Method for high-yield production of difficult-to-express recombinant proteins in plants using albumin fusion

Abstract

The present invention relates to a method for producing difficult-to-express recombinant proteins in plants at high yields. According to the present invention, it is possible to mass-produce various growth factors and cytokines, which have high utility in biopharmaceuticals and stem cell engineering industries, in plants at high yields as bioactive recombinant proteins.

Description

High-yield production method of recombinant proteins in plants using albumin fusion

[0001] The present invention relates to a method for producing a recombinant protein that is poorly expressed in plants at high yield by fusing albumin to the recombinant protein, which is difficult to produce due to its low expression level in plants.

[0002] Growth factors are substances that induce cell proliferation and differentiation. They are primarily proteins or steroid hormones. Some cytokines, which induce cell proliferation, also fall into this category. These recombinant growth factors have been used for various medical purposes, such as wound healing through tissue regeneration and bone tissue regeneration.

[0003] With recent advancements in stem cell engineering, the market for culture media essential for stem cell culture and induction is expanding, leading to a corresponding increase in the industrial use of growth factors. A key element in the stem cell culture media industry are the various growth factors that induce cell proliferation and differentiation.

[0004] Previously, cells were cultured by adding fetal bovine serum (FBS), a high-protein mixture containing growth and attachment factors, hormones, antioxidants, and lipids, to the cell culture medium. However, it was difficult to qualitatively and quantitatively identify the components contained in the serum, and since fetal bovine serum was applied equally to the culture and differentiation of various cells of different origins and species, there was a problem in that it was not possible to establish conditions optimized for culture and differentiation tailored to the unique characteristics of each cell.

[0005] In addition, as awareness of the use of fetal bovine serum has changed from recent environmental and ethical perspectives, regulations and policies on the use of fetal bovine serum have been changing worldwide. Recently, the paradigm has shifted toward completely excluding the use of fetal bovine serum, and the development of next-generation culture media such as 'serum-free chemical composition culture media' is actively underway.

[0006] In this case, the serum-free chemical composition culture medium must contain growth factors suitable for cell growth and differentiation, and therefore, the development of a technology to safely and inexpensively produce such growth factors is required.

[0007] Currently, recombinant growth factors are primarily produced in microorganisms such as Escherichia coli or animal cell lines using human-derived genes for research purposes. However, when producing recombinant growth factors using microorganisms such as E. coli, there is a high risk of endotoxin contamination. In many cases, inclusion bodies are formed or disulfide bonds do not form properly, which increases production costs during the separation and purification process or requires a complex refolding process. In addition, when producing recombinant growth factors in animal cells, there is a high risk of viral infection, etc.

[0008] Recently, there have been active attempts to produce recombinant proteins in plants to solve these problems, but when producing growth factors or cytokines in plants, there was a problem of low production yield (Yao J., Weng Y., Dickey A., Wang K. Plants as factories for human pharmaceuticals: applications and challenges. Int. J. Mol. Sci. 2015;16:28549-28565; Margolin E., Chapman R., Williamson AL, Rybicki EP, Meyers AE Production of complex viral glycoproteins in plants as vaccine immunogens. Plant Biotechnol. J. 2018;16:1531-1545. doi: 10.1111 / pbi.12963; Abiri R., Valdiani A., Maziah M., Shaharuddin NA, Sahebi M., Yusof ZNB, Atabaki N., Talei D. A critical review of the concept of transgenic plants: insights into pharmaceutical biotechnology and molecular farming. Curr. Issues Mol. Biol. 2015;18:21-42).

[0009] Accordingly, in the present invention, while searching for a method for producing growth factors or cytokines in plants, it was confirmed that when albumin is fused to growth factors or cytokines and expressed in plants, a recombinant protein can be stably produced with high expression efficiency, thereby completing the present invention.

[0010]

[0011] The present invention aims to provide a DNA construct for producing a promiscuous protein in a plant at a high yield and a method for producing a promiscuous protein in a plant at a high yield using the same.

[0012] To achieve the above purpose, a DNA construct or recombinant vector is provided, which comprises a sequence encoding albumin and a sequence encoding a non-expressed protein.

[0013] In the present invention, the hyper-expressed protein may be characterized as being a protein having a molecular weight of 5 to 20 kDa.

[0014] In the present invention, the hyper-expressed protein may be characterized as being a growth factor or cytokine.

[0015] In the present invention, the overexpressed protein may be characterized by being selected from the group consisting of FGF-1 (fibroblast growth factor), GMCSF (granulocyte macrophage colony stimulating factor), LIF (leukemia inhibitory factor), IGF (insulin-like growth factor), interleukin, interferon, EGF (epidermal growth factor), human stem cell factor, glial cell line-derived neurotropic factor, vascular endothelial growth factor, activin A, bone morphogenetic proteins, SHH (sonic hedgehog protein), and TGF (transforming growth factors).

[0016] In the present invention, the albumin may be characterized as being serum albumin.

[0017] In the present invention, the serum albumin is bovine serum albumin, and the bovine serum albumin may be characterized as being a wild type (bSA), a mutant (bSA110) including domain 1 from the N-terminus, and a mutant (bSA300) including domains 1 to 3 from the N-terminus.

[0018] In the present invention, it may be characterized by further including a sequence encoding a peptide linker between the sequence encoding the albumin and the sequence encoding the infrequently expressed protein.

[0019] In the present invention, it may be characterized by further including a sequence encoding an enzyme-cleaved peptide between the sequence encoding the albumin and the sequence encoding the infrequently expressed protein.

[0020] In the present invention, the DNA construct or recombinant vector may be characterized by further comprising at least one sequence selected from the group consisting of a sequence encoding a GB1 domain, a sequence encoding an endoplasmic reticulum leader peptide, and a sequence encoding an endoplasmic reticulum retention peptide.

[0021] In the present invention, the DNA construct or recombinant vector

[0022] (i) the first intron sequence of the modified ubiquitin10;

[0023] (ii) 5' UTR sequence; and

[0024] (iii) It may be characterized by further comprising at least one sequence selected from the group consisting of sequences encoding a purification tag.

[0025] The present invention also provides a transformed plant cell or a transformed plant into which the DNA construct or recombinant vector has been introduced.

[0026] In the present invention, the transformed plant cell or transformed plant may be characterized in that a recombinant vector for human calreticulin (CRT) expression and a p38 recombinant vector are additionally introduced.

[0027] The present invention also provides a method for producing a highly expressible protein, comprising the following steps:

[0028] (a) a step of culturing or growing the transformed plant cell or transformed plant; and

[0029] (b) A step of recovering a non-expressed protein from a lysate obtained by crushing the transformed plant cell or transformed plant that has been cultured or grown, or from a cultured solution.

[0030] In the present invention, the step (b) may be characterized by purifying the albumin and the fusion protein of the in vitro protein and treating it with a peptide cleavage enzyme to recover the liberated in vitro protein.

[0031] According to the present invention, various growth factors and cytokines with high utility in biopharmaceutical and stem cell engineering industries can be mass-produced in plants in the form of recombinant proteins with physiological activity at high yields.

[0032]

[0033] Figure 1 shows the results of constructing a recombinant vector containing a GB1 domain to express bovine serum albumin fusion protein in plants and transforming the vector into plants, and confirming the expression of bovine serum albumin fusion protein (red arrow) and CRT (green asterisk).

[0034] Figure 2 shows the results of constructing a recombinant vector excluding the GB1 domain to express a bovine serum albumin fusion protein, transforming the vector into a plant, and confirming the expression of bovine serum albumin using CBB staining.

[0035] Figure 3 illustrates the structure of a recombinant vector in which bovine serum albumin of various sizes (wild type, 110aa, 300aa) is fused to the N-terminus of a growth factor or cytokine according to an embodiment of the present invention. (L sequentially represents Linker 1 and Linker 2)

[0036] Figure 4 shows the results of comparing the expression levels of fusion proteins according to the presence or absence of bSA fusion and the type of fused bSA by CBB staining and Western blotting after introducing FGF-1 as a growth factor (GF) and IL4 as a cytokine (CK) into the recombinant vector of Figure 3, respectively, and transforming these fusion proteins into plants.

[0037] Figure 5 shows the results of constructing a recombinant vector in which wild type or 300aa bovine serum albumin is fused to the N-terminus of various growth factors according to an embodiment of the present invention, transforming the vector into a plant, and confirming the expression of bovine serum albumin by Western blotting.

[0038] Figure 6 shows the results of constructing a recombinant vector in which wild type or 300aa bovine serum albumin is fused to the N-terminus of various cytokines according to an embodiment of the present invention, transforming the vector into a plant, and confirming the expression of bovine serum albumin by Western blotting.

[0039] Fig. 7 is Ni 2+ -The results confirmed that a 17 kDa sized growth factor located at the C-terminus was released through enterokinase treatment in the recombinant protein of growth factor bound to bSA or bSA300 (bSA binding - 95 kDa, bSA300 binding - 63 kDa) separated by NTA affinity resin, as confirmed by Coomassie brillent blue (CBB) staining.

[0040]

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein and the experimental methods described below are well known and commonly used in the art.

[0042] In the present invention, it should be understood that terms such as “include” or “have” are intended to specify the presence of a feature, number, step, operation, component, part or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, parts or combinations thereof.

[0043] In the present invention, the term "about" is used to mean approximately, nearly, approximately, or to a certain degree. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower boundaries of the indicated values. Generally, the term "about" is used to modify the upper and lower values ​​of the values ​​mentioned herein by a 10 percent variation (upper or lower).

[0044]

[0045] In the present invention, a technology was developed for mass-producing in plants, with high yield, recombinant proteins in the mature form or active fusion form of various growth factors or cytokines that have high utility in clinical and stem cell engineering industries but have low expression levels in plant cells, making it difficult to produce recombinant proteins in plants. In addition, it was confirmed that when a recombinant vector is used that fuses the full-length sequence of bovine serum albumin or a mutant lacking a partial sequence to a growth factor or cytokine and expresses it, the desired poorly expressed protein (particularly, a growth factor or cytokine) can be produced with high yield.

[0046]

[0047] Accordingly, the present invention relates to a DNA construct or recombinant vector comprising, in one aspect, a sequence encoding albumin and a sequence encoding a non-expressed protein.

[0048] Specifically, the present invention relates to a DNA construct or recombinant vector for plant expression of a fusion recombinant protein of albumin and an unexpressed protein.

[0049] In the present invention, the hyper-expressed protein may be a protein having a molecular weight of about 5 to about 20 kDa, and in some embodiments, may be a growth factor or cytokine.

[0050] In the present invention, the overexpressed protein may be selected from the group consisting of FGF-1 (fibroblast growth factor), GMCSF (granulocyte macrophage colony stimulating factor), LIF (leukemia inhibitory factor), IGF (insulin-like growth factor), interleukin, interferon, EGF (epidermal growth factor), human stem cell factor, glial cell line-derived neurotropic factor, vascular endothelial growth factor, activin A, bone morphogenetic proteins, SHH (sonic hedgehog protein), and TGF (transforming growth factors), but is not limited thereto.

[0051] In the present invention, the hyper-expressed protein may be in an activated form (mature form).

[0052] In the present invention, the albumin may be serum albumin.

[0053] For example, the serum albumin may be a mammalian serum albumin such as human serum albumin (hASA), bovine serum albumin (bSA), or canine serum albumin, but is not limited thereto.

[0054] In one embodiment, the serum albumin is bovine serum albumin, and the bovine serum albumin may be a wild type (bSA) or a variant thereof comprising a total of six domains, and the variant may be, for example, a fragment comprising domain 1 from the N-terminus (bSA110) or a fragment comprising domains 1 to 3 from the N-terminus (bSA300).

[0055] In the present invention, a sequence encoding an enzyme-cleaved peptide may be additionally included between the sequence encoding the albumin and the sequence encoding the infrequently expressed protein.

[0056] The above enzyme-cleaved peptide is a peptide that is expressed as part of a fusion protein in a plant and can be cleaved by an enzyme during a purification or separation process. Through the enzyme-cleavage process, only the target protein can be separated from the fusion protein, and this is particularly useful for producing an activated form of the target protein.

[0057] In the present invention, the enzyme capable of cleaving the enzyme-cleaved peptide is a peptide endopeptidase, which may be TEV, C3 protease, or enterokinase, but is not limited thereto. The enzyme-cleaved peptide sequences cleaved by each of the above enzymes are known in the art.

[0058] In one embodiment, the enzyme-cleaved peptide used in the present invention can be represented by the amino acid sequence of SEQ ID NO: 25, which can be cleaved by enterokinase.

[0059] In the present invention, a sequence encoding a peptide linker may be additionally included between the sequence encoding the albumin and the sequence encoding the infrequently expressed protein.

[0060] In the present invention, the DNA construct or recombinant vector may be characterized by further comprising at least one sequence selected from the group consisting of a sequence encoding a GB1 domain, a sequence encoding an endoplasmic reticulum leader peptide, and a sequence encoding an endoplasmic reticulum retention peptide.

[0061] In the present invention, when the fusion protein comprises an endoplasmic reticulum leader peptide, production efficiency can be improved through endoplasmic reticulum accumulation of the target protein in the plant, and the endoplasmic reticulum leader peptide can be encoded by a BiP leader sequence.

[0062] In the present invention, when the fusion protein comprises an endoplasmic reticulum-retaining peptide, production efficiency can be improved through endoplasmic reticulum accumulation of the target protein in the plant. In one embodiment, the endoplasmic reticulum-retaining peptide may be HDEL.

[0063] When the recombinant protein according to the present invention additionally includes a GB1 domain, the production stability of the target protein can be increased and the production efficiency can be improved.

[0064] In the present invention, the DNA construct or recombinant vector

[0065] (i) the first intron sequence of the modified ubiquitin10;

[0066] (ii) 5' UTR sequence; and

[0067] (iii) It may be characterized by further comprising at least one sequence selected from the group consisting of sequences encoding a purification tag.

[0068] When the first intron sequence of the modified ubiquitin10 is introduced into the above DNA construct or recombinant vector, it can induce an intron-mediated enhancement effect that increases the transcription level by affecting not only the binding of transcription factors but also the transcription rate, nuclear export, and transcription stability, and can improve the transcription efficiency of a gene that encodes a ubiquitin10 protein fused at the 3' end.

[0069] When the 5' UTR sequence is introduced into the above DNA construct or recombinant vector, the translation efficiency of the fusion protein in the plant can be improved.

[0070] Meanwhile, the DNA construct or recombinant vector contains a promoter and terminator for expression of the fusion protein.

[0071] Meanwhile, the fusion protein according to the present invention may additionally include a peptide tag at the N-terminus or C-terminus for an efficient separation and / or purification process. The peptide tag may be any tag commonly introduced in a recombinant protein production process, including, but not limited to, His Tag, EPEA Tag, GST Tag, and HA Tag. The peptide tag may be removed after the separation and / or purification process is completed.

[0072] In the present invention, in addition to the albumin and the unexpressed protein, a peptide linker may be additionally included when one or more domains or peptides are fused among the GB1 domain, the Tag peptide for purification, the endoplasmic reticulum leading peptide, and the endoplasmic reticulum retention peptide.

[0073] In this case, the peptide linker may be, but is not limited to, 2 to 20 peptide linkers composed of Gly, Pro, Arg and / or Ser. For example, the peptide linker may include, but is not limited to, GGGSPR, GGGSGGS, GGGGSGGGS, GGSG, GSGS, GSGSG, GGSGGS, GGSGGSGGS or GGSGGSGGSGGS, or may be, but is not limited to, GGSG, GSGS, GSGSG, GGSGGS, GGSGGSGGS or GGSGGSGGSGGS.

[0074] In the present invention, the linker may be represented by any one of the amino acid sequences of SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 37, and SEQ ID NO: 39, but is not limited thereto.

[0075] In one embodiment, the fusion protein according to the present invention may be a fusion protein in which an endoplasmic reticulum storage peptide, a GB1 domain, albumin (full length or fragment), a target protein (i.e., a hyper-expressed protein), and an endoplasmic reticulum retention peptide are sequentially fused.

[0076] In another embodiment, the fusion protein according to the present invention may be a fusion protein in which an endoplasmic reticulum accumulation peptide, a GB1 domain, albumin (full length or fragment), a peptide tag, an enzyme-cleaved peptide, and a target protein (i.e., a hyper-expressed protein) are sequentially fused.

[0077] In another embodiment, the fusion protein according to the present invention may be a fusion protein in which an endoplasmic reticulum storage peptide, a GB1 domain, albumin (full length or fragment), a peptide tag, an enzyme-cleavable peptide, an endoplasmic reticulum retention peptide, and a target protein (i.e., a hyper-expressed protein) are sequentially fused.

[0078] Any peptide or any domain of the fusion protein may comprise a linker.

[0079] Various DNA constructs for expression of fusion proteins according to the present invention are disclosed in FIGS. 3, 5, 6, 7, and 8, but these are only some embodiments of the present invention, and the DNA constructs according to the present invention are not limited to these DNA constructs.

[0080] As used herein, "vector" refers to a DNA construct containing a DNA sequence operably linked to suitable regulatory sequences capable of expressing the DNA in a suitable host. The vector may be a plasmid, a phage particle, or simply a potential genomic insert. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, can integrate into the genome itself. Since plasmids are currently the most commonly used form of vector, the terms "plasmid" and "vector" are sometimes used interchangeably herein. For the purposes of the present invention, the use of plasmid vectors is preferred. A typical plasmid vector that can be used for this purpose has a structure that includes (a) an origin of replication that allows efficient replication, such that several to several hundred plasmid vectors can be contained per host cell, (b) an antibiotic resistance gene that allows selection of host cells transformed with the plasmid vector, and (c) a restriction enzyme cleavage site into which a foreign DNA fragment can be inserted. Even if suitable restriction enzyme cleavage sites do not exist, ligation of the vector and the foreign DNA can be easily achieved using conventional synthetic oligonucleotide adaptors or linkers. After ligation, the vector must be transformed into a suitable host cell. Transformation can be easily achieved using the calcium chloride method or electroporation (Neumann et al., EMBO J., 1:841, 1982).

[0081] The vector used for overexpression of the gene according to the present invention may be an expression vector known in the art. In the present invention, a binary vector commonly used for plant transformation was used.

[0082] As is well known in the art, in order to increase the expression level of a transgene in a host cell, the gene must be operably linked to transcription and translation expression control sequences. Preferably, the expression control sequence and the gene are contained within a single recombinant vector that also contains a bacterial selection marker and an origin of replication. Preferably, the recombinant vector further includes an expression marker useful in plant cells. A plant or plant cell transformed with the above-described recombinant vector constitutes another aspect of the present invention. The term "transformation" as used herein refers to the introduction of DNA into a host cell so that the DNA becomes replicable as an extrachromosomal element or by chromosomal integration. On the other hand, "transfection" refers to the introduction of DNA into a host cell so that the DNA becomes replicable within the host cell.

[0083] It should be understood that not all vectors are equally effective in expressing DNA sequences within the system of the present invention. However, those skilled in the art can select appropriate vectors and expression control sequences without undue experimental burden and without departing from the scope of the present invention. The copy number of the vector, its ability to control copy number, and the expression of other proteins encoded by the vector, such as antibiotic markers, should also be considered.

[0084] A preferred example of a recombinant vector of the present invention is a Ti-plasmid vector, which, when present in a suitable host such as Agrobacterium tumefaciens, is capable of transferring part of itself, the so-called T-region, into plant cells. Other types of Ti-plasmid vectors (see EP 0 116 718 B1) are currently used to transfer hybrid DNA sequences into plant cells or protoplasts from which new plants can be produced, wherein the hybrid DNA is suitably integrated into the genome of the plant. A particularly preferred form of Ti-plasmid vector is a so-called binary vector, as claimed in EP 0 120 516 B1 and U.S. Pat. No. 4,940,838. Other suitable vectors that can be used to introduce the DNA according to the present invention into a plant host include viral vectors, such as those derived from double-stranded plant viruses (e.g., CaMV) and single-stranded viruses, geminiviruses, etc., and non-complete plant viral vectors. The use of such vectors can be particularly advantageous when it is difficult to properly transform the plant host.

[0085] The expression vector will preferably include one or more selectable markers. These markers are typically nucleic acid sequences with properties that can be selected chemically, and include any gene that can distinguish transformed cells from untransformed cells. Examples include, but are not limited to, herbicide resistance genes such as glyphosate or phosphinothricin, and antibiotic resistance genes such as kanamycin, G418, bleomycin, hygromycin, and chloramphenicol.

[0086] In the present invention, a gene encoding a full-length wild-type albumin may be represented by a base sequence of SEQ ID NO: 1, and a full-length wild-type albumin protein may be represented by an amino acid sequence of SEQ ID NO: 2, and the gene sequence of albumin according to the present invention may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence of SEQ ID NO: 1, and the protein sequence of albumin may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more with the amino acid sequence of SEQ ID NO: 2. It can have a homology of 96% or more, 97% or more, 98% or more, or 99% or more, or 100% or more.

[0087] In the present invention, an albumin variant, for example, an albumin fragment including domains 1 to 3 or domain 1, may be used in place of the full-length wild-type albumin, and the gene sequence encoding the albumin fragment may be represented by the base sequence of SEQ ID NO: 14 or SEQ ID NO: 16, respectively, and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, or 100% with the base sequence, and the albumin fragment may be represented by the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 17, respectively, and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more with the amino acid sequence, It may have a homology of 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100%.

[0088] In one embodiment, the hyper-expressed protein according to the present invention may be FGF1 as a growth factor, and the sequence encoding the FGF1 may be represented by the base sequence of SEQ ID NO: 28 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence, and the FGF1 protein may be represented by the amino acid sequence of SEQ ID NO: 29 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, or 97% with the amino acid sequence. It can have a homology of 98% or more, or 99% or more, or 100% or more.

[0089] In another aspect, the protein that is overexpressed according to the present invention may be IL4 as a cytokine, and the sequence encoding the IL4 may be represented by the base sequence of SEQ ID NO: 30 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence, and the IL4 protein may be represented by the amino acid sequence of SEQ ID NO: 31 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, or 97% with the amino acid sequence. It can have a homology of 98% or more, or 99% or more, or 100% or more.

[0090] In another aspect, the overexpressed protein according to the present invention may be GMCSF as a cytokine or growth factor, and the sequence encoding the GMCSF may be represented by the base sequence of SEQ ID NO: 40 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence, and the GMCSF protein may be represented by the amino acid sequence of SEQ ID NO: 41 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% It can have a homology of 97% or more, 98% or more, 99% or more, or 100% or more.

[0091] In another aspect, the hyper-expressed protein according to the present invention may be LIF as a growth factor, and the sequence encoding the LIF may be represented by the base sequence of SEQ ID NO: 42 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence, and the LIF protein may be represented by the amino acid sequence of SEQ ID NO: 43 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more with the amino acid sequence, It can have a homology of 98% or more, or 99% or more, or 100%.

[0092] In another aspect, the protein that is overexpressed according to the present invention may be bIGF as a growth factor, and the sequence encoding the bIGF may be represented by the base sequence of SEQ ID NO: 44 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence, and the bIGF protein may be represented by the amino acid sequence of SEQ ID NO: 45 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more with the amino acid sequence, It can have a homology of 97% or more, 98% or more, 99% or more, or 100%.

[0093] In another aspect, the overexpressed protein according to the present invention may be IL15 as a cytokine, and the sequence encoding the IL15 may be represented by the base sequence of SEQ ID NO: 46 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence, and the IL15 protein may be represented by the amino acid sequence of SEQ ID NO: 47 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more with the amino acid sequence, It can have a homology of 97% or more, 98% or more, 99% or more, or 100%.

[0094] In another aspect, the overexpressed protein according to the present invention may be IFNa as a cytokine, and the sequence encoding the IFNa may be represented by the base sequence of SEQ ID NO: 48 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence, and the IFNa protein may be represented by the amino acid sequence of SEQ ID NO: 49 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more with the amino acid sequence, It can have a homology of 97% or more, 98% or more, 99% or more, or 100%.

[0095] In another aspect, the overexpressed protein according to the present invention may be IFNb as a cytokine, and the sequence encoding the IFNb may be represented by the base sequence of SEQ ID NO: 50 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence, and the IFNb protein may be represented by the amino acid sequence of SEQ ID NO: 51 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more with the amino acid sequence, It can have a homology of 97% or more, 98% or more, 99% or more, or 100%.

[0096] In the present invention, the sequence encoding the GB1 domain may be represented by the base sequence of SEQ ID NO: 3 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the base sequence, and the GB1 domain may be represented by the amino acid sequence of SEQ ID NO: 4 and may have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with the amino acid sequence. It can have homology.

[0097] In the present invention, a gene encoding an endoplasmic reticulum-retained peptide may be represented by a base sequence of SEQ ID NO: 26, and the endoplasmic reticulum-retained peptide may be represented by an amino acid sequence of SEQ ID NO: 27.

[0098] In the present invention, the gene encoding the endoplasmic reticulum leader peptide is gBiP or cBiP, which can be represented by the base sequence of SEQ ID NO: 5 or SEQ ID NO: 6, respectively, and can have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more or 100% with each of the base sequences, and the endoplasmic reticulum leader peptide can be represented by the amino acid sequence of SEQ ID NO: 7, and can have a homology of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more with the amino acid sequence. It can have a homology of 97% or more, 98% or more, 99% or more, or 100%.

[0099] The tag peptide used as an embodiment in the present invention may be an amino acid sequence represented by SEQ ID NO: 9 or an amino acid sequence represented by SEQ ID NO: 19, but the embodiment of the present invention is not limited thereto, and it is obvious in the art that various tag peptides used to separate or purify proteins can be applied to the present invention.

[0100] In the present invention, the 5' UTR sequence can be represented by the base sequence of sequence number 10.

[0101] In the present invention, the first intron sequence of the modified ubiquitin10 can be represented by the base sequence of SEQ ID NO: 11.

[0102] The gene used in the present invention may undergo various modifications to the coding region within a range that does not change the amino acid sequence of the protein expressed from the coding region, and may undergo various modifications or alterations in a portion other than the coding region within a range that does not affect the expression of the gene, and such modified genes are also included in the scope of the present invention.

[0103] Accordingly, the present invention also encompasses polynucleotides having substantially the same base sequence as the gene, as well as fragments of the gene. A substantially identical polynucleotide refers to a gene encoding an enzyme having the same function as that used in the present invention, regardless of sequence homology. A fragment of the gene also refers to a gene encoding an enzyme having the same function as that used in the present invention, regardless of the length of the fragment.

[0104] In addition, the amino acid sequence of the protein, which is the expression product of the gene of the present invention, can be secured from various biological resources such as microorganisms within a range that does not affect the potency and activity of the corresponding enzyme, and proteins secured from such other biological resources are also included in the scope of the present invention.

[0105] Accordingly, the present invention also encompasses polypeptides having an amino acid sequence substantially identical to the protein described above, as well as fragments of the polypeptide. A "substantially identical polypeptide" refers to a protein having the same function as the protein described herein, regardless of amino acid sequence homology. A fragment of the polypeptide also refers to a protein having the same function as the protein described herein, regardless of its length.

[0106] The protein used in the present invention may have some of its amino acids substituted, and the amino acid substitutions of the present application may be non-conserved substitutions. The non-conserved substitutions may include altering amino acid residues of the target protein or polypeptide in a non-conservative manner, such as, for example, replacing an amino acid residue having a specific side chain size or specific property (e.g., hydrophilicity) with an amino acid residue having a different side chain size or different property (e.g., hydrophobicity).

[0107] The amino acid substitutions may also be conserved substitutions. Conserved substitutions may involve altering amino acid residues of a target protein or polypeptide in a conserved manner, such as replacing an amino acid residue having a particular side chain size or a particular characteristic (e.g., hydrophilicity) with an amino acid residue having the same or similar side chain size or the same or similar characteristic (e.g., still hydrophilicity). Such conserved substitutions generally do not significantly affect the structure or function of the resulting protein. In the present application, the amino acid sequence variants, fragments thereof, or variants thereof with one or more amino acid substitutions, which are mutants of a fusion protein, may comprise conserved amino acid substitutions that do not significantly alter the structure or function of the protein.

[0108] For example, mutual substitutions between amino acids in each of the following groups may be considered conservative substitutions in the present application:

[0109] A group of amino acids with nonpolar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine.

[0110] A group of uncharged amino acids with polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.

[0111] A group of negatively charged amino acids with polar side chains: aspartic acid and glutamic acid.

[0112] A group of positively charged basic amino acids: lysine, arginine, and histidine.

[0113] A group of amino acids containing phenyl: phenylalanine, tryptophan, and tyrosine.

[0114] The proteins, polypeptides and / or amino acid sequences encompassed by the present invention may also be understood to include at least the following scope: variants or homologues having the same or similar function as said proteins or polypeptides.

[0115] In the present invention, the variant may be a protein or polypeptide generated by substitution, deletion, or addition of one or more amino acids compared to the amino acid sequence of the protein and / or the polypeptide. For example, the functional variant may comprise a protein or polypeptide having an amino acid change by substitution, deletion, and / or insertion of at least one amino acid, for example, substitution, deletion, and / or insertion of 1-30, 1-20, or 1-10, alternatively, for example, 1, 2, 3, 4, or 5 amino acids. The functional variant may substantially retain the biological properties of the protein or the polypeptide prior to the change (e.g., substitution, deletion, or addition). For example, the functional variant may retain 60%, 70%, 80%, 90%, or 100% or more of the biological activity of the protein or the polypeptide prior to the change.

[0116] In the present invention, the homologue may be a protein or polypeptide having at least about 80% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology with the amino acid sequence of the protein and / or the polypeptide.

[0117] In the present invention, the homology generally refers to the similarity, analogousness or association between two or more sequences. The "percentage of sequence homology" can be calculated by comparing two aligned sequences in a comparison window to determine the number of positions in which the same nucleic acid base (e.g., A, T, C, G, I) or the same amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) exists, and dividing the number of matching positions by the total number of positions to provide the number of matching positions in the comparison window (i.e., window size), and multiplying the result by 100 to provide the percentage of sequence homology. Alignment to determine the percentage of sequence homology can be performed in a variety of ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for sequence alignment, including any algorithm necessary to achieve maximal alignment within the full-length sequences being compared or within the target sequence region. The homology can also be determined by the following methods: FASTA and BLAST. The FASTA algorithm is described, for example, in W.R. Pearson and D.J. Lipman's "Improved Tool for Biological Sequence Comparison", Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D, J. Lipman and W.R.For a description of the BLAST algorithm, see Pearson's "Fast and Sensitive Protein Similarity Search", Science, 227:1435-1441, 1989, and S. Altschul, W. Gish, W. Miller, E.W. Myers and D. Lipman, "A Basic Local Alignment Search Tool", Journal of Molecular Biology, 215: 403-410, 1990.

[0118] In the plant expression vector of the present invention, the promoter may be, but is not limited to, CaMV 35S, double enhancer CaMV, MacT, CsVMV, actin, ubiquitin, pEMU, MAS, or histone promoters. The term "promoter" refers to a region of DNA upstream from a structural gene and refers to a DNA molecule to which RNA polymerase binds to initiate transcription. A "plant promoter" is a promoter capable of initiating transcription in plant cells. A "constitutive promoter" is a promoter that is active under most environmental conditions and developmental states or cell differentiation. A constitutive promoter may be preferred in the present invention because selection of transformants can be performed by various tissues at various stages. Therefore, a constitutive promoter does not limit the selectability.

[0119] In the present invention, preferably, the promoter may be MacT, which may be represented by the base sequence of SEQ ID NO: 12.

[0120] In the recombinant vector of the present invention, conventional terminators can be used, and examples thereof include, but are not limited to, nopaline synthase (NOS), rice α-amylase RAmy1 A terminator, HSP18.2 terminator, intro removal terminator of Nicotiana tabacum extensin, protease inhibitor II terminator, RD19B terminator, phaseoline terminator, terminator of the Octopine gene of Agrobacterium tumefaciens, rrnB1 / B2 terminator of Escherichia coli, and the like. Regarding the necessity of terminators, it is generally known that such regions increase the certainty and efficiency of transcription in plant cells. Therefore, the use of terminators is highly preferred in the context of the present invention.

[0121] In the present invention, most preferably, the terminator may be a 3PR transcription terminator fused with a 35S transcription terminator, a PINII transcription terminator of soybean, or an RB7 matrix attachment domain, which may be represented by the base sequence of SEQ ID NO: 13.

[0122]

[0123] The present invention also provides a transformed plant cell or a transformed plant into which the DNA construct or recombinant vector has been introduced.

[0124] In the present invention, the transformed plant cell or transformed plant may be characterized in that a recombinant vector for human calreticulin (CRT) expression and a p38 recombinant vector are additionally introduced.

[0125] In the present invention, the structure of the recombinant vector for expressing human calreticulin (CRT) is clearly diagrammed in Fig. 2a of Song el al. (Plant Biotechnol J. 2022 Dec;20(12):2298-2312. doi: 10.1111 / pbi.13908), which is incorporated herein by reference in its entirety.

[0126] In the present invention, the method for producing the p38 recombinant vector is disclosed in detail in Kumari M et al. (Plant Cell Rep. 39, 1317-1329, 10.1007 / s00299-020-02566-4), which is incorporated herein by reference in its entirety.

[0127]

[0128] In another aspect, the present invention relates to a method for producing a highly expressible protein, comprising the following steps:

[0129] (a) a step of culturing or growing the transformed plant cell or transformed plant; and

[0130] (b) A step of recovering a non-expressed protein from a lysate obtained by crushing the transformed plant cell or transformed plant that has been cultured or grown, or from a cultured solution.

[0131] In the present invention, the step (b) may be characterized by separating or purifying an albumin and a fusion protein of an in vitro protein, and recovering the liberated in vitro protein by treating it with a peptide cleavage enzyme.

[0132] In the present invention, the process of isolating or purifying the fusion protein and the process of recovering the liberated target protein by treating it with a peptide cleavage enzyme can be performed by methods known in the art.

[0133] In the present invention, methods for crushing plant cells or transgenic plants include mechanical methods such as homogenizer, bead mill, or grinding and flaking, chemical methods such as enzyme treatment (e.g., cellulase, pectinase, etc.) or surfactant (e.g., Triton X-100, SDS, NP-40), and physical methods such as liquid nitrogen crushing or ultrasonic crushing, which are well known in the art and can be easily applied to the present invention by a person skilled in the art.

[0134] In the present invention, a method for isolating and purifying a fusion protein includes removing insoluble cell debris from crushed plant cells or transformed plants, recovering soluble proteins from the supernatant, and then using methods such as a Nickel-NTA agarose column, glutathione affinity chromatography, immunoprecipitation, ion exchange chromatography, and size exclusion chromatography, which are well known in the art, and can be easily applied to the present invention by a person skilled in the art.

[0135]

[0136] In the present invention, the plant is a plant selected from the group consisting of Arabidopsis, soybean, tobacco, eggplant, pepper, potato, tomato, cabbage, radish, cabbage, lettuce, peach, pear, strawberry, watermelon, melon, cucumber, carrot, celery, rice, barley, wheat, rye, corn, sugarcane, oats, and onion, and the plant cell may be characterized as being derived from the plant.

[0137] In the present invention, the plant cell may be characterized as being a plant cell derived from a dicotyledonous plant or a monocotyledonous plant, but is not limited thereto.

[0138] In the present invention, the dicotyledonous plant may be characterized by being selected from the group consisting of soybeans, tobacco, eggplants, peppers, potatoes, tomatoes, cabbages, radishes, cabbages, lettuce, peaches, pears, strawberries, watermelons, cantaloupes, cucumbers, carrots, and celery, but is not limited thereto.

[0139] In addition, in the present invention, the monocotyledonous plant may be characterized by being selected from the group consisting of rice, barley, wheat, rye, corn, sugarcane, oats, and onions, but is not limited thereto.

[0140] In some embodiments, the plant or plant cell may be characterized as being derived from Nicotiana benthamiana, Nicotiana tabacum, or Arabidopsis thaliana.

[0141] In the present invention, a gene encoding a recombinant fusion protein can be transiently expressed or stably transformed in a transformed plant or plant cell through a vector.

[0142] In addition to transient expression of a gene encoding a recombinant fusion protein in a transgenic plant or plant cell, a gene encoding a target protein can be introduced into the genome of the transgenic plant or plant cell and exist as a chromosomal element, thereby achieving stable transformation. It will be apparent to those skilled in the art that inserting a gene targeting the target protein into a plant genome chromosome would achieve the same effect.

[0143] In the present invention, introduction of a vector containing a gene encoding a recombinant fusion protein or chromosomal insertion of a gene encoding a target protein can be performed by co-cultivating a population of plant cells with Agrobacterium containing a vector containing a gene encoding a target protein.

[0144] In one embodiment, the co-cultivation may be characterized as being performed under dark conditions. The co-cultivation may be performed by stirring and culturing a culture of Agrobacterium containing a plant cell and a vector containing a gene encoding the recombinant fusion protein, and may further include a stationary culture step.

[0145] In this way, a gene encoding a recombinant fusion protein can be transiently expressed or stably transformed in plant cells through a vector.

[0146] The above static culture is a method of culturing in a stationary state in a container without stirring the culture medium, and can be used interchangeably with sedimentation without stirring in this invention.

[0147] The above-mentioned static culture may be included in a single or intermittent culture form. If a single static culture is included, it may be characterized by, for example, co-cultivating plant cells and Agrobacterium cultures while stirring, performing static culture, and then performing agitation culture again. If an intermittent static culture is included, the culture form of co-cultivating plant cells and Agrobacterium cultures while stirring, performing static culture, and then performing agitation culture again may be repeated several to several dozen times.

[0148] At this time, in detail, the culturing may be characterized by co-cultivating a culture of Agrobacterium containing the plant cell and a vector including a gene encoding the target protein with stirring for 1 minute to 48 hours, followed by stationary culturing for 1 minute to 96 hours, and then stirring culturing again for 1 to 10 days. The OD600 of Agrobacterium added for co-culturing may be 0.00001 to 2.0.

[0149] OD of Agrobacterium 600 If the OD is too low, there is a problem that the transfection rate for transient expression is low, and if it is too high, there is a problem that the survival rate of the host cell is rapidly reduced. Therefore, in the above-defined range, the OD 600 It is desirable to co-culture by adding Agrobacterium.

[0150] At this time, Agrobacterium can be used as Agrobacterium commonly used for plant transformation, and examples thereof include Agrobacterium tumefaciens or Agrobacterium rhizogenes.

[0151] Transformation of plants refers to any method for transferring DNA into plants. Such transformation methods do not necessarily require regeneration and / or tissue culture. Transformation of plant species is now commonplace, including both dicotyledonous and monocotyledonous plants. In principle, any transformation method can be used to introduce the hybrid DNA according to the present invention into suitable progenitor cells. Methods can be suitably selected from the calcium / polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant elements, particle bombardment of various plant elements (DNA or RNA-coated), Agrobacterium tumefaciens-mediated gene transfer by infiltration of plants or transformation of mature pollen or microspores, infection by (incomplete) viruses, etc. Preferred methods according to the present invention include Agrobacterium-mediated DNA transfer.

[0152]

[0153] In another aspect, the present invention can provide a kit comprising the recombinant vector.

[0154] The above kit may additionally include a description of reagents required for transforming the above recombinant vector and / or experimental methods or precautions using the above recombinant vector.

[0155]

[0156] Example

[0157] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0158]

[0159] Example 1. Vector design for expression of bSA fusion recombinant protein

[0160] We aimed to construct a recombinant gene for high expression of low molecular weight recombinant proteins in plants.

[0161]

[0162] 1-1. macT(P)-5'UTR:gBiP:GB1:bSA:EPEA-3PR(T) recombinant expression vector

[0163] A DNA fragment (1) was chemically synthesized to include the 5' UTR from the 5' end, the genomic DNA fragment of Arabidopsis BiP1 encoding the BiP leader sequence, and the GB1 domain (commissioned by Gene Universal). An XbaI cleavage sequence was added to the 5' end of this DNA fragment, and a BamHI cleavage sequence was added to the 3' end.

[0164] Next, the 1749 bp wild type base sequence of bovine serum albumin excluding the stop codon was chemically synthesized. Here, a 12 bp base sequence encoding a C-tag (EPEA) for separation and purification was added to the position where the 3 bp stop codon was removed, and DNA fragment (2) was synthesized (commissioned by Gene Universal). In this case, BamHI and XhoI cleavage sequences were added to the 5'- and 3'-ends of DNA fragment (2), respectively, and synthesized.

[0165] DNA fragment (1) was introduced into the expression vector pTEX1L (see Song et al., J. Integr. Plant Biol. 63, 1505-1520. doi: 10.1111 / jipb.13141) having a MacT promoter cut with the same restriction enzymes by cutting DNA fragment (1) with XbaI and BamHI, and then DNA fragment (2) cut with BamHI and XhoI was additionally introduced to construct a recombinant vector.

[0166] In the above recombinant vector, a 3PR chimeric transcription terminator (5S terminator, soybean protease inhibitor gene (PI-II) terminator, and matrix attachment domain RB7 sequence fused) that was confirmed to exhibit high transcription efficiency in a previous study (Yun et al., 2023https: / doi.org / 10.3389 / fpls.2023.1138089) is introduced in place of the existing RD29B transcription terminator.

[0167]

[0168] 1-2. macT(P)-mU10In:5'UTR:cBiP:bSA:EPEA-3PR(T) recombinant expression vector

[0169] From the recombinant gene constructed in this way, a recombinant gene was additionally produced by removing the GB1 domain. For this purpose, a DNA fragment (3) was chemically synthesized to include the CDS DNA fragment of Arabidopsis BiP1 encoding the ubiquitin 10 intron, 5' UTR, and BiP leader sequence modified from the 5' end (commissioned by Gene Universitaire). In this case, the DNA fragment (3) was synthesized by adding XbaI and BamHI cleavage sequences to the 5'- and 3'-ends, respectively.

[0170] DNA fragment (3) was cut with XbaI and BamHI, and the recombinant vector produced in Example 1-1 was cut with the same restriction enzymes to produce a recombinant vector in which the existing 5'UTR:gBiP:GB1 region was replaced with DNA fragment (3), mU10In:5'UTR:cBiP.

[0171]

[0172] 1-3. P38 expression vector

[0173] To achieve high expression of the bSA fusion recombinant protein, a previously constructed and reported P38 expression vector was used to express the Turnip crinkle virus P38 protein, which has been reported to suppress the antiviral silencing response of foreign genes (derived from viruses) in the host plant and help mass-produce the recombinant protein when co-expressed. (See Kumari et al., Plant Cell Rep. 39, 1317-1329. 10.1007 / s00299-020-02566-4)

[0174]

[0175] 1-4. CRT expression vector

[0176] To achieve high expression of bSA fusion recombinant proteins, a previously constructed and reported CRT expression vector was used to enable expression in the endoplasmic reticulum (ER) of CRT, which has been reported to help plants produce large quantities of recombinant proteins in a healthy manner by reducing ER stress when co-expressed with the bSA fusion recombinant protein. (See Plant Biotechnology Journal (2022) 20, pp. 2298-2312 / Figure 2a / doi: 10.1111 / pbi.13908.)

[0177]

[0178] The base sequences used in Example 1 are as shown in Table 1.

[0179]

[0180] [Table 1]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186] Example 2. Expression of bSA fusion recombinant protein

[0187] In order to express the recombinant vector constructed in Example 1 in the leaves of a plant (N. benthamiana), Agrobacterium transformed with the recombinant vector constructed above was secured and cultured overnight in a 5 ml medium. 1 ml of this was taken and added to 50 ml of LB medium containing 50 μg kanamycin and 50 μg rifampicin, and cultured for 16 hours.

[0188] Afterwards, the cells were pelleted by centrifugation at 4,500 xg for 8 minutes at 4℃. The cells were suspended in a buffer solution (10 mM MES, 10 mM MgSO4, pH 5.7), diluted to an OD600 of 0.8, and acetosyringone was added to 400 μM, and incubated for 3 hours to prepare an Agrobacterium suspension containing bovine serum albumin.

[0189] Agrobacterium suspensions transformed with P38 or CRT were also prepared in the same manner and mixed with Agrobacterium suspensions containing bovine serum albumin in a volume ratio of 1:1 (bSA:P38, -) ​​or 4:4:2 (bSA:P38:CRT, CRT). Expression of the recombinant protein was induced through transient expression by infiltrating the leaves of 4-5 week-old Nicotiana benthamiana plants using a 1 ml syringe with the needle removed.

[0190] Infiltrated leaves were harvested 5 and 7 days post-infiltration (dpi), completely ground in liquid nitrogen, and a 5-volume buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 1% protease inhibitor cocktail) was added to prepare total soluble protein extracts. The extracted total soluble proteins were developed by SDS-PAGE and stained with Coomassie brilliant blue (CBB) staining solution (CBB, 0.1%; methanol, 50%; glacial acetic acid, 10%) for 20 min. After staining, the stained proteins were destained with a washing solution containing 40% methanol and 10% glacial acetic acid, and the expression levels of the stained proteins were compared using an LAS3000 imaging system (Fuji, Japan).

[0191] As a result, a clear GB1:bSA:EPEA band at 75 kDa according to Example 1-1 (see Fig. 1) and a bSA:EPEA band of a slightly smaller size than 70 kDa according to Example 1-2 (see Fig. 2) were confirmed. In particular, bovine serum albumin showed high expression even under conditions without the GB1 domain, and it was found that this expression showed a synergistic effect when the GB1 domain was present (G).

[0192]

[0193] Example 3. Vector design for expression of bSA and FGF-1 or IL4 fusion recombinant proteins.

[0194] To improve production efficiency in plants, a recombinant gene was constructed by fusing the growth factor FGf-1 or cytokine IL4, known as a protein overexpressed in plants, with bSA.

[0195] In this case, a recombinant gene was constructed using wild-type bovine serum albumin (bSA) consisting of six domains, variant 1 (bSA110) consisting of 110 amino acid residues including one N-terminal domain of wild-type bovine serum albumin, and variant 2 (bSA300) consisting of 300 amino acid residues including three N-terminal domains of wild-type bovine serum albumin, in which the 5'UTR, gBiP, and GB1 domains were sequentially fused at the N-terminus, and an 8xhis tag, two linkers, and an enterokinase recognition sequence and either FGF-1, one of the growth factors, or interleukin-4 (IL4), one of the cytokines, were sequentially fused at the C-terminus. As a control, a recombinant gene without bovine serum albumin protein fusion was also constructed.

[0196] Specifically, a 1752 bp wild-type base sequence of bovine serum albumin including the stop codon was chemically synthesized (by request from Gene universal) and used as a template, with a forward primer having a BamHI restriction enzyme sequence at the 5' end and a reverse primer having a XmaI restriction enzyme sequence at the 3' end, to PCR amplify bovine serum albumin of various lengths. At this stage, the transcription stop codon of bovine serum albumin was replaced with a base sequence encoding an 8xHis tag and a linker (GGGSPR) (indicated as L in the vector map).

[0197] The amplified DNA fragment was inserted into the vector constructed in Example 1-2 using BamHI and XmaI restriction enzymes. Thereafter, FGF-1 and IL4 gene sequences with XmaI and XhoI cleavage sequences added to the 5' and 3', respectively, were chemically synthesized (commissioned by Gene universal) and inserted into the bSA C-tem position using the two restriction enzymes to construct a recombinant vector. The base sequences additionally used for constructing the recombinant gene are as shown in Table 2 below.

[0198]

[0199] [Table 2]

[0200]

[0201]

[0202]

[0203]

[0204] Example 4. Confirmation of bSA and growth factor or cytokine fusion recombinant protein

[0205] In Example 3, the recombinant vector produced was mixed in a 4:4:2 ratio with an Agrobacterium suspension transformed with a recombinant vector for expressing each of growth factors or cytokines, P38, and CRT in the same manner as in Example 2, and then the mixture was infiltrated into the mesophyll tissue of N. benthamiana to temporarily induce expression. Seven days after infiltration, the leaves were harvested, and the total soluble protein was extracted. 1 μg of this was separated from a 10% SDS-PAGE gel, transferred to a PVDF membrane, and blocked with 5% skim milk. Thereafter, the mixture was reacted with an anti-His-Peroxidase antibody (Sigma A7058). The ECL solution was then treated according to the manufacturer's instructions. In addition, CBB staining was performed in the same manner as in Example 2.

[0206] As a result, expression of FGF-1 not bound to bovine serum albumin was not confirmed, whereas expression of FGF-1 bound to full-length or fragment bSA was confirmed in plants, and among them, the expression level of FGF-1 fused to full-length bSA was significantly higher. In addition, expression of IL4 not bound to bovine serum albumin was not confirmed, but expression of IL4 fused to full-length or fragment bSA was confirmed in plants, and among them, the expression level of the fusion protein bound to bSA300 was the highest.

[0207] In particular, the expression level of these two fusion proteins was so high that expression could be confirmed with the naked eye even through CBB staining, and this showed that when bSA is fused to produce growth factors and cytokines in plants, the production efficiency can be significantly improved (Fig. 4).

[0208]

[0209] Example 5. Vector design for expression of bSA and various growth factor or cytokine fusion recombinant proteins.

[0210]

[0211] We further verified whether other various growth factors and cytokines could improve production efficiency in plants when fused with bSA.

[0212] To this end, genes encoding growth factors or cytokines, as described in Table 3 below, were chemically synthesized by adding a linker and an enterokinase recognition sequence to the N-terminus and optionally adding a base sequence encoding an ER retention sequence (HDEL) to the C-terminus (Gene Universal). In this case, XmaI and XhoI cleavage sequences were added to the 5'- and 3'-terminals of the DNA fragments, respectively, for synthesis.

[0213]

[0214] [Table 3]

[0215]

[0216]

[0217] [Table 4]

[0218]

[0219]

[0220]

[0221]

[0222] The DNA fragment produced in this way was cut with XmaI and XhoI and inserted into the vector produced in Example 3, which was cut with the same restriction enzymes.

[0223]

[0224] In the same manner as in Example 2, the Agrobacterium suspension transformed with a recombinant vector for expressing each of the growth factors, P38, and CRT was mixed in a 4:4:2 ratio, and then infiltrated into N. benthamiana mesophyll tissue to temporarily induce expression. Leaves were harvested on days 3, 5, and 7 after infiltration, and 5 μg of total soluble protein was subjected to SDS-PAGE analysis, followed by Western blotting analysis in the same manner as in Example 4.

[0225]

[0226] As a result, it was confirmed that all growth factors (FGF-1, GMCSF, LIF, bIGF) and cytokines (IL4, IL15, IFNa, IFNb) used in the experiment could be expressed with high efficiency in plants when fused with bSA (Fig. 5, Fig. 6).

[0227]

[0228] Example 7. Confirmation of growth factor or cytokine release through enterokinase treatment

[0229] We sought to determine whether a 17 kDa growth factor could be released from multiple domains linked to the N-terminus of the recombinant protein by treating the recombinant protein with enterokinase.

[0230] For this purpose, the recombinant protein of growth factor (bSA binding-95 kDa, bSA300 binding-63 kDa) bound to bSA or bSA300 was Ni 2+-Then, the protein was separated according to the manufacturer's method using NTA affinity resin (Qiagen, 30230) and suspended in enterokinase buffer (20 mM Tris-HCl, pH 7.4, 2 mM CaCl2, 50 mM NaCl). Then, 0.01 or 0.1 unit of enterokinase (Genecell Biotech, Jeonju, E001) was added, and the cleavage reaction was performed at 37℃ for 1 hour. After the cleavage reaction, the protein was developed by SDS-PAGE, and Coomassie brilliant blue (CBB) staining confirmed that it was accurately cleaved by EK at the EK site between bSA or bSA300 and the target protein (Fig. 7).

[0231]

[0232] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A DNA construct or recombinant vector comprising a sequence encoding albumin and a sequence encoding a non-expressed protein.

2. A DNA construct or recombinant vector according to claim 1, wherein the in vitro expressed protein is a protein having a molecular weight of 5 to 20 kDa.

3. A DNA construct or recombinant vector according to claim 1, wherein the overexpressed protein is a growth factor or cytokine.

4. In the third paragraph, the overexpressed protein is a DNA construct or recombinant vector selected from the group consisting of FGF-1 (fibroblast growth factor), FGF-21, GMCSF (granulocyte macrophage colony stimulating factor), LIF (leukemia inhibitory factor), IGF (insulin-like growth factor), interleukin, interferon, EGF (epidermal growth factor), human stem cell factor, glial cell line-derived neurotropic factor, vascular endothelial growth factor, activin A, bone morphogenetic proteins, SHH (sonic hedgehog protein), and TGF (transforming growth factors).

5. A DNA construct or recombinant vector according to claim 1, wherein the albumin is serum albumin.

6. A DNA construct or recombinant vector in claim 5, wherein the serum albumin is bovine serum albumin, and the bovine serum albumin is a wild type (bSA), a mutant (bSA110) including domain 1 from the N-terminus, or a mutant (bSA300) including domains 1 to 3 from the N-terminus.

7. A DNA construct or recombinant vector according to claim 1, further comprising a sequence encoding a peptide linker between the sequence encoding the albumin and the sequence encoding the in vitro expressed protein.

8. A DNA construct or recombinant vector according to claim 1, further comprising a sequence encoding an enzyme-cleaved peptide between the sequence encoding the albumin and the sequence encoding the in vitro expressed protein.

9. A DNA construct or recombinant vector according to claim 1, wherein the DNA construct or recombinant vector further comprises at least one sequence selected from the group consisting of a sequence encoding a GB1 domain, a sequence encoding an endoplasmic reticulum leader peptide, and a sequence encoding an endoplasmic reticulum retention peptide.

10. In the first paragraph, the DNA construct or recombinant vector (i) the first intron sequence of the modified ubiquitin10; (ii) 5' UTR sequence; and (iii) A DNA construct or recombinant vector further comprising at least one sequence selected from the group consisting of sequences encoding a purification tag.

11. A transformed plant cell or transformed plant into which the DNA construct or recombinant vector of any one of claims 1 to 10 has been introduced.

12. In claim 11, the transformed plant cell or transformed plant is a transformed plant cell or transformed plant into which a recombinant vector for expressing human calreticulin (CRT) and a p38 recombinant vector are additionally introduced.

13. A method for producing a highly expressed protein, comprising the following steps: (a) a step of culturing or growing the transformed plant cell or transformed plant of Article 11; and (b) A step of recovering a non-expressed protein from a lysate obtained by crushing the transformed plant cell or transformed plant that has been cultured or grown, or from a cultured solution.

14. In paragraph 13, A method for producing a non-expressed protein, wherein the step (b) above is to purify an albumin and non-expressed protein fusion protein and treat it with a peptide cleavage enzyme to recover the free non-expressed protein.

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