Patent Grant
11845945
The Sequence Listing submitted in text format (.txt) filed on Apr. 19, 2022, named “SequenceListing.txt”, created on Apr. 18, 2022 (28.2 KB), is incorporated herein by reference.
The present invention relates to a technique for highly expressing a target protein in a plant cell by using a glycosylation domain, a recombinant vector comprising a gene encoding a fusion protein of a glycosylation domain and a target protein, a recombinant cell, a transgenic plant, and a method of producing a target protein using these.
The remarkable development of molecular biology and genetic engineering techniques has also been applied to the plant field, and efforts to produce useful physiologically active substances from plants are steadily continuing. When producing useful substances in plants, production costs may be dramatically reduced, various contaminants such as viruses, oncogenes, and enterotoxins that may be generated in a conventional method of separating and purifying a protein through synthesis in animal cells or microorganisms may be fundamentally excluded, and unlike animal cells or microorganisms, such useful substances may be stored and managed as seeds for a long period of time even in the commercialization stage. In addition, when demand for the corresponding useful substance surges, the above system is absolutely advantageous compared to existing animal cell systems in terms of equipment technology or costs required for mass production, and thus supply corresponding to the increased demand is possible within the shortest time.
Despite these advantages, however, a relatively low level of protein expression is the biggest drawback in protein production in plant cells, compared to other hosts including animal cells. Thus, many studies have been conducted and there have been attempts to increase a protein expression level in plant cells by using various methods.
Previous studies to increase a level of target protein expression in a plant cell have been focused mainly on a transcription stage prior to a translation stage where a protein is produced from mRNA during a protein expression process, and few studies have been conducted on a method of increasing a protein expression level when a protein is translated from mRNA.
Meanwhile, it is well known that conventional N-glycosylation affects protein stability. In this case, N-glycans were thought to enhance stability because they protect proteins from proteases. It is also known that other mechanisms provide an additional binding force for protein three-dimensional structures to thereby provide stability.
As a result of having made intensive efforts to increase an expression level of a target protein in the translation stage in producing a target protein in a plant cell, the inventors of the present invention confirmed that, when a small domain causing glycosylation is fused to a target protein, an expression level of the protein was increased, and verified that production efficiency of the target protein could be increased in a transgenic plant by using the above finding, thus completing the present invention.
The present invention relates to a use of an N-glycosylation domain in target protein expression.
An embodiment provides a composition for expressing a target protein, which comprises one or more selected from the group consisting of a gene encoding an N-glycosylation domain, a recombinant vector comprising the gene, and a recombinant cell comprising the recombinant vector.
The composition for expressing a target protein may further comprise a gene encoding the target protein or a recombinant vector comprising the gene. In this regard, the gene encoding the target protein and the gene encoding an N-glycosylation domain may be comprised in the form of a gene encoding a fusion protein comprising the N-glycosylation domain and the target protein or a recombinant vector comprising the gene.
The N-glycosylation domain may be an N-glycosylation domain (e.g., multiple N-glycosylation domains) comprising one or more N-glycosylation sites or two or more N-glycosylation sites. In one embodiment, the N-glycosylation domain may comprise a CD45-derived M domain, such as a human CD45-derived M domain or a portion thereof. The target protein expression may be performed in a eukaryotic cell (e.g., a plant cell) or a eukaryotic organism (e.g., a plant).
Another embodiment provides a recombinant vector comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein.
Another embodiment provides a recombinant cell into which the recombinant vector is introduced. The recombinant cell may be a eukaryotic cell, for example, a plant cell.
The recombinant vector comprising a gene encoding a fusion protein and/or the recombinant cell may be used for enhancing production of the target protein. Therefore, another embodiment provides a composition for producing a target protein or enhancing production of a target protein, which comprises a recombinant vector comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein and/or a recombinant cell.
Another embodiment provides a transgenic organism into which the recombinant vector comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein is introduced. The transgenic organism may be a transgenic eukaryotic organism, for example, a transgenic plant.
Another embodiment provides a method of producing a target protein or enhancing production of a target protein, comprising introducing, into a cell, the composition for producing a target protein or enhancing production of a target protein. The method may increase an expression level or productivity of the target protein, compared to a case in which a gene encoding the target protein is introduced alone into a cell (i.e., introduced via an N-glycosylation domain-free recombinant vector).
The present invention has been made to address the above-described problems, and provides a use of an N-glycosylation domain for expressing a target protein in a plant, more particularly, a technique for increasing an expression level of a target protein by expressing a fusion gene produced by fusing a gene encoding the target protein and a gene encoding an N-glycosylation domain to a C-terminal-corresponding site (3′-terminal) or N-terminal-corresponding site (5′-terminal) of the gene.
An embodiment provides a composition for expressing a target protein, which comprises one or more selected from the group consisting of a gene encoding an N-glycosylation domain, a recombinant vector comprising the gene, and a recombinant cell comprising the recombinant vector.
The composition for expressing a target protein may further comprise a gene encoding the target protein or a recombinant vector comprising the gene. In this regard, the gene encoding the target protein and the gene encoding an N-glycosylation domain may be comprised in the form of a gene encoding a fusion protein comprising the N-glycosylation domain and the target protein or a recombinant vector comprising the gene.
The N-glycosylation domain may be an N-glycosylation domain comprising one or more N-glycosylation sites or two or more N-glycosylation sites. In one embodiment, the N-glycosylation domain may comprise a CD45-derived M domain or a portion thereof, for example, a human CD45-derived M domain or a portion thereof. The target protein expression may be performed in a eukaryotic cell (e.g., a plant cell) or a eukaryotic organism (e.g., a plant).
Another embodiment provides a recombinant vector comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein. The recombinant vector may be used for expression in a eukaryotic cell, for example, a plant cell.
Another embodiment provides a recombinant cell comprising the gene encoding a fusion protein comprising an N-glycosylation domain and a target protein. The recombinant cell may be a cell into which a recombinant vector comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein is introduced. The cell may be a eukaryotic cell, for example, a plant cell.
Another embodiment provides a transgenic organism comprising the gene encoding a fusion protein comprising an N-glycosylation domain and a target protein. The transgenic organism may be an organism into which a recombinant vector comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein is introduced. The transgenic organism may be a transgenic eukaryotic organism, for example, a transgenic plant. The transgenic organism may be a eukaryotic organism (e.g., a plant) comprising the above-described recombinant cell.
The recombinant vector comprising a gene encoding a fusion protein and/or the recombinant cell and/or the transgenic organism may be used for producing a target protein or enhancing the production of a target protein.
Therefore, another embodiment provides a composition for producing a target protein or enhancing the production of a target protein, the composition comprising one or more selected from the group consisting of a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein, a recombinant vector comprising the gene, a recombinant cell comprising the recombinant vector, and a transgenic organism comprising the recombinant vector.
Another embodiment provides a method of producing a target protein or enhancing the production of a target protein, comprising culturing a recombinant cell comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein. The recombinant cell may be a cell into which a recombinant vector comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein is introduced. The cell may be a eukaryotic cell, for example, a plant cell. The method may further comprise, before the culturing process, introducing, into a cell, a recombinant vector comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein. The method may further comprise, after the culturing process, isolating (or extracting) and/or purifying a target protein from the cultured cell (a cell, cell debris, or a cell lysate) and/or a culture medium.
Another embodiment provides a method of producing a target protein or enhancing the production of a target protein, comprising growing a transgenic organism comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein. The organism may be a eukaryotic organism, for example, a plant. The method may further comprise, before the growing process, introducing, into an organism, a recombinant vector comprising a gene encoding a fusion protein comprising an N-glycosylation domain and a target protein. The method may further comprise, after the growing process, isolating (or extracting) and/or purifying a target protein from the eukaryotic organism (e.g., a plant), or a cell of the eukaryotic organism (a cell, cell debris, cell lysate or a culture of the cell).
The method may increase an expression level or productivity of the target protein, compared to a case in which a gene encoding the target protein is introduced alone into a cell or an organism (i.e., introduced via an N-glycosylation domain-free recombinant vector), a case in which a target protein fused with an O-glycosylation domain is used, and/or a case in which the target protein intrinsically contains an N-glycosylation site without being fused with a separate N-glycosylation domain.
In one embodiment, the N-glycosylation domain may be a polypeptide having one or more N-glycosylation sites or two or more N-glycosylation sites (N-glycosylated amino acid; asparagine (Asn)), for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 N-glycosylation sites and having a total number of amino acids of 10 to 100 or 20 to 80. In one embodiment, the N-glycosylation domain may be a polypeptide comprising 10 to 100 or 20 to 80 consecutive amino acids comprising at least a human CD45-derived M domain (having 4 N-glycosylation sites) in human CD45 or a portion thereof. For example, the human CD45 protein may have an amino acid sequence represented by SEQ ID NO: 5 (UniProt No. P08575). The human CD45-derived M domain may be a polypeptide consisting of a total of 60 amino acids from Ala (residue 231) to Asp (residue 290) of the human CD45 protein (SEQ ID NO: 5) (60aa; ANITVDYLYN KETKLFTAKL NVNENVECGN NTCTNNEVHN LTECKNASVS ISHNSCTAPD; SEQ ID NO: 2; underlined and bold characters denote N-glycosylation sites). In one embodiment, a gene encoding the human CD45-derived M domain may comprise a nucleic acid sequence of SEQ ID NO: 1. A portion of the human CD45-derived M domain may be a fragment of a polypeptide comprising 10 or more, 15 or more, or 20 or more consecutive amino acids having one or more N-glycosylation sites or two or more N-glycosylation sites, for example, 1, 2, 3, or 4 N-glycosylation sites in the CD45-derived M domain (e.g., selected from N(Asn) at residue 2, N(Asn) at residue 30, N(Asn) at residue 40, and N(Asn) at residue 46 of SEQ ID NO: 2). The fragment of the polypeptide may be a polypeptide comprising 10 or more, 15 or more, or 20 or more consecutive amino acids having one or more amino acid residue selected from N at residue 40 and N at residue 46 of the amino acid sequence of SEQ ID NO: 2, and may be, for example, “LTECKNASVS ISHNSCTAPD (SEQ ID NO: 6)” or “NVNENVECGN NTCTNNEVHN LTECKNASVS ISHNSCTAPD (SEQ ID NO: 7)”, but the present invention is not limited thereto. In one embodiment, the N-glycosylation domain may be a polypeptide (e.g., SEQ ID NO: 6, 7, or 8) comprising 10 to 100 or 20 to 80 consecutive amino acids having an M domain (SEQ ID NO: 2) or an M domain portion comprising 10 or more, 15 or more, or 20 consecutive amino acids of the M domain (SEQ ID NO: 2), in the human CD45 protein (SEQ ID NO: 5). In one embodiment, a gene encoding the M domain may have a nucleic acid sequence represented by SEQ ID NO: 1.
In one embodiment, the recombinant vector may further comprise one or more selected from the group consisting of a transcriptional regulatory factor, a translational regulatory factor, and a marker for confirming gene expression.
In one embodiment, the transcriptional regulatory factor may be one or more selected from all transcription factors commonly used for transcriptional regulation in a cell, for example, a plant cell and may be, for example, one or more selected from the group consisting of a cauliflower mosaic virus 35S RNA promoter, a cauliflower mosaic virus 19S RNA promoter, a figwort mosaic virus-derived full-length transcription promoter, and a tobacco mosaic virus coat protein promoter, but the present invention is not limited thereto.
The translational regulatory factor may be one or more selected from all translational regulatory factors commonly used for translational regulation in a cell, for example, a plant cell and may be, for example, an M17 factor, but the present invention is not limited thereto. The M17 factor may have a nucleic acid sequence represented by SEQ ID NO: 3.
In another embodiment, the recombinant vector may further comprise a signal sequence for targeting (migration and/or retention) to a specific intracellular organelle. In one embodiment, the recombinant vector may be engineered to target the endoplasmic reticulum (ER), and to this end, may further comprise an endoplasmic reticulum (e.g., a cell membrane surface) transfer signal (e.g., a BiP (chaperone binding protein)-encoding gene or the like) and/or an endoplasmic reticulum retention signal (e.g., HDEL (His-Asp-Glu-Leu) (SEQ ID NO: 54) peptide-encoding gene). The signal sequence for targeting an intracellular organelle (e.g., an endoplasmic reticulum) may be linked to the N-terminal (5′-terminal of a gene encoding a fusion protein) or C-terminal (3′-terminal of the gene encoding a fusion protein), for example, N-terminal (5′-terminal of a gene encoding a fusion protein) of the fusion protein. As such, the recombinant vector may be targeted to the endoplasmic reticulum (e.g., inside the endoplasmic reticulum) of the intracellular organelle, thereby further increasing a protein expression level (see
The BiP (chaperone binding protein) may have a nucleic acid sequence represented by SEQ ID NO: 4.
In one embodiment, the present invention provides a recombinant vector for transforming a plant to increase an expression level of a target protein, the recombinant vector comprising a gene encoding the target protein, a gene encoding a human CD45-derived M domain, a transcriptional regulatory factor, an M17 factor operably linked to the transcriptional regulatory factor, and a gene encoding BiP (chaperone binding protein) and/or a HDEL (His-Asp-Glu-Leu) peptide.
In one embodiment, the recombinant vector may further comprise a marker for confirming gene expression. In one embodiment of the present invention, a HA epitope sequence was used to confirm the presence or absence of expression by western blotting, but the present invention is not limited thereto.
The eukaryotic cell described herein may be one or more selected from the group consisting of a fungus, an animal cell, and a plant cell, and may be, for example, a plant cell. The eukaryotic organism may be one or more selected from the group consisting of all unicellular eukaryotic organisms and multicellular eukaryotic organisms (plants or animals) and may be, for example, a plant. The plant described herein may be one or more plant selected from all algae, monocotyledonous plants, and dicotyledonous plants, or a cell thereof and may be, for example, a dicotyledonous plant selected from the group consisting of Arabidopsis, soybeans, tobacco, eggplants, peppers, potatoes, tomatoes, Korean cabbage, radish, cabbage, lettuce, peaches, pears, strawberries, watermelons, melons, cucumbers, carrots, and celery; a monocotyledonous plant selected from rice, barley, wheat, rye, corn, sugarcane, oats, and onions; or a cell thereof, but the present invention is not limited thereto.
The introduction of the recombinant vector into a eukaryotic organism (e.g., a plant) or a eukaryotic cell (e.g., a plant cell) may be performed using a general transduction method, for example, using one or more methods selected from the group consisting of an Agrobacterium sp.-mediated method, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and polyethylene glycol (PEG)-mediated transformation, but the present invention is not limited thereto.
As described above, the term “N-glycosylation” as used herein refers to a series of processes for binding glycans, which are sugar molecule oligosaccharides, to the nitrogen atom of an amino acid of a protein, and is distinguished from 0-glycosylation, which binds sugar molecules to the oxygen atom of an amino acid residue of a protein. In the present specification, N-glycosylation may occur in an endoplasmic reticulum (e.g., inside an endoplasmic reticulum).
The term “N-glycosylation domain” as used herein refers to a polypeptide comprising an N-glycosylation site (amino acid residue), which may be non-naturally occurring, e.g., chemically or recombinantly synthesized, or naturally occurring.
In one embodiment, a recombinant vector comprising a gene encoding a fusion protein produced by fusing a human CD45-derived M domain to the C-terminal of a target protein was prepared to be used in experiments, but the present invention is not limited thereto, and the human CD45-derived M domain may also be fused to the N-terminal of a target protein.
The fusion protein comprising a target protein and an N-glycosylation domain may comprise a suitable linker (e.g., a 1-50 aa, 1-30 aa, 1-20 aa, 2-50 aa, 2-30 aa, or 2-20 aa peptide linker) between the target protein and the N-glycosylation domain. The peptide linker may be a sequence in which glycine-serine is repeated, but the present invention is not limited thereto.
The amino acid sequences and nucleic acid sequences described herein may be construed as extending to a sequence with at least 70% homology, at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 91% homology, at least 92% homology, at least 93% homology, at least 94% homology, at least 95% homology, at least 96% homology, at least 97% homology, at least 98% homology, or at least 99% homology, to the provided sequences.
The “% sequence homology” may be confirmed by comparing two optimized sequences using a comparison region, and some of the polynucleotide sequences in the comparison region may comprise an addition or deletion (i.e., a gap) compared to reference sequences (additions or deletions excluded) for the optimal alignment of two sequences.
The term “recombinant vector or recombinant cell” as used herein refers to a cell that replicates a heterologous nucleic acid (polynucleotide) or expresses the nucleic acid, or a vector or cell that expresses a protein encoded by a peptide, a heterologous peptide, or a heterologous nucleic acid. The recombinant cell may express a gene or gene segment that is not found in a natural form of the cell in one of a sense form and/or an antisense form. In addition, the recombinant cell may express a gene found in a cell in its natural state, but the gene is a modified form and a gene reintroduced into a cell by an artificial means.
The “recombinant vector” may be one or more selected from the group consisting of all plasmids, phage, yeast plasmids, plant cell viruses, mammalian cell viruses, and other media known in the art into which a gene sequence or nucleotide sequence can be inserted or introduced. Generally, any plasmid and vector may be used without particular limitation as long as it is capable of replicating and being stabilized in a plant cell or a plant host. In one embodiment, the recombinant vector may be for use in transforming a plant or a plant cell. The gene sequence or nucleotide sequence according to the present invention may be operably linked to an expression regulatory factor, and the expression regulatory factor operably linked to the gene sequence may be comprised in a single expression vector comprising both a selectable marker and a replication origin.
Examples of known vectors comprise pBI121, pHellsgate8, pROKII, pBI76, pET21, pSK(+), pLSAGPT, pUC, and pGEM. In addition, examples of vectors expressed in plants, which comprises a CMV35s promoter, comprise the pCAMBIA series (pCAMBIA1200, 1201, 1281, 1291, 1300, 1301, 1302, 1303, 1304, 1380, 1381, 2200, 2201, 2300, 2301, 3200, 3201, and 3300), pMDC32, and pC-TAPapYL436, but the present invention is not limited thereto.
The term “operably linked” as used herein may refer to a gene and an expression regulatory factor that are linked in such a way to enable gene expression when an appropriate molecule is bound to the expression regulatory factor.
The term “expression regulatory factor” as used herein refers to a DNA sequence that regulates the expression of a polynucleotide sequence operably linked in a particular host cell. Such regulatory factors comprise a transcriptional regulatory factor comprising a promoter for performing transcription and any operator sequence, a translational regulatory factor comprising a sequence encoding an appropriate mRNA ribosome-binding site and a sequence that regulates protein synthesis, and a sequence that regulates the termination of transcription and translation.
In the present invention, the transcriptional regulatory factor may be selected from the group consisting of a cauliflower mosaic virus 35S RNA promoter, a cauliflower mosaic virus 19S RNA promoter, a figwort mosaic virus-derived full-length transcription promoter, and a tobacco mosaic virus coat protein promoter, but the present invention is not limited thereto.
In the present invention, the translational regulatory factor may be an M17 sequence, which serves to increase the amount of a target protein synthesized in a plant. In the present invention, preferably, the M17 sequence may be represented by SEQ ID NO: 3.
The recombinant vector of the present invention may further comprise a nucleic acid encoding BiP (chaperone binding protein) or a HDEL (His-Asp-Glu-Leu) peptide, which may be operably linked to the transcriptional regulatory factor.
The BiP, which is a luminal binding protein, was identified as an immunoglobulin heavy chain binding protein and a glucose regulated protein, and is a member of the HSP70 chaperone family located in the endoplasmic reticulum and temporarily binds to a protein newly synthesized in the endoplasmic reticulum. In addition, BiP serves to enable target proteins to be targeted to the endoplasmic reticulum since it has, at the N-terminal thereof, a signal sequence that determines targeting to the endoplasmic reticulum. For example, a nucleic acid encoding the BiP may have a nucleic acid sequence represented by SEQ ID NO: 4.
In addition, the recombinant vector for transforming a plant may comprise a nucleotide sequence encoding an ER retention signal peptide such as HDEL. In the case of a HDEL signal peptide, a target protein is retained in the ER such that folding and assembly by a molecular chaperone are increased, resulting in further minimization of protein degradation. As an example, it is known that, in a case in which a target protein is retained more in the ER when sent to the secretory pathway, a yield of the target protein is increased about 10-fold to about 100-fold.
An embodiment also provides a recombinant vector for transforming a plant to increase an expression level of a target protein, the recombinant vector comprising a target protein, a gene encoding a human CD45-derived M domain or a portion thereof, a transcriptional regulatory factor, M17 operably linked to the transcriptional regulatory factor, and a nucleic acid encoding BiP (chaperone binding protein) and a HDEL (His-Asp-Glu-Leu) peptide.
The recombinant vector described herein may further comprise a marker for confirming gene expression. In one embodiment, a HA epitope sequence was used to confirm expression by western blotting, but the present invention is not limited thereto.
The term “target protein” as used herein refers to a protein for producing or a fragment thereof, and the target protein is not limited to a specific protein. Specifically, the target protein may be any one or more selected from the group consisting of an antigen, an antibody, an antibody fragment, a structural protein, a regulatory protein, a transcription factor, a toxin protein, a hormone, a hormone analog, a cytokine, an enzyme, an enzyme inhibitor, a transport protein, a receptor (e.g., tyrosine kinase receptor and the like), a receptor fragment, a biological defense inducer, a storage protein, a movement protein, an exploitive protein, a reporter protein, and the like.
In one embodiment, leptin, GLP-1, Exendin-4, aprotinin, a green fluorescent protein (GFP), and the like have been described as examples of the target protein, but these are provided for illustrative purposes only to achieve a protein production-enhancing effect provided in the present specification, but the target protein is not limited to the above-listed proteins.
The present invention also provides a plant transformed with the recombinant vector. The transformed plant comprises an M domain sequence and is devised to be operably linked to transcriptional and translational regulatory factors and controlled thereby. The transformed plant described herein may be a whole plant, a plant cell (e.g., a cell such as a leaf, a stem, a root, and the like), or plant tissue (e.g., a leaf, a stem, a root, and the like). The plant tissue may comprise a plant seed. The plant may be an herbaceous or textured plant, and may be a dicotyledonous plant or a monocotyledonous plant. In particular, the dicotyledonous plant may be selected from the group consisting of Arabidopsis, soybeans, tobacco, eggplants, peppers, potatoes, tomatoes, Korean cabbage, radish, cabbage, lettuce, peaches, pears, strawberries, watermelons, melons, cucumbers, carrots, and celery, and the monocotyledonous plant may be selected from the group consisting of rice, barley, wheat, rye, corn, sugarcane, oats, and onions, but the present invention is not limited thereto.
A method of introducing the recombinant vector of the present invention into a plant may be selected from an Agrobacterium sp.-mediated method, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and polyethylene glycol (PEG)-mediated transformation, but the present invention is not limited thereto.
The transformed plant may be obtained through a sexual propagation method or an asexual propagation method, which is a conventional method in the art. More specifically, the plant of the present invention may be obtained through sexual propagation, which is a process of producing seeds through pollination and propagating from the seeds. In addition, the plant may be transformed with the recombinant vector according to the present invention and then obtained through asexual propagation, which is a process of inducing callus, rooting, and acclimatizing soil, according to a conventional method. That is, an explant of the plant transformed with the recombinant vector according to the present invention is placed in a suitable medium known in the art, and then cultured under appropriate conditions to induce callus formation, and when shoots are formed, they are transferred to a hormone-free medium and cultured. After about 2 weeks, the shoots are transferred to a rooting medium to induce roots. Thereafter, the roots may be transplanted into the soil and acclimatized, thereby obtaining the plant according to the present invention. The transformed plant of the present invention may comprise tissues, cells, or seeds obtainable therefrom.
The present invention also provides a method of producing a target protein, comprising: constructing a recombinant vector for transforming the plant; introducing the recombinant vector into a plant to produce a transgenic plant; culturing the transgenic plant; and isolating and purifying a target protein from the transgenic plant or a culture solution.
The introduction of the recombinant vector into a plant cell or a plant may be performed using one or more methods selected from the group consisting of an Agrobacterium sp.-mediated method, particle gun bombardment, silicon carbide whiskers, sonication, electroporation, and polyethylene glycol (PEG)-mediated transformation. In one embodiment of the present invention, PEG-mediated transformation was used.
A recombinant vector for transforming a plant, according to the present invention, has overcome the difficulty of obtaining a highly expressed transformant, which was the biggest problem in protein production using existing plant transformation, by increasing an expression level of a target protein in a plant cell, and thus is expected to be great help in the production of useful proteins using a plant.
Hereinafter, the present invention will be described in further detail with reference to the following examples.
It will be obvious to those of ordinary skill in the art that these examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention.
Arabidopsis (Arabidopsis thaliana ecotype, Col-0) plants were grown on B5 plates in a growth chamber at 20° C. to 22° C. under a 16 h/8 h light/dark cycle. Leaf tissues from 2-week-old plants were used for protoplast isolation.
The mature peptide region of mouse leptin cDNA (NM_008493.3) was used. A DNA fragment (SEQ ID NO: 1) encoding an M domain was synthesized by repetitive PCR, and mutants of the N-glycosylation (Asn-Gln substitution) were generated by PCR-based site-directed mutagenesis. A DNA fragment (SEQ ID NO: 11) encoding aprotinin was produced by chemical synthesis (Bioneer, Daejeon, Korea). Enterokinase and furin cleavage sites were included in the primer used for leptin amplification (5′-GGATCCAAGATGATGATGATAAGGTGCCTATCCAGAAAGTCCAGGAT-3′ (SEQ ID NO: 18)). A HA epitope and an ER retention signal HDEL were introduced using the primers used for amplification of the M domain. PCR conditions were as follows: 94° C. for 5 minutes, (94° C. for 30 seconds, 52° C. for 1 minute, and 72° C. for 30 seconds) repeated 30 times, 72° C. for 7 minutes. The primer sequences used are summarized in Table 1 below:
The mature region of leptin and the M domain or Asn-to-Gln-substituted mutant M domain were sequentially ligated into the vector BiP: HA: CBD: HDEL.
To accumulate fusion proteins in chloroplasts and mitochondria, a Cab transit peptide or F1-ATPase gamma subunit presequence was amplified by PCR and substituted with BiP in EeLepf and EeLepfM vectors (see Lee, D., et al. W. et al., Arabidopsis nuclear-encoded plastid transit peptides contain multiple sequence subgroups with distinctive chloroplast-targeting sequence motifs. Plant Cell 20, 1603-1622 (2008); Lee, S. et al., Mitochondrial targeting of the Arabidopsis F1-ATPase gamma-subunit via multiple compensatory and synergistic presequence motifs. Plant Cell 24, 5037-5057 (2012)).
The constructs were all constructed from the same vector and therefore have the same 5′-UTRs. Nucleotide sequences of all PCR products were confirmed by nucleotide sequencing.
The plasmid prepared in Reference Example 2 was introduced into a protoplast of plant cells prepared in Reference Example 1 by polyethylene glycol (PEG)-mediated transformation. After transformation, proteins were extracted at 24 hours or at a predetermined time to prepare protein extracts. Immediately after transformation, protoplasts were treated with tunicamycin (10 μg/mL; Sigma-Aldrich, St. Louis, MO) and then treated with cycloheximide (50 μg/mL; Sigma-Aldrich, St. Louis, MO) 12 hours after transformation. Western blotting analysis was performed on protein extracts using an anti-HA antibody (Roche Diagnostics, Indianapolis, IN), an anti-actin antibody (MP Biomedicals, Solon, OH), an anti-GFP antibody (Bio-Application, Pohang, Korea), or an anti-BiP antibody. Protein blots were developed with an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ) and images were acquired using a LAS4000 image analyzer (Fujifilm, Tokyo, Japan).
Total RNA was extracted from PEG-mediated transformed plant protoplasts using an Ambion phenol-free total RNA isolation kit and treated with TURBO DNase (Ambion). cDNA was synthesized from the extracted total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems). Transcript levels were detected using the Power SYBR Green PCR Master Mix (Applied Biosystems). ACTIN2 was used as an endogenous control. A PCR mixture (20 μl) contained 50 ng of a template, 0.5 mM forward and reverse primers, and 1×SYBR Mix.
PCR conditions were as follows: initial denaturation at 95° C. for 10 min, followed by 40 cycles of 95° C. for 15 s and 60° C. for 1 min.
To confirm specific amplification, a melting curve was generated by heating at 95° C. for 15 s and then at 60° C. for 1 min, and then increasing the temperature 0.3° C. every 15 s up to 95° C.
To highly express a target protein in a plant, a recombinant vector for plant transformation which comprised a gene encoding a fusion protein in which the M domain of human CD45 was fused to the target protein was constructed. As the target protein, Leptin in which an enterokinase cleavage site and a furin cleavage site were fused to the N-terminal and the C-terminal, respectively (hereinafter referred to as “eLepf”) was used. A CaMV 35S promoter was used in the vector pCAMBIA1300 as the recombinant vector, which is a commonly used vector, an M17 sequence (SEQ ID NO: 3) was added to increase the amount of protein synthesized, the target protein was transferred to an endoplasmic reticulum using a genomic DNA sequence (SEQ ID NO: 4) corresponding to a signal peptide of BiP (chaperone binding protein), and HDEL (His-Asp-Glu-Leu) was added to the C-terminal to be retained in the endoplasmic reticulum so that the final target protein could be accumulated in the endoplasmic reticulum. In addition, the HA epitope was used to confirm the presence or absence of fusion protein expression by western blotting. A diagram of the recombinant vector constructed in the present example is shown in
A nucleic acid sequence (SEQ ID NO: 10) of the recombinant vector shown in
The N-glycosylation residues of an M domain of SEQ ID NO: 2 are as follows:
For comparison with an expression level of the fusion protein eLepfM produced by the recombinant vector comprising the M domain, constructed in Example 1, a recombinant vector was constructed as follows.
A vector EeLepf in which the M domain was removed from the recombinant vector (EeLepfM; see
As illustrated in
To more clearly verify whether the increase in protein expression level due to the fusion of the M domain was induced by N-glycosylation, a recombinant vector was constructed such that the M domain-free target protein (eLepf) and the M domain-fused protein (eLepfM) were targeted to each of the endoplasmic reticulum, chloroplast, and mitochondria. To target the fusion proteins to each of the endoplasmic reticulum, chloroplast, and mitochondria, BiP (SEQ ID NO: 4), a Cab transit peptide (SEQ ID NO: 12), or F1-ATPase gamma subunit presequence (SEQ ID NO: 13) was fused to the N-terminal of the target protein, and an M domain-encoding nucleic acid sequence (SEQ ID NO: 1) was fused to the C-terminal of the target protein, thereby completing the construction of the recombinant vectors. Each recombinant vector was introduced into a plant cell using the above-described method, followed by culturing to express the corresponding fusion protein. Protein expression levels were confirmed by western blotting.
The results of the obtained protein expression levels are illustrated in
As illustrated in
When the results of
As confirmed in Example 2, to understand the mechanism for the N-glycosylation-induced increase in protein expression, an expression rate of the M domain-fused protein was examined. Each of the M domain-fused recombinant vector EeLepfM and the vector EeLepfM1234 in which the N-glycosylation sites of the M domain were mutated was transformed into a plant cell, and after 12 hours, each vector was treated with cycloheximide or dimethyl sulfoxide (DMSO), which blocks protein synthesis, and then proteins were extracted at an interval of 12 hours to perform western blotting thereon.
The results thereof are illustrated in
As a result, as illustrated in
To confirm whether the expression increase effect due to the M domain is applicable to proteins other than the target protein (eLepf) used in the examples, the M domain-encoding gene (SEQ ID NO: 1) was fused to another target protein, e.g., a gene encoding Exendin4 (SEQ ID NO: 51) or a gene encoding GLP-1 (SEQ ID NO: 52), and a G domain, which is a translation enhancer domain, was fused thereto, thereby completing the construction of a recombinant vector (see
In addition, a recombinant vector comprising leptin (Lep), aprotinin (Apr; SEQ ID NO: 11), or GFP (Gfp; SEQ ID NO: 15) at the position Xxx of each of the recombinant vectors illustrated in
The results thereof are illustrated in
The expression level of a target protein (Leptin) was measured using recombinant vectors comprising genes encoding mutants (one mutation, two mutations, three mutations, and all four mutations of the four N-glycosylation sites) into which Asn-Gln substitution mutation(s) was/were introduced to various combinations of the four N-glycosylation sites of the M domain (see the drawing of Example 1) (see
As illustrated in
It was tested whether the low expression level of an unglycosylated protein is related to ER-associated degradation. In conclusion, it was confirmed that the low expression level of the unglycosylated protein was not due to ER-associated degradation.
More specifically, each of the EeLepfM vector (expression of a fusion protein of Leptin and wild-type M domain) and EeLepfM1234 vector (expression of a fusion protein of Leptin and a mutant M domain in which all four glycosylation sites were mutated (substituted with Gln) was transformed into a plant protoplast, and then 20 μM MG132 (IUPAC name: Benzyl N-[(2S)-4-methyl-1-[[(2S)-4-methyl-1-[[(2S)-4-methyl-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopentan-2-yl]carbamate), which is an inhibitor of 26S proteasome-mediated degradation, was added to a protoplast culture medium at 18 h or 21 h, followed by further culturing of each protoplast for 6 hours or 3 hours.
(HAT: Hours after Transformation)
For comparison (positive control), each protoplast was transformed with Rbcs[T4, 7A]: GFP, and then treated with MG132 at 18 h and further cultured for 6 hours. Rbcs[T4, 7A]: GFP, which is a GFP fusion construct, expresses a mutant form of the RbcS delivery peptide defective in the introduction of a protein into the chloroplast, and is ubiquitinated and degraded by the cytoplasmic 26S proteasome.
At 24 hours after transformation, proteins were extracted from each transformed protoplast, and then the protein extracts were analyzed by western blotting using an anti-HA antibody or an anti-GFP antibody.
The obtained western blotting results are illustrated in
It was examined whether the expression level of the target protein is increased even in the case of N-glycosylation of the target protein itself that was not fused with the M domain, that is, a case in which N-glycosylation of the target protein itself was possible due to its intrinsic inclusion of N-glycosylation sites.
To this end, with reference to the previous examples, an LIF (EeLiff)-expressing recombinant vector to which the M domain was fused or not fused was constructed using a leukemia inhibitory factor (LIF) (SEQ ID NO: 17; nucleic acid-encoding sequence: SEQ ID NO: 16; EeLiff; intrinsically having 6 N-glycosylation sites) instead of the Leptin protein, each recombinant vector was transformed into a plant protoplast, and after 24 hours, proteins were extracted and protein expression levels were measured by western blotting.
The obtained protein expression levels are illustrated in
The expression level of a fusion protein in which the target protein (Leptin) was fused with a portion or extension portion of the M domain was tested. To this end, with reference to the method of Example 2, a fusion protein in which the target protein and a fragment (SEQ ID NO: 6) having a length of 20 amino acids at positions 41-60 of the M domain (SEQ ID NO: 2; total 60aa) (EeLepfM20; comprising 1 N-glycosylation site (N4: Asn at position 46), a fusion protein in which a fragment (SEQ ID NO: 7) having a length of 40 amino acids at positions 21-60 was fused (EeLepfM40; comprising 3 N-glycosylation sites of the M domain (N2: Asn at position 30; N3: Asn at position 40; and N4: Asn at position 46), and a fusion protein (E3LepfM80) in which a fragment (SEQ ID NO: 8) having a length of a total of 80 amino acids which extends by 10 amino acids towards each of the N-terminal and C-terminal of the M domain of SEQ ID NO: 2 in CD45 (SEQ ID NO: 5) were each introduced into a protoplast of a plant cell and expressed, and then proteins were extracted and expression levels thereof were measured by western blotting. For comparison, the expression level of EeLepf to which the M domain was not fused was also measured.
The obtained protein expression levels are illustrated in
A plant cell (Reference Example 1) was transformed with each of recombinant vectors comprising a wild-type (not mutated) M domain or various M domain mutants in which each of the N-glycosylation sites was mutated and leptin, and total RNA was extracted after 1 day to perform quantitative RT-PCR. For a detailed method, refer to Reference Example 4.
The obtained RNA levels are illustrated in
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0008160 | Jan 2017 | KR | national |
This application is a divisional application of U.S. patent application Ser. No. 16/478,592, filed Jul. 17, 2019, which was a 371 of PCT/KR2018/0000807, filed Jan. 17, 2018, which claims the benefit of priority from Korean Patent Application No. 10-2017-0008160, filed Jan. 17, 2017, the contents of each of which are incorporated herein by reference in its entirety.
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| Number | Date | Country | |
|---|---|---|---|
| 20220235366 A1 | Jul 2022 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16478592 | US | |
| Child | 17650975 | US |
