KR20160123145A - - Human IgG Fc-fusion human -glucocerebrosidase and preparation thereof - Google Patents

Abstract

The present invention relates to a polynucleotide fused with a protein secretion signal sequence, human beta-glucocerebrosidase, and a human IgG Fc fragment, and to a method for mass-producing beta-glucocerebrosidase and human IgG Fc fragment fused proteins by using the same. According to the present invention, a protein secretion signal sequence, human beta-glucocerebrosidase, and human IgG Fc are fused, and thus human beta-glucocerebrosidase can be mass-produced in a plant expression system. Accordingly, fused proteins can be easily qualitatively and quantitatively analyzed, and blood half-life thereof can be improved. Thus, the present invention can be useful in producing improved human beta-glucocerebrosidase.

Description

Human beta-glucocerebrosidase and preparation thereof, which comprises fusing a human IgG Fc fragment,

The present invention relates to a method for mass production of human beta-glucocerebrosidase and human IgG Fc fragment fusion protein using a polynucleotide fused with a protein secretion signal sequence, human beta-glucocerebrosidase and human IgG Fc fragment, .

A lysosomal storage disorder is a collective term for diseases in which the remaining substrate is not degraded by the genetic abnormality of lysosomal enzymes and accumulates in the lysosomes, resulting in impaired cell function. The treatment of lysosomal accumulation diseases is done with enzyme replacement therapy. Enzyme replacement therapy refers to a therapy that replaces a specific enzyme deficient in a lysosomal disease patient with a functional enzyme synthesized in vitro. These diseases are known to be more effective when treated in infancy. Currently, lysosomal storage diseases that can be substituted for enzymes include Gaucher disease, Fabry disease, Pompe disease, Mucopolysaccharidosis (MPS) type 1, 2 and 6. Among them, Gaucher disease is characterized by a deficiency of the lysozyme enzyme glucocerebrocidase.

Glucocerebrosidase is a membrane-bound lysosomal enzyme that produces glucose and ceramide by hydrolyzing glucoserine glucocerebroside (glucosylceramide, GlcCer), and is a thermoregulatory disorder (chromosome 1 q21-q31 ) Causes deficiency. Therefore, if glucocerebrosidase can be mass produced by recombinant technology, it is likely to be used as an alternative treatment for Gaucher's disease. In commercial terms, it is preferable to produce low cost eukaryotic cells such as prokaryotic cells such as E. coli or yeast, but in this case, it has been a limitation that rGCRs which are non-glycosylated or changed in sugar pattern are induced. Therefore, there is still a need for a new technique for obtaining large quantities of glucocerebrosidase.

In the past 15 to 20 years, intensive studies have been conducted on culturing microorganisms and animal cells. However, expression systems capable of mass production of proteins using plant cell cultures are being studied. The use of transgenic plant or plant cell cultures for the production of pharmaceuticals, functional proteins, industrial enzymes and functional secondary metabolites has recently been referred to as molecular farming.

Plant cell culture has several advantages when compared with animal cell culture and microorganisms. Compared to microorganisms, since plant cells are eukaryotic cells, they can perform almost any post-translational modification and can maintain the biological activity of the produced recombinant protein similar to the native form. Particularly superior to animal cell systems is that there is no risk of contamination, as plant cells do not have any human pathogens. In addition, the cell culture conditions of the plant cells are simpler and cheaper than those of the animal cells. Thus, proteins produced by plant cell cultures have proven to be safer than prokaryotic or animal cell systems, particularly in the production of proteins used in the treatment of human diseases (Doran, 2000. Curr. Opin. Biotech. 11: 199-204).

In the case of plant cell culturing, when the plant protein is secreted out of the plant cell using the plant suspension cell culture, the yield of the recombinant protein secreted out of the plant cell is 0.05-0.5 of the total cell exocrine protein (James et al., 2000, Protein Expr. Purif. 19: 131-138). These low yields are generally known to be caused by the denaturation of proteins released from plant cells by physical factors such as pH, salinity, etc. in the medium or degradation of protein by proteolytic enzymes. Therefore, there is a need for a method for culturing a plant cell capable of mass-producing a recombinant protein by overcoming such a problem. There is a need for a culture method for effectively mass-producing and obtaining glucocerebrosidase in plant cells.

When the protein secretion signal sequence, the human IgG Fc fragment, and the human beta-glucocerebrosidase are fused and expressed in a plant expression system, the present inventors have found that human beta-glucocerebrosidase fused with human IgG-Fc fragments And the present invention has been completed.

Accordingly, an object of the present invention is to provide a polynucleotide fused with a protein secretion signal sequence, human beta-glucocerebrosidase, and human IgG Fc fragment.

Another object of the present invention is to provide a method for producing a vector comprising the polynucleotide, a plant transformed cell transformed with the vector, a human beta-glucocerebrosidase and a human IgG Fc fusion protein in the plant transformed cell .

It is another object of the present invention to provide a recombinant fusion protein produced by the above method.

In order to achieve the above object, the present invention provides a polynucleotide in which a protein secretion signal sequence, human beta-glucocerebrosidase, and human IgG Fc fragment are fused.

The present invention also provides a vector comprising the polynucleotide.

The present invention also provides a plant transformed cell transformed with said vector.

In addition,

1) preparing a vector comprising a protein secretion signal sequence, a polynucleotide fused with human beta-glucocerebrosidase and a human IgG Fc fragment;

2) preparing a plant transformed cell transformed with the vector of step 1); And

3) obtaining a human beta-glucocerebrosidase fused with a human IgG Fc fragment from the plant transformed cells of step 2), wherein the human beta-glucocerebrosidase is fused with human beta-glucocerebrosidase, beta-glucocerebrosidase).

The present invention also provides human beta-glucocerebrosidase and human IgG Fc fusion recombinant protein produced by the above method.

The present invention enables the mass production of human beta-glucocerebrosidase in a plant expression system by fusing a protein secretion signal sequence, human beta-glucocerebrosidase, and human IgG Fc, so that the qualitative and quantitative analysis And its half life of blood can be improved, so that it can be usefully used for the production of improved human beta-glucocerebrosidase.

1 is a diagram showing the structure of a recombinant vector comprising a fusion gene of a protein secretion signal sequence (RAmy1ASP), human beta-glucocerebrosidase (GCD), and a human IgG Fc fragment.
FIG. 2 is a diagram showing a backbone of the expression vector of FIG. 1; FIG.
FIG. 3 is a graph showing the results of PCR to determine whether the fusion polynucleotide was introduced into an expression vector and whether the expression vector was introduced into Agrobacterium (13-1: cell line).
FIG. 4 is a graph showing the results of quantitative analysis of total protein amount and target protein amount obtained from the transformed cell line (13_2, 13_4, 13_5, 13_6, 13_7, 13_8, 13_12, 13_14, 13_20, 13_22, 13_25: .
FIG. 5 is a graph showing the results of measurement of volumetric productivity of recombinant proteins in seven cell lines selected from the results of FIG. 4 (13-5, 13-6, 13-7, 13-8, 13-8, 12, 13-14, 13-20: cell line).
FIG. 6 is a diagram showing a whole process for producing a recombinant protein fused with human IgG Fc fragment and human beta-glucocerebrosidase through transgenic rice cell culture.

The present invention provides a polynucleotide fused with a protein secretion signal sequence, human β-glucicerebrosidase (hereinafter referred to as "GCD"), and human IgG Fc fragment (human IgG Fc fragment).

The fusion polynucleotide fuses human IgG Fc fragment and protein secretion signal sequence to enable mass production of recombinant protein in plant cells.

The " protein secretion signal sequence " of the present invention is a signal sequence that causes the recombinant protein to be secreted out of the cell. Because the recombinant protein is secreted out of the cell after the expression of the signal sequence, the cell can be efficiently purified without rupturing the cell, and can be located in front of the gene encoding the recombinant protein. The protein secretion signal sequence may include a signal peptide gene sequence of Rice α-amylase 1A (RAmy1A), which is the sequence described in SEQ ID NO: 1, but is not limited thereto.

In the present invention, " GCD " is a membrane-bound lysosomal enzyme which hydrolyzes glucosin and glucocerebroside (glucocel ceramide, GlcCer) to produce glucose and ceramide. The GCD sequence of the present invention may include, but is not limited to, the sequence described in SEQ ID NO: 2.

The " Fc fragment " in the human IgG Fc fragment of the present invention is one of the functional fragments of the antibody molecule, and the gene encoding the human IgG Fc fragment may be a sequence known in the art.

The invention also provides a vector comprising a polynucleotide fused with a protein secretory signal sequence, a GCD and a human IgG Fc fragment.

The term "vector" in the present invention is used to refer to a DNA fragment (s), a nucleic acid molecule, which is transferred into a cell. The vector replicates the DNA and can be independently regenerated in the host cell. Vector refers to a recombinant DNA molecule comprising a desired coding sequence and a suitable nucleic acid sequence necessary for expressing a coding sequence operably linked in a particular host organism. Promoters, enhancers, termination signals, and polyadenylation signals available in eukaryotic cells are known.

The vector may further comprise a promoter, 3 'UTR (untranslated region).

In the present invention, the promoter may be native, homologous, foreign or heterologous to the host. Outpatient means a transcription initiation region not found in a wild-type host into which a transcription initiation region is introduced. The promoter can be selected according to the desired result and suitable plant promoters known in the art can be used. The plant promoter may be a promoter of Cauliflower Mosaic Virus 35S promoter, opine synthetase promoter (nos, mas, ocs etc.), ubiquitin promoter, actin promoter, ribulose bisphosphate (RubP) carboxyl 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 26, 85), tomato spotted wilt virus, tobacco rattle virus, tobacco necrotic virus, tobacco ring spot virus, tomato ring spot virus, cucumber mosaic virus, peanut stump virus peanut stump virus, alfalfa mosaic virus, sugarcane-like viral virus (sugarca amylase 3D (RAmy3D) promoter of rice, but is not limited thereto, and preferably includes a promoter of Rice alpha-amylase 3D (RAmy3D) promoter of rice, Lt; / RTI > The alpha-amylase may be alpha-amylase 3D of rice, alpha-amylase 3E of rice, or alpha-amylase IA of rice, but is not limited thereto.

In the present invention, the 3 'UTR region means an mRNA region that is not translated into a protein after the stop codon on the mRNA.

The present invention also provides a plant transformed cell transformed with said vector.

The plants used for the production of plant transformed cells according to the present invention may be any plants regardless of dicots or monocotyledons. Preferably, the plants are selected from tobacco, rice, wheat, barley, potatoes, tomatoes, lettuce, Lt; RTI ID = 0.0 > and / or < / RTI >

The plant cells used in the production of plant transformed cells in the present invention may be any plant cells, but may include cultured cells, cultured tissues, culture or whole plants, and preferably cultured cells, cultured tissues or culture media , More preferably any form of cultured cells is possible.

The present invention also provides a method for mass production of GCD and human IgG Fc fusion proteins in the plant transformed cells.

The mass-

1) preparing a vector comprising a protein secretory signal sequence, a GCD and a polynucleotide fused with a human IgG Fc fragment;

2) preparing a plant transformed cell transformed with the vector of step 1); And

3) obtaining a GCD in which the human IgG Fc fragment is fused from the plant transformed cells of step 2); .

The mass production method of the present invention facilitates the secretion by the protein secretory signal sequence and facilitates the mass production and purification of the recombinant protein fused with human IgG Fc fragment and GCD despite the use of the plant cell culture system .

The present invention also provides a recombinant fusion protein produced by the above method.

The recombinant fusion protein is a fusion protein of GCD and human IgG Fc fragment. The recombinant protein can be obtained by purifying the recombinant protein expressed in a conventional suspension cell, and can be obtained by various purification methods depending on the kind of the recombinant protein.

The mass-produced recombinant protein in the present invention can be usefully used as an enzyme replacement therapy for lysosomal accumulation diseases.

The recombinant protein may be characterized in that the C-terminal of the protein secretion signal sequence is fused to the N-terminus of GCD and the N-terminus of the IgG Fc fragment is fused to the C-terminus.

Terms not otherwise defined herein have meanings as commonly used in the art to which the present invention belongs.

Hereinafter, the present invention will be described in detail with reference to examples. However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the following.

Example 1 - Construction of a vector for fusion protein expression

A recombinant vector comprising a fusion protein of the protein secretory signal sequence, GCD and human IgG Fc fragment was prepared and its structure is shown in FIG. 1 briefly. As a backbone of the expression vector, the pMYN409 vector was used and it is shown in Fig. The pMYN409 vector is an inducible promoter induced by glucose deprivation.

More specifically, the signal sequence gene of Rice α-amylase 1A (RAmy1A) was inserted in the N-terminal part of the GCD gene to promote the secretion of the protein, and the overlapping PCR of the human IgG Fc fragment was performed at the C- To construct a plant expression vector. Fusion of human IgG Fc fragments was performed for the purpose of facilitating purification and analysis of GCD and enhancing blood half-life and stability of GCD. The plant expression vector was used for further experiments.

The primers used for the cloning are shown in Table 1 below.

Example 2 - Preparation of recombinant plant transformed cells and suspension culture

2.1 Preparation and Screening of Recombinant Plant Transformed Cells

The seeds of rice (Oryza sativa L. cv Dongjin) were treated with 70% ethanol and 2% lactose and placed on callus induction medium. Agrobacterium tumefaciens transformed with a polynucleotide fused with a protein secretion signal sequence, GCD, and a human IgG Fc fragment were co-cultured with callus to induce callus transformation, thereby transforming the recombinant plant Cells were prepared. It was confirmed by a PCR method that the fused polynucleotide was introduced and is shown in Fig.

(2 mg / L), sucrose (30 g / L), MES (0.5 g / L) and L-proline (0.5 g / L) in order to confirm the introduction of the fusion gene. L-glutamine (0.5 g / l), casamino acid (0.3 g / l), myo-inositol (0.01 g / l), hygromycin B (40 mg / Lt; RTI ID = 0.0 > N6 < / RTI > Thereafter, a callus into which a polypeptide having a fusion of a protein secretory signal sequence, GCD and IgG Fc fragment was introduced was selected by selecting a callus resistant to hygromycin.

2.2 Suspension Culture

N6 medium (Duchefa) was used for the growth and maintenance of the transformed rice cells obtained in Example 2.1 above. (2,4-D), 0.2 mg / l kinetin, 0.1 mg / l gibberellic acid (gibberellic acid) as a growth regulator, (0.5 g / l), L-glutamine (0.5 g / l), casamino acid (0.3 g / l) After addition of myo-inositol (0.01 g / l), the pH of the medium was adjusted to 5.8. The prepared medium was dispensed into a 500 ml Erlenmeyer flask and autoclaved at 121 占 폚 and 1 atm. 40 mg / l hygromycin was filter sterilized with a 0.22 占 퐉 membrane filter (Millipore, USA). Rice cells suspended in N6 medium were cultured in shaking incubator at 28 ° C, 120 rpm, and dark, and subculture was carried out at intervals of 9 days to cultivate plant transformed cells.

Example 3 - Analysis of human beta-glucocerebrosidase and human IgG Fc fusion recombinant protein

The callus resistant to hygromycin B was selected from the culture broth obtained in Example 2.2, recovered in a sucrose-free N6 medium for 7 days, and then recovered. Cells were disrupted using liquefied nitrogen and resuspended in PBS medium containing protease inhibitor, followed by centrifugation to collect only the supernatant. For the quantitative analysis of GCD and human IgG Fc fusion recombinant proteins, Bradford assay was used for the total protein content and ezyme-linked immunosorbent assay (ELISA) for the target protein content. In order to perform the sandwich ELISA, goat anti-human IgG (Fc) (KPL, USA) was used as the primary antibody and secondary antibody was peroxidase-labeled goat anti-human IgG KPL, USA) was used. Substrates were used as ABTS peroxidase substrate (KPL, USA) and the degree of color development was measured by absorbance at 405 nm. ImmunoPure® human IgG (Pierce, USA) was used as a standard.

The recombinant proteins were quantitatively analyzed in various cell lines as described above, and cell lines having a high amount of the target protein were selected according to the total protein amount. The results are shown in FIG. The volumetric productivity of the seven selected cell lines was measured and the results are shown in FIG.

As shown in FIGS. 4 and 5, although the plant cell culture method was used, it was confirmed that the recombinant protein of GCD-human IgG Fc could be mass produced by fusion of the protein secretory signal sequence and the human IgG Fc fragment. In addition, it was confirmed that quantification and qualitative analysis of the target protein were facilitated by fusion of the human IgG Fc fragment.

<110> INHA-INDUSTRY PARTNERSHIP INSTITUTE <120> Human IgG Fc-fusion human beta-glucocerebrosidase and preparation          the <130> 1_155P <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 93 <212> DNA <213> Oryza sativa <400> 1 atgcaggtgc tgaacaccat ggtgaacaaa cacttcttgt ccctttcggt cctcatcgtc 60 ctccttggcc tctcctccaa cttgacagcc ggg 93 <210> 2 <211> 2193 <212> DNA <213> Homo sapiens <400> 2 gcccgcccct gcatccctaa aagcttcggc tacagctcgg tggtgtgtgt ctgcaatgcc 60 cctccgctat 120 ggagtacac gcagtgggcg acggatggag ctgagtatgg ggcccatcca ggctaatcac 180 acgggcacag gcctgctact gaccctgcag ccagaacaga agttccagaa agtgaaggga 240 tttggagggg ccatgacaga tgctgctgct ctcaacatcc ttgccctgtc accccctgcc 300 caaaatttgc tacttaaatc gtacttctct gaagaaggaa tcggatataa catcatccgg 360 gtacccatgg ccagctgtga cttctccatc cgcacctaca cctatgcaga cacccctgat 420 gatttccagt tgcacaactt cagcctccca gaggaagata ccaagctcaa gatacccctg 480 attcaccgag ccctgcagtt ggcccagcgt cccgtttcac tccttgccag cccctggaca 540 tcacccactt ggctcaagac caatggagcg gtgaatggga aggggtcact caagggacag 600 cccggagaca tctaccacca gacctgggcc agatactttg tgaagttcct ggatgcctat 660 gctgagcaca agttacagtt ctgggcagtg acagctgaaa atgagccttc tgctgggctg 720 ttgagtggat accccttcca gtgcctgggc ttcacccctg aacatcagcg agacttcatt 780 gcccgtgacc taggtcctac cctcgccaac agtactcacc acaatgtccg cctactcatg 840 ctggatgacc aacgcttgct gctgccccac tgggcaaagg tggtactgac agacccagaa 900 gcagctaaat atgttcatgg cattgctgta cattggtacc tggactttct ggctccagcc 960 aaagccaccc taggggagac acaccgcctg ttccccaaca ccatgctctt tgcctcagag 1020 gcctgtgtgg gctccaagtt ctgggagcag agtgtgcggc taggctcctg ggatcgaggg 1080 atgcagtaca gccacagcat catcacgaac ctcctgtacc atgtggtcgg ctggaccgac 1140 tggaaccttg ccctgaaccc cgaaggagga cccaattggg tgcgtaactt tgtcgacagt 1200 cccatcattg tagacatcac caaggacacg ttttacaaac agcccatgtt ctaccacctt 1260 ggccacttca gcaagttcat tcctgagggc tcccagagag tggggctggt tgccagtcag 1320 aagaacgacc tggacgcagt ggcactgatg catcccgatg gctctgctgt tgtggtcgtg 1380 ctaaaccgct cctctaagga tgtgcctctt accatcaagg atcctgctgt gggcttcctg 1440 gagacaatct cacctggcta ctccattcac acctacctgt ggcatcgcca ggcagagccc 1500 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 1560 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 1620 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 1680 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 1740 agcacgtacc gggtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1800 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1860 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 1920 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 1980 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 2040 ctggactccg acggctcctc cttcctctac agcaagctca ccgtggacaa gagcaggtgg 2100 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 2160 cagaagagcc tctccctgtc tccgggtaaa tga 2193

Claims (9)

A protein secretion signal sequence, a polynucleotide fused with human beta-glucocerebrosidase and a human IgG Fc fragment.
2. The polynucleotide of claim 1, wherein the protein secretion signal sequence is SEQ ID NO:
2. A polynucleotide according to claim 1, wherein the human beta-glucocerebrosidase is SEQ ID NO: 2.
A vector comprising the polynucleotide of claim 1.
A plant transformed cell transformed with the vector of claim 4.
The plant transfected cell according to claim 5, wherein the plant is at least one plant selected from the group consisting of tobacco, rice, wheat, barley, potato, tomato, lettuce, corn and Arabidopsis.
1) preparing a vector comprising a protein secretion signal sequence, a polynucleotide fused with human beta-glucocerebrosidase and a human IgG Fc fragment;
2) preparing a plant transformed cell transformed with the vector of step 1); And
3) obtaining a human beta-glucocerebrosidase fused with a human IgG Fc fragment from the plant transformed cells of step 2), wherein the human beta-glucocerebrosidase is fused with human beta-glucocerebrosidase, beta-glucocerebrosidase).
Human beta-glucocerebrosidase and human IgG Fc fusion recombinant protein produced by the method of claim 7.
9. The method according to claim 8, wherein the C-terminal of the protein secretion signal sequence is fused to the N-terminus of the human beta-glucocerebrosidase and the N-terminus of the IgG Fc fragment is fused to the C-terminus of the human beta-glucocerebrosidase , Recombinant protein.

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