Artificial forisome bodies with seo-f fusion proteins, plant or yeast cells with vectors for encoding these proteins and vectors for encoding seo-f fusion proteins

Abstract

Artificial forisome bodies include a fusion protein of at least one SEO-F protein or an at least 50-amino acid portion of an SEO-F protein, and at least one additional protein or peptide, with the exception of GFP and the Venus protein. The additional protein or peptide has a mass of at most 30 kDa, or the forisome body further includes an unfused SEO-F protein or a form of the protein having C-terminal deletions of up to 45 amino acids and/or N-terminal deletions of up to 13 amino acids, in which the unfused SEO-F protein is a protein capable of forming homomeric forisome bodies in the absence of additional SEO-F proteins.

What is claimed is: 
     
         1 . An artificial forisome body comprising a fusion protein of (A) a SEO-F (Sieve Element Occlusion by Forisome) protein having an amino acid sequence of SEQ ID NO: 4, 5, 6, 7 or 8, or a fragment of the amino acid sequence of SEQ ID NO: 4, 5, 6, 7 or 8, wherein said fragment results from deleting up to 45 amino acids from the C-terminus and/or up to 13 amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 4, 5, 6, 7 or 8, and (B) at least one additional protein, wherein the at least one additional protein is not a fluorescent protein,
 (a) wherein the additional protein has a mass of at most 30 kDa, and wherein the artificial forisome body does not contain an unfused SEO-F protein or an unfused SEO-F protein having C-terminal deletions of up to 45 amino acids and/or N-terminal deletions of up to 13 amino acids,   or   (b) wherein the forisome body further comprises an unfused SEO-F protein, wherein said unfused SEO-F protein is   (i) a protein having the amino acid sequence of SEQ ID NO: 4, 5, 6, 7 or 8,   or   (ii) a protein comprising a fragment of the amino acid sequence of SEQ ID NO: 4, 5, 6, 7 or 8, wherein said fragment results from deleting up to 45 amino acids from the C-terminus and/or up to 13 amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 4, 5, 6, 7 or 8,   and wherein the unfused SEO-F protein has ability to form homomeric forisome bodies in the absence of additional SEO-F proteins.   
     
     
         2 . The artificial forisome body according to  claim 1 , with the exception of forisome bodies that are comprised entirely or partly of a fusion protein that contains an artificial fluorescent variant or a portion of a fluorescent portion of a green fluorescent protein (GFP) or Venus protein. 
     
     
         3 . The artificial forisome body according to  claim 1 , wherein the artificial forisome body comprises a fusion protein of (A) a SEO-F (Sieve Element Occlusion by Forisome) protein having the amino acid sequence of SEQ ID NO: 4, or a fragment of the amino acid sequence of SEQ ID NO: 4, wherein said fragment results from deleting up to 45 amino acids from the C-terminus and/or up to 13 amino acids from the N-terminus of the amino acid sequence of SEQ ID NO: 4, and (B) the at least one additional protein. 
     
     
         4 . The artificial forisome body according to  claim 1 , wherein the additional protein is selected from the group consisting of proteins that contribute to metabolism, proteins capable of triggering an immune response and/or proteins having a therapeutic benefit, and proteins that are useful for biotechnological applications. 
     
     
         5 . The artificial forisome body according to  claim 1 , wherein the additional protein is selected from the group consisting of enzymes, antibodies, and antigens, wherein the additional protein can be immobilized on a substrate due to their affinity reaction with a substrate-bound biological or biochemically produced material. 
     
     
         6 . The artificial forisome body according to  claim 1 , wherein the fusion protein contains an enzyme fused to the N-terminal end of the SEO-F protein or a portion thereof, or wherein the fusion protein comprises a protein that has a therapeutic benefit or is useful for biotechnological applications, and which is fused to the C-terminal end of the SEO-F protein or portion thereof. 
     
     
         7 . The artificial forisome body according to  claim 1 , comprising at least two fusion proteins, each comprising an enzyme, such that a product of a reaction of one substrate with a first of said enzymes can serve as a substrate for a second of said enzymes. 
     
     
         8 . A plant cell or yeast cell comprising:
 a first vector encoding a fusion protein of at least one SEO-F protein or a portion thereof comprising at least 50 amino acids and at least one additional protein, with the exception of a green fluorescent protein (GFP) and Venus protein and artificial fluorescent variants or fluorescent portions of the GFP protein or the Venus protein, and optionally   a second vector encoding a SEO-F protein or a form of said protein having C-terminal deletions of up to 45 amino acids and/or N-terminal deletions of up to 13 amino acids, wherein the SEO-F protein is capable of forming forisome bodies in the absence of further homomeric SEO-F proteins, with the proviso that optionally any number of the four cysteines that are located in the native SEO-F proteins in the C-terminal region between aa 600 and aa 670, may be replaced by amino acids that are not capable for forming an SS-bond.   
     
     
         9 . The plant cell or yeast cell according to  claim 8 , wherein the additional protein has a mass of at most 30 kDa. 
     
     
         10 . The plant cell or yeast cell according to  claim 8 , wherein the additional protein is a portion of a second SEO-F protein, with the proviso that one of the two SEO-F proteins in unfused form is capable of forming homomeric forisome bodies and the fusion protein is comprised of an N-terminal SEO-F protein portion and a C-terminal SEO-F protein portion, wherein the portions represent an SEO-F protein that is complete or C-terminally deleted by up to approximately 50 amino acids and/or N-terminally by up to 13 amino acids. 
     
     
         11 . The plant cell or yeast cell according to  claim 8 , wherein the additional protein is selected from the group consisting of proteins that contribute to metabolism, proteins capable of triggering an immune response and/or proteins having a therapeutic benefit, and proteins that are useful for biotechnological applications. 
     
     
         12 . The plant cell or yeast cell according to  claim 8 , wherein the additional protein is selected from the group consisting of enzymes, antibodies, and antigens, wherein the additional protein can be immobilized on a substrate due to their affinity reaction with a substrate-bound biological or biochemically produced material. 
     
     
         13 . The plant cell or yeast cell according to  claim 8 , wherein the fusion protein comprises an enzyme which is fused to the N-terminal end of the SEO-F protein or a portion thereof, or wherein the fusion protein comprises a protein that has a therapeutic benefit or is useful for biotechnological applications, and is fused to the C-terminal end of the SEO-F protein or portion thereof. 
     
     
         14 . The plant cell or yeast cell according to  claim 8 , wherein the first vector encodes a fusion protein comprising an amino acid sequence of a first enzyme, characterized in that the cell contains at least one further vector encoding a fusion protein comprising the amino acid sequence of a second enzyme, wherein a reaction product of a substrate with the first enzyme is suitable as a substrate for the second enzyme. 
     
     
         15 . A vector capable of being amplified in the yeast cell according to  claim 8 , comprising a region encoding a fusion protein comprised of at least one SEO-F protein or a portion thereof comprising at least 50 amino acids and at least one further protein or peptide, with the exception of the GFP and the Venus protein. 
     
     
         16 . The vector according to  claim 15 , wherein the additional protein is selected from the group consisting of proteins that contribute to metabolism, proteins capable of triggering an immune response and/or proteins having a therapeutic benefit, and proteins that are useful for biotechnological applications. 
     
     
         17 . The vector according to  claim 15 , wherein the additional protein or peptide is selected from the group consisting of enzymes, antibodies, and antigens, wherein the additional protein can be immobilized on a substrate due to their affinity reaction with a substrate-bound biological or biochemically produced material. 
     
     
         18 . The vector according to  claim 15 , wherein the fusion protein comprises an enzyme fused to the N-terminal end of the SEO-F protein or a portion thereof, or wherein the fusion protein comprises a protein that has a therapeutic benefit or can be used for biotechnological applications, and is fused to the C-terminal end of the SEO-F protein or the portion thereof. 
     
     
         19 . A method for producing an artificial forisome body comprising a fusion protein of at least one SEO-F (Sieve Element Occlusion by Forisome) protein or an at least 50-amino acid portion thereof, and at least one additional protein, with the exception of a green fluorescent protein (GFP) and Venus protein, wherein
 (a) the additional protein or peptide has a mass of at most 30 kDa, and the artificial forisome body does not contain an unfused SEO-F protein or a form of said protein having C-terminal deletions of up to 45 amino acids and/or N-terminal deletions of up to 13 amino acids, or   (b) the forisome body further comprises an unfused SEO-F protein or a form of said protein having C-terminal deletions of up to 45 amino acids and/or N-terminal deletions of up to 13 amino acids, wherein the unfused SEO-F protein is selected from proteins having the capability of forming homomeric forisome bodies in the absence of additional SEO-F proteins, or   (c) the further protein or peptide is a portion of a second SEO-F protein, with the proviso that one of the two SEO-F proteins in its unfused form is capable of forming homomeric forisome bodies, and the fusion protein is comprised of an N-terminal SEO-F protein portion and a C-terminal SEO-F protein portion, wherein the portions represent an SEO-F protein that is complete or C-terminally deleted by up to 50 amino acids and/or N-terminally deleted by up to 13 amino acids, with the proviso that optionally any number of the four cysteines located in the C-terminal portion between aa 600 and aa 670 of the native SEO-F proteins, may be replaced by amino acids that are not capable of forming an SS-bond.   
     
     
         20 . The method according to  claim 19 , wherein the fusion protein does not contain any artificial fluorescent variant and no fluorescent portion of the GFP protein or the Venus fluorescent protein. 
     
     
         21 . The artificial forisome body according to  claim 2 , with the exception of forisome bodies that are composed exclusively or partly of a fluorescent fusion protein. 
     
     
         22 . The method according to  claim 20 , wherein the fusion protein does not comprise fluorescent protein portions.