be switched on at will. Recombinant viral switches as described in WO02/068664 address all these problems, but do not guarantee tight environmental safety requirements, as the heterologous nucleic acid in the viral vector can recombine.
There is an abundant literature including patent applications which describe the design of virus resistant plants by the expression of viral genes or mutated forms of viral RNA (e.g. U.S. Pat. Nos. 5,792,926; 6,040,496). However, there is an environmental risk associated with the use of such plants due to the possibility of forming novel viruses by recombination between the challenging virus and transgenic viral RNA or DNA (Adair & Kearney, 2000, Arch. Virol, 145, 1867-1883).
Hooykaas and colleagues (2000, Science, 290, 979-982; WO01/89283) described the use of a translational fusion of Cre recombinase with vir gene fragments for Agrobacterium-mediated recombinase translocation into plant cells. Cre-mediated in planta recombination events resulted in a selectable phenotype. The translocation of Cre recombinase is the first use of a translocated protein as a switch to trigger a process of interest in plant cells. However, despite the translocation is not necessarily accompanied by DNA transfer, this approach does not guarantee high level safety, as the phytopathogenic genetically-modified microorganism (Agrobacterium) posesses a complete coding sequence of the switching protein Cre recombinase. Further, the process of interest can only be triggered in cells that receive the switching protein. If large ensembles of cell are to be treated, the ratio of cells receiving switching protein to the total number of cells becomes very small. The method of Hooykaas can therefore not be applied to entire plants. Instead, its usefulness is limited to cells in tissue culture or cell culture.
It is therefore object of this invention to provide a method of switching on a cellular process of interest in entire plants. It is another object of the invention to provide an environmentally safe method of switching on a cellular process of interest in plants, whereby the cellular process may be selectively switched on at any predetermined time. It is another object of this invention to provide a method for producing a product in a transgenic plant, wherein the production of the product may be selectively switched on after the plant has grown to a desired stage, whereby the process is environmentally safe in that genetic material necessary for said cellular process and genetic material coding for the control function are not spread in the environment together.
The above objects are achieved by a method of controlling a genetically-modified plant, comprising
The invention also provides genetically-modified plants or parts thereof obtained or obtainable by the method of the invention. Preferred parts of said plants are leaves and seeds. Seeds are most preferred examples for parts of a plant.
The invention also provides a genetically-modified plant containing a heterologous nucleic acid in cells thereof, wherein said plant is inactive with regard to a cellular process of interest, wherein said heterologous nucleic acid is adapted such that said cellular process of interest can be switched on by directly introducing a polypeptide into cells containing said heterologous nucleic acid, wherein said polypeptide and said heterologous nucleic acid are mutually adapted such that said polypeptide is capable of switching on said cellular process of interest.
Further, the invention provides a system for controlling a cellular process of interest in a genetically-modified plant, comprising a plant as defined above and a polypeptide for switching on said cellular process of interest in the genetically-modified plant, whereby said plant and said polypeptide are mutually adapted such that said polypeptide is capable of switching on said cellular process of interest.
The present invention allows to switch on a cellular process of interest in a plant by directly introducing a polypeptide into cells that contain said heterologous nucleic acid. Directly introducing said polypeptide means that said introducing does not comprise applying nucleic acids to said plant that code for said polypeptide or for a functional part of said polypeptide. A part of said polypeptide is functional if it is capable of switching on the cellular process of the invention. By said direct application of said polypeptide to said plant, a very high level of biological safety is achieved by the invention, since the plant does not come into contact with genetic material that could switch on said cellular process of interest. Instead, at least one necessary component for said cellular process is provided to the plant as a polypeptide without genetic material coding for said polypeptide. A major advantage of the invention is that genetic material necessary for the cellular process of interest and genetic material coding for said polypeptide cannot both be transferred to progeny of said plant or otherwise spread together in the environment.
The method of Hooykaas (2000, Science, 290, 979-982; WO01/89283) allows switching on a cellular process of interest in plant cells, whereby a switching protein is introduced using pathogenic bacteria. As this method is limited to cell culture (laboratory scale), biological safety concerns due to the use of Agrobacteria that code for the switching protein do not arise. The present invention provides for the first time a method of controlling a cellular process of interest that is efficient in whole plants and that is at the same time environmentally safe even when used on a large scale like in a green-house or on a farm field.
In step (a) of the method of the invention, a genetically-modified plant is provided. Higher plants, notably higher crop plants, are preferred. Said plant is genetically-modified in that cells of said plant contain a heterologous nucleic acid that is involved in switching on said cellular process of interest. In many cases, said heterologous nucleic acid may code for a protein to be expressed. Said plant provided in step (a) may be a transgenic plant, whereby most or all of the cells of said plant contain said heterologous nucleic acid stably integrated in the genome of said cells. Said heterologous nucleic acid may be stably integrated into the nuclear genome or in the genome of organelles like mitochondria or, preferably, plastids. Integration of said heterologous nucleic acid in the plastid genome is advantageous in terms of biological safety. The method of the invention is preferably carried out with transgenic plants. Alternatively, however, said plant may be transiently modified and/or said heterologous nucleic acid may be present in a fraction of cells but not in other cells. A heterologous nucleic acid in a transiently modified plant may be stably integrated in the genome of said fraction of cells or it may be present episomally. Incorporation of said heterologous nucleic acid in a fraction of cells of said plant may be achieved by transiently transfecting said organism e.g. using viral transfection or Agrobacterium-mediated transformation. In any case, the genetically-modified plant provided in step (a) is inactive with regard to the cellular process of interest before step (b) has been carried out.
In step (b) of the method of the invention, said polypeptide is introduced from a cell-free composition into at least some of said cells containing said heterologous nucleic acid. If said plant is transgenic, said polypeptide may in principal be applied to any part or to any cells of the plant. If only a fraction of the cells of said plant contains said heterologous nucleic acid, said polypeptide is applied to the plant such that said polypeptide can reach cells containing said heterologous nucleic acid for switching on the cellular process of interest. As noted above, said polypeptide is directly introduced into cells of said plant from a cell-free composition. A cell-free composition does not contain viable cells that could replicate nucleic acids coding for said polypeptide. Preferably, said cell-free composition contains no viable cells. A cell-free composition may be a cell extract obtained by lysing cells (e.g. cells like bacterial cells used for expressing said polypeptide), provided there are no viable cells in said composition that could replicate nucleic acids coding for said polypeptide. Other examples of cell-free compositions are solutions, preferably buffered aqueous solutions, of said polypeptide or said polypeptide in solid or dry form, provided there are no viable cells as defined above.
Directly introducing may be done by (i) particle (microprojectile) bombardment, (ii) application of said polypeptide on at least a part of said plant, or (iii) by injecting a solution containing said polypeptide in tissue of said plant. In methods (ii) and (iii), said polypeptide is typically contained in a liquid, preferably aqueous, cell-free composition (or solution) that is applied to parts of the plant. Such a composition may be applied e.g. by spraying said plant with said composition containing the polypeptide. Further, said composition may be injected according to (iii).
For methods (ii) and (iii), said polypeptide preferably comprises a membrane translocation sequence (MTS) that enables entering of said polypeptide into cells of said plant. Said membrane translocation sequence may be covalently or non-covalently bound to said polypeptide. Preferably, it is covalently bound to said polypeptide. Said membrane translocation sequence may be a peptide that endows said polypeptide with the capability of crossing the plasma membrane of cells of said organism. Many such membrane translocation sequences are known in the art. Frequently, they comprise several basic amino acids, notably arginines. The size of membrane translocation sequences may vary largely, however, they may typically have 3 to 100 amino acids, preferably 5 to 60 amino acids. Said polypeptide may be produced by standard protein expression techniques e.g. in E. coli. Purification of said polypeptide after its expression is preferably done, notably removal or destruction of nucleic acids coding for said polypeptide. Nucleic acids may be removed or destroyed by hydrolysis, preferably catalysed by an enzyme like a (DNase) or a ribonuclease (RNase). Further or additionally, chromatographic techniques may be used for removing nucleic acids from said polypeptide. Said polypeptide may be applied to a plant e.g. by spraying said plant with a liquid composition, preferably an aqueous solution, containing said polypeptide. Preferably, measures are taken to facilitate entering of said polypeptide into cells of a plant, notably measures that allow crossing of the plant cell wall and/or the outer plant layer. An example of such measures is slight wounding of parts of the plant surface e.g. by mechanical scratching. Another example is the use of cellulose-degrading enzymes to weaken or perforate the plant cell wall.
Switching on of the cellular process of interest (step (b)) requires directly introducing said polypeptide from a cell-free composition into cells that contain said heterologous nucleic acid. Said polypeptide and said heterologous nucleic acid are mutually adapted such that said polypeptide is capable of switching on said cellular process of interest.
With respect to said cellular process of interest, there are no particular limitations and the invention is of very broad applicability. Said cellular process of interest may be or may comprise formation of a DNA, an RNA or a protein from said heterologous nucleic acid or involving said heterologous nucleic acid. There are numerous possibilities for achieving formation of said DNA, said RNA or said protein. Said polypeptide may for example comprise a segment having a binding activity to said heterologous nucleic acid, e.g. to a promoter. Said segment may then e.g. act as a transcription factor inducing transcription of said hetereologous nucleic acid, thus triggering formation of said RNA and or said protein.
Preferably, said polypeptide has a segment having an enzymatic activity capable of triggering formation of said DNA, said RNA or said protein. Examples of such activities are DNA or RNA-modifying activities like the activity of a site-specific recombinase, flippase, resolvase, integrase, polymerase, or a transposase. Said enzymatic activity may modify said heterologous nucleic acid leading to expression of said protein e.g. by recombination. In an embodiment wherein said polypeptide has polymerase activity, said segment may be a DNA-dependent RNA polymerase that acts on a promoter of said heterologous nucleic acid. Said promoter is preferably not recognized by native polymerases of said plant. Examples of such promoter-polymerase systems are bacterial, viral, or bacteriophage promoter-polymerase systems like the T7 promoter-T7 polymerase.
Moreover, said switching on of said cellular process of interest may comprise formation of a DNA, an RNA or a protein from said heterologous nucleic acid or involving said heterologous nucleic acid. As an example, the formation of an expressible operon from said heterologous nucleic acid or from an RNA expression product of said heterologous nucleic acid may be mentioned.
A sequence portion of said heterologous nucleic acid (or of said additional nucleic acid described below) may be operably linkable to a transcription promoter by the action of said protein, which allows to switch on expression of a protein of interest or transcription of an RNA-viral amplicon from said additional heterologous nucleic acid, e.g. by operably linking a sequence encoding said protein of interest or an RNA amplicon with a promoter. There are several ways of reducing this embodiment to practice. One option is to separate, in said (additional) heterologous nucleic acid, the sequence encoding an RNA amplicon and a promoter by a sequence block that precludes an operable linkage therebetween. Said sequence block may be flanked by recombination sites such that said block can be cut out by a recombinase recognizing said recombination sites. Thereby, operable linkage for transcription of the sequence encoding an RNA amplicon can be established and expression may be switched on. Another option is to have a portion of a sequence necessary for transcription (e.g. a promoter or promoter portion) in flipped orientation and flanked by recombination sites. Providing a suitable recombinase (e.g. with said polypeptide) may flip said sequence portion back in correct orientation, whereby an operable linkage can be established.
Further, said DNA, said RNA or said protein may be capable of spreading to other cells of said plant (e.g. a DNA or RNA viral vector). An important example of such a cellular process is the formation of an expressible amplicon from said heterologous nucleic acid or from an RNA expression product of said heterologous nucleic. Said amplicon is capable of amplifying within cells of its activation or formation (amplifying vector). Said amplicon may be an expressible amplicon that contains a gene of interest to be expressed in said cellular process of interest. Further, said amplicon may be capable of cell-to-cell or systemic movement in the plant of the invention. An amplicon may be based on a plant DNA or RNA virus. Plant RNA viruses like tobamoviruses are preferred. The amplification properties of said protein capable of spreading (see below) and said amplicon may behave synergistically, thus allowing an extremely strong cellular process of interest that spreads over significant parts of said plant (e.g. leading to extremely strong expression of a protein of interest from said amplicon). Engineering of amplicons based on Tobamoviruses is known in the art (see e.g. Dawson et al., 1989, Virology, 172, 285-293; Yusibov et al., 1999, Curr. Top. Microbiol. Immunol., 240, 81-94; for review, see “Genetic Engineering With Plant Viruses”, 1992, eds. Wilson and Davies, CRC Press, Inc.).
In a major embodiment of the invention, said switching on of said cellular process of interest involves formation of a protein from said heterologous nucleic acid, whereby said protein is capable of spreading within the plant, i.e. capable of leaving a cell of its formation and entering other cells of said plant (such a protein is also referred to as “protein switch” herein). In other cells, said protein may switch on a cellular process of interest, notably by controlling an additional heterologous nucleic acid (see below). Said leaving a cell and entering other cells preferably comprises cell-to-cell-movement or systemic movement in said plant or in a part thereof. Said protein (also referred to herein as “protein switch”) preferably contains a protein portion enabling said leaving a cell and entering other cells of said protein switch. Said protein portion may be a domain of a viral movement protein or of a viral coat protein. Further, said protein portion may be a plant or an animal transcription factor, or a domain of a plant or animal transcription factor capable of cell-to-cell or systemic movement. Further, said protein portion may be a plant or animal peptide intercellular messenger, or a domain of a plant or an animal peptide intercellular messenger. Moreover, said protein portion may be an artificial peptide capable of enabling cell-to-cell or systemic movement. Preferably, however, said protein portion is or comprises a viral movement protein or viral coat protein, or a domain of a viral movement or coat protein.
When said protein capable of spreading enters other cells that contain said heterologous nucleic acid, it is preferably capable of switching on (inducing) expression of said protein from said heterologous nucleic acid. By making use of this protein (protein switch), the method of the invention allows to amplify and propagate the switching signal provided externally with said polypeptide in said plant. Particularly, if the number of cells initially reached by said polypeptide is small, the switching signal is efficiently carried to further cells in said plant. There are many ways how said protein can be made to control its own expression from said heterologous nucleic acid. These ways correspond to those that may be employed for said polypeptide of step (b) given above.
Apart from the capability of controlling its own expression, the protein has preferably the capability of switching on a cellular process of interest. Although switching on of a cellular process is preferred, it is clear to those skilled in the art that the end result of a cellular process that was switched on may also be a suppression or a switching off of a process in cells of the plant. For being capable of switching on said cellular process of interest, said protein may have a segment that is capable of controlling said cellular process. Said segment may have a binding activity or an enzymatic activity that controls a nucleic acid (notably said additional heterologous nucleic acid) necessary for said cellular process of interest. In the method of the invention, said switching on of said cellular process may be achieved analogously to the control of its own expression from said heterologous nucleic acid. For this purpose, said protein may have a segment for controlling said cellular process and a segment for causing said expression of said protein, whereby the control mechanisms of said two segments may be different. Preferably, the two control mechanisms are similar or identical, wherein one segment of said protein may be sufficient for switching on said cellular process and for controlling the expression of said protein. Thus, for simplicity, said protein contains most preferably said one segment and a portion endowing said protein with the capability of leaving a cell and entering other cells of said plant.
In the invention, said polypeptide of the invention can have the same switching function as said protein switch, e.g. have the same enzymatic activities as said protein switch. Said polypeptide is applied externally and can switch on a cellular process of interest in cells it enters. Said protein switch is produced inside cells of said genetically-modified plant, preferably in response to the switching function of said polypeptide. Said protein switch can in turn, after its production in cells of said plant, switch on a cellular process of interest in cells where it is produced and/or in other cells of said plant. If said polypeptide and said protein switch exert their switching function by the same of a related enzymatic activity, they may differ in that said polypeptide preferably has a membrane translocation sequence, whereas said protein switch preferably has a protein portion endowing said protein switch with the capability of leaving a cell and entering other cells.
Said cellular process of interest may require, as mentioned above, the presence of an additional heterologous nucleic acid in cells of said plant where said cellular process is to be controlled. Said additional heterologous nucleic acid may be present in all cells or in a fraction of cells of said plant. It may be stably incorporated in nuclear or organellar genomes of cells of said organism. What has been said regarding said heterologous nucleic acid of the invention generally applies also to said additional heterologous nucleic acid. Preferably, said plant is transgenic regarding said additional heterologous nucleic acid and regarding said heterologous nucleic acid.
Said additional heterologous nucleic acid will e.g. be made use of, if said heterologous nucleic acid is used for forming a protein capable of spreading in the plant. The spreading protein may then switch on a cellular process of interest encoded in said additional heterologous nucleic.
In a further important embodiment of this invention, a protein expressed from said heterologous nucleic acid and said directly introduced polypeptide jointly generate a predetermined function leading to switching on said cellular process of interest only when said protein and said polypeptide are jointly present (cf.
The cellular process of interest that was switched on as described herein does, however, not have to affect the entire plant. Instead, said cellular process of interest may be limited to a part of said plant like leaves or seeds. A cellular process of interest in seeds may be the production of a protein of interest in seeds, whereby the protein of interest can be easily harvested by conventional methods and stored in said seeds. Preferably, however, the cellular process of interest affects substantial parts of said plant. The part of a plant where said cellular process is switched on depends inter alia on the place(s) of application of said polypeptide. Generally, said cellular process of interest may be strongest in the vicinity of the place of application of said polypeptide and may decrease with increasing distance from said place. Said decrease may in general be anisotropic and depend on the structure of the tissue of said plant where said polypeptide was applied. If, for example, a cellular process of interest is to be switched on (e.g. expression of a gene of interest is to be switched on) and said polypeptide is applied to a fraction of a leaf of the plant, said cellular process of interest typically occurs within said fraction of said leaf and in the vicinity of said fraction of said leaf. Preferably, said cellular process of interest occurs in the major part of said leaf. More preferably, said cellular process of interest occurs also in the shoot and in other leaves. Most preferably, said cellular process of interest occurs in the major part of said plant. The extent of said cellular process of interest (e.g. expression of a gene of interest) may vary within said plant e.g. with the cell type or tissue type. Obviously, application of said polypeptide is normally not limited to a single point on the surface of a plant. Preferably, said polypeptide is applied to several parts of said plant (see further below).
The cellular process according to the invention may comprise or give rise to a whole biochemical cascade of interest like a multi-step biosynthetic pathway in cells of the plant. The cellular process or biochemical cascade of interest is not operable in the plant prior to exposure to said polypeptide. The method of the invention may provide control over a cellular process or biochemical cascade of interest with a hitherto unattainable technical precision and environmental safety. Thereby, novel applications in biotechnology in general, specifically in plant biotechnology, are available for solving problems which cannot be solved by conventional technologies like basal transgene expression activity in a plant, particularly when producing toxic substances or biodegradable polymers. Moreover, the precise control according to the invention allows to grow a transgenic plant to a desired stage where, for example, the plant is best suited for performing the cellular process of interest without burdening the plant with a basal expression activity slowing down the growth of the plant. Once the plant is ready for efficiently performing the cellular process of interest, the process of interest may be switched on and performed with high efficiency. Accordingly, the method of the invention allows to safely decouple the growth phase and the production phase of a multicellular organism, specifically a transgenic plant. Moreover, it is possible to design multi-component systems for multiple cellular processes or biochemical cascades of interest, whereby one or more desired processes or cascades can be selectively switched on.
(A) depicts a heterologous nucleic acid encoding PS:TP and an additional heterologous nucleic acid hNA. No external polypeptide is introduced, thus the protein switch is not expressed and no cellular process of interest is switched on.
(B) an external (cell-permeable) polypeptide is introduced causing expression of the protein-switch PS:TP. The protein switch can control a cellular process by acting on hNA in the cell of its expression. Further, the protein switch can leave the cell that was triggered by said externally introduced polypeptide and enter other cells. In other cells, the protein switch can induce its own expression and also control the cellular process by acting on hNA.
(C) depicts a heterologous nucleic acid encoding two protein switches: PS1 and PS2. Expression of PS1 is caused by an externally applied signal (the polypeptide of the invention), leading to PS1:TP. As above, PS1:TP can spread to other cells and activate expression of PS2. PS2 in turn can control a cellular process by acting on hNA.
At the basis of this invention is the use of a polypeptide capable of entering cells of a plant, leading to switching on a cellular process of interest without delivery of nucleic acids encoding said polypeptide or a functional part of said polypeptide into said cells. Preferably, said switching on a cellular process of interest comprises forming a protein that is capable of causing its own expression. The general principle of the method according to the invention is schematically shown in
Choice of Protein for “Switch” Function
The same switching functions that are described herein for said protein switch may also be used for the switching function of said polypeptide and vice versa. There are countless numbers of cellular processes of interest which can be irreversibly triggered by said protein of the invention (protein switch). The protein switch (marked as AB in
There are numerous reactions that affect RNA molecules that may be used as efficient triggering device for the cellular process according to the present invention. These include, inter alia, reactions such as RNA replication, reverse transcription, editing, silencing, or translation. There is abundant prior art describing in detail how, for example, a site-specific recombinase, integrase or transposase can trigger a process of interest by DNA excision, inversion or insertion in cells, notably in plant cells (Zuo, Moller & Chua, 2001, Nat Biotech., 19, 157-161; Hoff, Schnorr & Mundy, 2001, Plant Mol. Biol., 45, 41-49; U.S. Pat. No. 5,225,341; WO9911807; WO9925855; U.S. Pat. Nos. 5,925,808; 6,110,736 WO0140492; WO 0136595). Site-specific recombinases/integrases from bacteriophages and yeasts are widely used for manipulating DNA in vitro and in plants and animals. Preferred recombinases-recombination sites for the use in this invention are the following: Cre recombinase-LoxP recombination site, FLP recombinase-FRT recombination sites, R recombinase-RS recombination sites, phage C31 integrase recognising attP/attB sites etc. Transposons are widely used for the discovery of gene function in plants. Preferred transposon systems for use in the present invention include Ac/Ds, En/Spm, transposons belonging to the “mariner” family, etc.
Heterologous transcription factors and RNA polymerases may also be used in a protein switch according to the invention. For example, the delivery of T7 polymerase into cells of a plant carrying a transgene under the control of the T7 promoter may induce the expression of such a transgene.
The expression of a plant transgene (e.g. the additional heterologous nucleic acid of the invention) that is under control of a bacteriophage promoter (e.g. T3, T7, SP6, K11) with the corresponding DNA/RNA polymerase delivered into cells of a plant may be another efficient approach for the development of protein switches contemplated in this invention. Another useful approach may be the use of heterologous or chimaeric or other artificial promoters which require heterologous or engineered transcription factors for their activation. Heterologous transcription factors also can be used in order to induce expression of the transgene of interest under control of said transcription factor-recognizable promoter. Examples of such transcription factors are inter alia yeast metalloresponsive ACE1 transcription factor binding specific sequences in the yeast MT (metallothionein) promoter (Meft et al., 1993, Proc. Natl. Acad. Sci., 90, 4567-4571), different chimaeric transcription factors having a sequence-specific DNA-binding domain and an activation domain like a transcription factor having a fusion six-zink finger protein 2C7 and herpes simplex virus VP16 transcription factor activation domain (Ordiz, Barbas & Beachy, 2002, Proc. Natl. Acad. Sci. USA, 99, 13290-13295), a transcription factor having a full length 434 repressor and the C-terminal 80 amino acids of VP16 transcriptional activator (Wilde et al., 1994, Plant Mol. Biol., 24, 381-388), a transcription factor used in steroid-inducible systems (Aoyama & Chua, 1997, Plant J., 11, 605-612; McNellis et al., 1998, Plant J., 14, 247-257; U.S. Pat. No. 6,063,985) or a tetracycline-inducible system (Weinmann et al., 1994, Plant J., 5, 559-569). In some cases, the existing inducible systems for transgene expression may be used. Alternatively, heterologous transcription factors may be modified such that no activating ligand-inducer will be required to drive the transcription factor into the active state. Chimaeric transcription factors would be of advantage for the use in this invention, as they allow to combine highly sequence-specific DNA binding domains and highly efficient activation domains, thus allowing a maximum desired effect after delivery of such a factor into the plant cell.
Another protein switch contemplated under the invention may rely on posttranslational modification of one or more expression product(s) of a heterologous nucleic acid, which may lead to the activation of the expression product. There are many possible implementations of such protein switches that could operate by controlling steps such as polypeptide folding, oligomer formation, removal of targeting signals, conversion of a pro-enzyme into an enzyme, blocking enzymatic activity, etc. For example, delivery of a site-specific protease into cells of a plant may trigger a cellular process of interest if a genetically-engineered host specifically cleaves a pro-enzyme, thus converting it into an active enzyme, if a product is targeted to a particular cellular compartment because of the host's ability to cleave or modify a targeting motif, or if a product is specifically mobilised due to the removal of a specific binding sequence. Cleavage of a translational fusion protein can be achieved via a peptide sequence recognized by a viral site-specific protease or via a catalytic peptide (Dolja et al., 1992, Proc. Natl. Acad. Sci. USA, 89 10208-10212; Gopinath et al., 2000, Virology, 267 159-173; U.S. Pat. No. 5,162,601; U.S. Pat. No. 5,766,885; U.S. Pat. No. 5,491,076). Other examples of site-specific proteases applicable to this invention are mammalian enterokinases, for example, human enterokinase light chain which recognizes the sequence DDDK-I (SEQ ID NO:1) (Kitamoto et al., 1994, Proc. Natl. Acad. Sci., 91, 7588-7592), and specifically cleaves Lys-Ile bonds; viral proteases, like Hc-Pro (Carrington JC & Herndon KL,1992, Virology, 187, 308-315) which catalyzes proteolysis between the Gly-Gly dipeptide but requires 4 amino acids for the recognition of the cleavage site; site-specific protease of Semliki Forest Virus (Vasiljeva et al., 2001, J Biol Chem., 276, 30786-30793); and proteases involved in polyubiquitin processing, ubiquitin-carboxy-terminal hydrolases (Osava et al., 2001, Biochem Biophys Res Commun., 283, 627-633).
Directly Introducing Said Polypeptide into Cells of a Plant
a) Microprojectile Bombardment (Particle Bombardment)
Different methods can be used for directly introducing (direct delivery) said polypeptide into cells of said plant. Among the simplest ones is the direct delivery with the help of mechanical interaction with plant tissue. For example, microprojectile bombardment of polypeptide-coated particles can deliver said polypeptide into the plant cell. The protocol can be similar to those described for DNA delivery in plant transformation protocols (U.S. Pat. No. 5,100,792; EP 00444882B1; EP 00434616B1). However, instead of DNA, said polypeptide may be used for coating the particles. There is a description of a biolistic process that uses particle coating methods which are reasonably gentle for preserving the activity of said polypeptide (Sanford, Smith & Russell, 1993, Methods in Enzymol., 217, 483-509). In principle, other plant transformation methods can also be used e.g. microinjection (WO 09209696; WO 09400583A1; EP 175966B1), or liposome-mediated delivery (for review see: Fraley & Papahadiopoulos, 1982, Curr. Top. Microbiol. Immunol., 96, 171-191).
b) Use of Membrane Translocation Amino Acid Sequences
The polypeptide of interest can be applied externally to target cells of said plant using a covalent fusion or non-covalent interaction with a membrane translocating sequence. Many examples of membrane translocating sequences (MTS), natural and synthetic, are known in the art. They are widely used as fusions with peptide drugs and therapeutic proteins in order to increase their cell membrane permeability. An MTS may be a simple amino acid repeat, for example a cationic peptide containing eleven arginines (SEQ ID NO:2) RRRRRRRRRRR (Matsushita et al., 2001, J. Neurosci., 21, 6000-6007). Another cationic MTS is a 27 amino acid long transportan (SEQ ID NO:3) GWTLNSAGYL LGKINLKALA ALAKKIL (Pooga et al., 1998, FASEB J., 12, 67-77). It is very likely that such peptides, for their penetration of the cell, exploit the asymmetry of the cellular plasma membrane where the lipid monolayer facing the cytoplasm contains anionic phospholipids (Buckland & Wilton, 2000, Biochim. Biophys. Acta/Mol. Cell. Biol. Of Lipids, 1483, 199-216). Certain proteins also contain subunits that enable their active translocation across the plasma membrane into cells. To such domains belongs the basic domain of HIV-1 Tat49-57 (SEQ ID NO:4) (RKKRRQRRR) (Wender et al., 2000, Proc. Natl. Acad. Sci. USA, 97 13003-13008), Antennapedia43-58 (SEQ ID NO:5) (RQIKIWFQNR RMKWKK) (Derossi et al., 1994, J. Biol. Chem., 269, 10444-10450), the Kaposi Fibroblast Growth Factor MTS (SEQ ID NO:6) (AAVALLPAVL LALLAP) (Lin et al., 1995, J. Biol. Chem., 270 14255-14258); the VP22 MTS (Bennet, Dulby & Guy, 2002, Nat. Biotechnol., 20, 20; Lai et al., 2000, Proc. Natl. Acad. Sci. USA, 97, 11297-302); homeodomains from the Drosophila melanogaster Fushi-tarazu and Engrailed proteins (Han et al., 2000, Mol Cells 10, 728-732). It was shown that all these positively charged MTSs are able to achieve cell entry by themselves and as fusions with other proteins like GFP (Zhao et al., 2001, J. Immunol. Methods, 254, 137-145; Han et al., 2000, Mol Cells, 10, 728-732), Cre recombinase (Peitz et al., 2002, Proc. Natl. Acad. Sci. USA, 4489-4494) in an energy-independent manner. However, the fusion is not necessarily required for protein transport into the cell. A 21-residue peptide carrier Pep-1 was designed (SEQ ID NO:7) (KETWWETWWTEWSQPKKKRKV) which is able to form complexes by mean of non-covalent hydrophobic interactions with different types of proteins, like GFP, b-Gal, or full-length specific antibodies. These complexes are able to efficiently penetrate cell membranes (Morris et al., 2001, Nature Biotechnol., 19, 1173-1176). The list of MTS can be continued and, in general, any synthetic or naturally occurring arginine-rich peptide can serve for practicing this invention (Futaki et al., 2001, J. Biol. Chem., 276, 5836-5840).
As there is no essential structural difference between plant and animal cell membranes affecting their general architecture and physico-chemical properties, said fusions of MTS with said polypeptide of the invention can also be efficiently used for penetrating plant cells. However, unlike animal cells, plant cells possess a tough cell wall (Varner & Linn, 1989, Cell, 56, 231-239; Minorsky, 2002, Plant Physiol., 128, 345-53). This obstacle can be overcome by using simple techniques. For example, injection of a (e.g. crude) protein extract containing said polypeptide having an MTS into a plant apoplast facilitates translocation of said polypeptide into the plant cells. Another approach to overcome the cell wall and to reach the cell membrane of plant cells can be the application of cellulytic enzymes many of which are commercially available. Once added to a composition containing said polypeptide, said enzymes help to remove or weaken the cell wall, but will leave the cell membrane intact and exposed for penetration by said polypeptide containing said MTS. Said cellulytic enzymes from bacteria and molds have been commercially available at industrial scale for a long time and are widely used (e.g. “Onozuka” R-10 enzyme preparation of Trichoderma harzianum, etc.) in plant cell tissue culture for obtaining plant protoplasts (Sidorov &Gleba, 1979, Tsitologia, 21, 441-446; Gleba & Gleba, 1978, Tsitol Genet, 12, 458-469; Ghosh et al., 1994, J. Biotechnol., 32, 1-10; Boyer, Zaccomer & Haenni, 1993, J. Gen. Virol, 74, 1911-1917; Hilbricht, Salamini & Bartels, 2002, Plant J., 31, 293-303). The approach of using cellulytic enzymes has potential for large scale applications of this invention. A mixture of cellulytic enzymes with a cell-permeable polypeptide can be sprayed over the genetically-modified plants or over parts thereof. Cellulases can make cell membranes accessible for membrane permeable polypeptides. Upon translocation into the cell, said polypeptide may trigger said cellular process of interest and the expression of said protein within the plant.
In addition to the above delivery methods for said polypeptide, efficient spreading of a protein switch inside the plant is preferably used for amplification purposes within said plant. Further, to make the overall method safe, strict control over the heterologous nucleic acid is required.
In order to address these issues it is proposed herein to use a “split genes” (or “split proteins”) approach for controlling the segregation of a transgene encoding the protein switch. In this embodiment, an active (functional) protein switch is assembled either by intein-mediated protein trans-splicing (
Intein-mediated trans-splicing of proteins with restoration of their activity is known in the prior art and is described in detail in many publications. Protein affinity interaction and/or trans-splicing can be achieved by using engineered inteins (
Trans-splicing of protein fragments (including covalent bond formation between exteins) is not necessarily required to restore the original function of the split protein. In many cases, affinity interaction between protein parts without peptide bond formation is sufficient to restore protein function (
Leucine zipper domains are of special interest for forming protein heterodimers once fused to a protein of interest (Riecker & Hu, 2000, Methods Enzymol., 328, 282-296; Liu et al., 2001, Curr. Protein Pept. Sci., 2, 107-121). An interesting example is the control of protein-protein interactions with a small molecule. For example, Cre recombinase was engineered in such a way that, when split in two inactive fragments, was able to restore 100% of its recombinase activity in the presence of the small molecule rapamycin that triggered activity complementation by heterodimerization between two inactive fragments (Jullien et al., 2003, Nucleic Acids Res., 31, e131). Rapamycin and, preferably non-toxic, analogues can also be used for conditional protein splicing, where they trigger a trans-splicing reaction (Mootz et al., 2003, J. Am. Chem. Soc., 125, 10561-10569). Similar approaches for regulation of protein-protein interactions with the help of small molecules, such as rapamycin or rapamycin analogues, are described in several papers (Amara et al., 1997, Proc. Natl. Acad. Sci. USA., 94, 10618-10623; Pollock et al., 2000, Proc. Natl. Acad. Sci. USA., 97, 13221-13226; Pollock et al., 2002, Nat. Biotechnol., 20, 729-733). Many other chemical dimerizers such as dexamethasone and methotrexate, can be used for assembling active homo- or heterodimers from inactive protein fragments (for review see: Pollock & Clackson, 2002, Curr. Opin. Biotechnol., 13, 459-467).
Affinity interactions can be efficiently engineered by using naturally occurring interacting protein domains or by identifying such domains with the help of two-hybrid (Fields & Son, 1989, Nature, 340, 245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 9578-9582; Yeast Protocol Handbook, Clontech Laboratories, Inc., 2000) or phage display systems. For example, phage display may be used to select a 5-12-mer oligopeptide with high affinity to a protein fragment of interest. Several such systems are now commercially available. Phage display is a selection technique in which a short variable 5-12-mer oligopeptide is inserted into a coat protein of bacteriophage. The sequence encoding this variable oligopeptide is included in the corresponding gene of the bacteriophage coat protein. Usually, a 7-mer phage display library has at least 109 independent clones bearing different combinations of 7-mer amino acids in variable oligopeptides. Phage display has been used to create affinity complexes between bacteriophage and a protein of interest, allowing rapid identification of peptide ligands for a given target protein by an in vitro selection process called “panning” (Parmley, Smith, 1988, Gene 73, 305-318; Cortese et al., 1995, Curr. Opin. Biotechnol., 6, 73-80). The phage-protein complex created after the panning procedure can be dissociated and a phage with affinity to a target protein can be amplified. Usually, one needs three panning cycles to get bacteriophage with high affinity. After three rounds, individual clones can be characterized by sequencing of variable region in genomic DNA. Said system can be efficiently adopted for identifying short interacting oligopeptides and using them as affinity tags in order to bring together protein fragments.
Another approach includes the use of naturally occurring interacting domains like leucine-rich repeats (Kobe & Deisenhofer, 1994, Trends Biochem Sci., 19, 415-421; Kobe & Kajava, 2001, Curr. Opin. Struct. Biol., 11, 725-732), zinc finger (Grossley, Merika & Orkin, 1995, Mol. Cell. Biol., 15, 2448-2456), ankyrin repeats (Thompson, Brown & McKnight, 1991, Science, 253, 762-768), chromo domains (Paro & Hogness, 1991, Proc. Natl. Acad. Sci. USA, 88, 263-267; Singh et al., 1991, Nucleic Acids Res., 19, 789-793) and many others involved in protein-protein interactions. However, the possibility of involving not only the engineered protein fragments containing the motive fusions in protein-protein interactions, but also endogenous proteins can be taken into account.
Involving protein-protein interactions for switching on a cellular process of interest like gene expression has inter alia the following advantages: Firstly, the system may be rendered highly specific, as the function of interest is a result of a highly specific protein-protein or protein-nucleic acid interaction, which is characterized by zero-level uninduced state and absence of non-specific leakiness. This is in contrast to prior art systems such as switches based on small molecules that are inherently less specific and invariably show a certain degree of leakiness. Secondly, said protein switch or a fragment thereof (or said polypeptide or a fragment thereof can be directly delivered into cells of a plant without a nucleic acid vector encoding said polypeptide, thus allowing precise dosage of said polypeptide. This makes direct delivery of said protein switch (or said polypeptide) into cells of a plant comparable with the use of small molecules for triggering a required process in cells. Thirdly, the system is inherently environmentally safer than prior art systems that contain full genetic information for the protein of interest (either in a form of linear nucleic acid or fragments of said nucleic acid), since it allows that the organism in question does not contain the full genetic information necessary for the expression of a protein of interest. According to the central dogma of molecular biology, biological systems cannot reverse translate proteins to nucleic acids. Thus, the ‘reverse engineering’ of the genetic information sufficient for expression of a functional trait by a living organism is impossible. Fourthly, the system provides a specific lock that could be used to prohibit unauthorized use of the system. The use of said polypeptide as a component of a crude protein extract from organism expressing said polypeptide or said polypeptide fragment makes it practically very difficult to identify the active component of said extract.
Spread of the Protein Switch within a Plant for Triggering a Cellular Process of Interest
Here, an approach for overcoming the problem of the low number of cells of a plant that can be reached by the externally-applied polypeptide is provided: said polypeptide may lead to the formation of an intracellular protein-switch molecule capable of cell-to-cell or systemic movement. Moreover, said polypeptide may lead to or may cause the formation of a virus-based vector (amplicon) expressing a gene of interest or a part thereof and being capable of cell-to cell or systemic movement in said plant. In these approaches, the movement of either viral vectors- or protein-switch molecules or both can lead to the spread of a cellular process and/or biochemical cascade over significant parts of said plant and even all over the genetically-modified plant.
In Example 1 of this invention, the protein-switch contains an integrase phiC31 to convert a precursor vector of a viral vector into the viral vector. The viral vector is capable of amplification, cell-to-cell and systemic movement. Integrase-mediated recombination between attP and attB sites of pICHGFPinv (
Our approach allows to overcome the limitations of the above-described viral vector systems, specifically their limited capacity for the size of the gene to be expressed and the lack of flexibility in controlling the expression. In our invention, the viral vector precursor (also referred to as provector) is preferably present in each cell of the transgenic plant. In the case of expression of large genes (above 1 Kb), protein-switch movement is preferred over viral vector movement. Viral vectors can efficiently amplify in cells and the size of the insert of a viral vector mostly affects the ability for cell-to-cell and systemic movement. Therefore, providing a moveable protein switch capable of activating a viral vector to many cells or even to all cells of the host plant will solve the above-mentioned problem. Additionally, to provide a system with an efficient switching function that is able to turn on the amplification of such a viral vector in most if not all cells of the host plant, protein switches capable of cell-to-cell/systemic movement are used in the present invention.
To this end, the protein switch may contain a protein portion that renders said protein capable of cell-to-cell and/or systemic movement. Examples of such protein portions capable to intercellular trafficking are known in prior art. There is evidence that plant transcription factors, defence-related proteins and viral proteins can traffic through plasmodesmata (for review see: Jackson & Hake, 1997, Curr. Opin. Genet. Dev., 7, 495-500; Ding, B. 1998, Plant Mol. Biol., 38, 279-310; Jorgensen R A., 2000, Sci STKE, 58, PE2; Golz & Hudson, 2002, Plant Cell, 14, S277-S288). It was shown that a fusion of 3a movement protein of Cucumber mosaic virus with GFP can traffic out via plasmodesmata to neighboring cells (Itaya et al., 2002, Plant Cell, 14, 2071-2083). Such fusions also showed movement through phloem from transgenic rootstock into non-transgenic scion. The movement protein of tobacco mosaic virus (TMV), P30, traffics between cells through plasmodesmata and, by affecting plasmodesmata size, facilitates the movement of many other large macromolecules not specified for such movement (Citovsky et al., 1999, Phil. Trans. R Soc. London B Biol Sci., 354, 637-443; Ding, Itaya & Woo, 1999, Int Rev. Cytol., 190, 251-316). The P30:GFP fusion showed movement between the cells independent of physiological conditions, while the non-targeted GFP diffusion through plasmodesmata at large depends of physiological state of the plant cells (Crawford & Zambryski, 2001, Plant Physiol, 125, 1802-1812). The fusion of GFP with transcription factor knotted1 also showed the ability for intercellular trafficking. The GFP:KN1 fusion protein demonstarted movement from internal tissues of the leaf to the epidermis, between epidermal cells and into the shoot apical meristem of tobacco plant (Kim et al., 2002, Proc. Natl. Acad. Sci. USA, 99, 4103-4108). Plasmodesmata play an important role in such trafficking and its physiologigal stage and structure are important for the efficiency of such trafficking. For example, simple plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves, while the branched ones do not (Oparka et al., 1999, Cell, 98, 5-8). Allowing trafficking of macromolecules including proteins appears to be a normal function of plasmodesmata, which was made use of by plant viruses for their cell-to-cell spread (Fujiwara et al., 1993, Plant Cell, 5, 1783-1794). In general, it is evident that plasmodesmata and the phloem play an important role in the transport and delivery of information macromolecules (proteins and nucleic acids) (Ruiz-Medrano et al., 2001, Curr. Opin. Plant Biol., 4, 202-209). Phloem sap proteins from Cucurbita maxima and Ricinum communis have the capacity of cell-to-cell trafficking through plasmodesmata (Balachandran et al., 1997, Proc. Natl. Acad. Sci. USA., 94, 14150-14155).
The ultimate purpose of the protein switch system contemplated herein is an operational control of a cellular process of interest or a cascade of biochemical reactions of interest in a plant production system. A biochemical cascade is a chain of biochemical reactions in a host production system that ultimately yields a specific product, effect, or trait of interest.
The approaches described herein, in addition to being versatile and leakage-proof, provide an efficient production control method. The two-component process described above is in essence a “key-lock” system, whereby a company can efficiently control access to production by selling the protein-switch component.
Preferred plants for the use in this invention include any plant species with preference given to agronomically and horticulturally important species. Common crop plants for the use in present invention include alfalfa, barley, beans, canola, cowpeas, cotton, corn, clover, lotus, lentils, lupine, millet, oats, peas, peanuts, rice, rye, sweet clover, sunflower, sweetpea, soybean, sorghum triticale, yam beans, velvet beans, vetch, wheat, wisteria, and nut plants. The plant species preferred for practicing of this invention are including but not restricted to: representatives of Gramineae, Compositeae, Solanaceae and Rosaceae, whereby Solanaceae are preferred.
Additionally, preferred species for use the invention, as well as those specified above, plants from the genera: Arabidopsis, Agrosts, Allium, Antirrhinum, Apium, Arachis, Asparagus, Atropa, Avena, Bambusa, Brassica, Bromus, Browaalia, Camellia, Cannabis, Capsicum, Cicer, Chenopodium, Chichorium, Citrus, Coffea, Coix, Cucumis, Curcubita, Cynodon, Dactylis, Datura, Daucus, Digitalis, Dioscorea, Elaeis, Eleusine, Festuca, Fragaria, Geranium, Glycine, Helianthus, Heterocallis, Hevea, Hordeum, Hyoscyamus, Ipomoea, Lactuca, Lens, Lilium, Linum, Lolium, Lotus, Lycopersicon, Majorana, Malus, Mangifera, Manihot, Medicago, Nemesia, Nicotiana, Onobrychis, Oryza, Panicum, Pelargonium, Pennisetum, Petunia, Pisum, Phaseolus, Phleum, Poa, Prunus, Ranunculus, Raphanus, Ribes, Ricinus, Rubus, Saccharum, Salpiglossis, Secale, Senecio, Setaria, Sinapis, Solanum, Sorghum, Stenotaphrum, Theobroma, Trifolium, Trigonella, Triticum, Vicia, Vigna, Vitis, Zea, and the Olyreae, the Pharoideae and many others.
Within the scope of this invention the plant species, which are not included into the food or feed chain are specifically preferred for pharmaceutical and technical proteins production. Among them, Nicotiana species are the most preferred, as the species easy to transform and cultivate with well developed expression vectors (especially viral vectors) systems.
Genes of interest, their fragments (functional or non-functional) and their artificial derivatives that can be expressed as the cellular process of interest and isolated using the present invention include, but are not limited to: starch modifying enzymes (starch synthase, starch phosphorylation enzyme, debranching enzyme, starch branching enzyme, starch branching enzyme II, granule bound starch synthase), sucrose phosphate synthase, sucrose phosphorylase, polygalacturonase, polyfructan sucrase, ADP glucose pyrophosphorylase, cyclodextrin glycosyltransferase, fructosyl transferase, glycogen synthase, pectin esterase, aprotinin, avidin, bacterial levansucrase, E. coli glgA protein, MAPK4 and orthologues, nitrogen assimilation/methabolism enzyme, glutamine synthase, plant osmotin, 2S albumin, thaumatin, site-specific recombinase/integrase (FLP, Cre, R recombinase, Int, SSVI Integrase R, Integrase phiC31, or an active fragment or variant thereof), isopentenyl transferase, Sca M5 (soybean calmodulin), coleopteran type toxin or an insecticidally active fragment, ubiquitin conjugating enzyme (E2) fusion proteins, enzymes that metabolise lipids, amino acids, sugars, nucleic acids and polysaccharides, superoxide dismutase, inactive proenzyme form of a protease, plant protein toxins, traits altering fiber in fiber producing plants, Coleopteran active toxin from Bacillus thuringiensis (Bt2 toxin, insecticidal crystal protein (ICP), CrylC toxin, delta endotoxin, polyopeptide toxin, protoxin etc.), insect specific toxin AalT, cellulose degrading enzymes, E1 cellulase from Acidothermus celluloticus, lignin modifying enzymes, cinnamoyl alcohol dehydrogenase, trehalose-6-phosphate synthase, enzymes of cytokinin metabolic pathway, HMG-CoA reductase, E. coli inorganic pyrophosphatase, seed storage protein, Erwinia herbicola lycopen synthase, ACC oxidase, pTOM36 encoded protein, phytase, ketohydrolase, acetoacetyl CoA reductase, PHB (polyhydroxybutanoate) synthase, acyl carrier protein, napin, EA9, non-higher plant phytoene synthase, pTOM5 encoded protein, ETR (ethylene receptor), plastidic pyruvate phosphate dikinase, nematode-inducible transmembrane pore protein, trait enhancing photosynthetic or plastid function of the plant cell, stilbene synthase, an enzyme capable of hydroxylating phenols, catechol dioxygenase, catechol 2,3-dioxygenase, chloromuconate cycloisomerase, anthranilate synthase, Brassica AGL15 protein, fructose 1,6-biphosphatase (FBPase), AMV RNA3, PVY replicase, PLRV replicase, potyvirus coat protein, CMV coat protein, TMV coat protein, luteovirus replicase, MDMV messenger RNA, mutant geminiviral replicase, Umbellularia californica C12:0 preferring acyl-ACP thioesterase, plant C10 or C12:0 preferring acyl-ACP thioesterase, C14:0 preferring acyl-ACP thioesterase (luxD), plant synthase factor A, plant synthase factor B, D6-desaturase, protein having an enzymatic activity in the peroxysomal b-oxidation of fatty acids in plant cells, acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, lipase, maize acetyl-CoA-carboxylase, 5-enolpyruvylshikimate-3-phosphate synthase (EPSP), phosphinothricin acetyl transferase (BAR, PAT), CP4 protein, ACC deaminase, protein having posttranslational cleavage site, DHPS gene conferring sulfonamide resistance, bacterial nitrilase, 2,4D monooxygenase, acetolactate synthase or acetohydroxyacid synthase (ALS, AHAS), polygalacturonase, Taq polymerase, bacterial nitrilase, many other enzymes of bacterial or phage including restriction endonucleases, methylases, DNA and RNA ligases, DNA and RNA polymerases, reverse trascryptases, nucleases (Dnases and RNAses), phosphatases, transferases etc.
Our invention also can be used for the purpose of molecular farming and purification of commercially valuable and pharmaceutically important proteins including industrial enzymes (cellulases, lipases, proteases, phytases etc.) and fibrous proteins (collagen, spider silk protein, etc.). Any human or animal health protein can be expressed and purified using described in our invention approach. Examples of such proteins of interest include inter alia immune response proteins (monoclonal antibodies, single chain antibodies, T cell receptors etc.), antigens including those derived from pathogenic microorganisms, colony stimulating factors, relaxins, polypeptide hormones including somatotropin (HGH) and proinsulin, cytokines and their receptors, interferons, growth factors and coagulation factors, enzymatically active lysosomal enzyme, fibrinolytic polypeptides, blood clotting factors, trypsinogen, a1-antitrypsin (AAT), human serum albumin, glucocerebrosidases, native cholera toxin B as well as function-conservative proteins like fusions, mutant versions and synthetic derivatives of the above proteins.
Binary vector pICHFPinv (
Transgenic Nicotiana benthamiana plants containing T-DNA of pICHGFPinv were obtained by Agrobacterium-mediated transformation of leaf discs as described by Horsch et al., (1985, Science, 227, 129-131). Leaf discs were incubated for 30 min with Agrobacterium strain GV3101 transformed with the construct of interest. After three days of incubation on medium (MS-medium 0.1 mgA NM, 1 mg/l BAP) without selective agent, selection of transformants was performed on the same MS-medium supplemented with 100 mg/L Kanamycin. In ordero reduce the growth of Agrobacterium, the medium was also supplemented with 300 mg/L carbenicilin and 300 mg/L cefataxime. Regenerants were incubated on selective MS-medium without hormones supplemented with the same concentration of the selective agents to induce the rooting. The presence of the transgene in segregating T2-populations was confirmed by PCR-analysis.
In order to produce a cell-permeable integrase or recombinase fused to the membrane translocation signal (MTS) of choice, the set of constructs for provector system (WO02088369) was made (see
Exposure of transgenic plant leaves to cell-permeable integrase causes site-specific recombination between attP and attB sites. Such recombination leads to the reversion of 3′ provector, thus creating a complete cDNA of a viral amplicon under the control of the actin 2 promoter (
| Number | Date | Country | Kind |
|---|---|---|---|
| 102 54 166 | Nov 2002 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP03/13018 | 11/20/2003 | WO | 00 | 6/22/2005 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2004/046360 | 6/3/2004 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20020046419 | Choo et al. | Apr 2002 | A1 |
| 20020143142 | Lin et al. | Oct 2002 | A1 |
| Number | Date | Country |
|---|---|---|
| 2000-500323 | Jan 2000 | JP |
| WO 9521248 | Aug 1995 | WO |
| WO 9837211 | Aug 1998 | WO |
| WO 9952563 | Oct 1999 | WO |
| WO0071701 | May 2000 | WO |
| WO0071701 | Nov 2000 | WO |
| WO 0138488 | May 2001 | WO |
| WO0189283 | May 2001 | WO |
| WO 0189283 | Nov 2001 | WO |
| WO 02088369 | Nov 2002 | WO |
| WO0189283 | Nov 2002 | WO |
| Number | Date | Country | |
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| 20060026718 A1 | Feb 2006 | US |
