Patent Application
20210108214
The present invention relates to a recombinant vector carrying a disulfide bond isomerase signal peptide and a use thereof.
This application claims priority to and the benefit of Korean Patent Application No. 10-2018-0010055 filed in the Korean Intellectual Property Office on Jan. 26, 2018, and all the contents disclosed in the specification and drawings of that application are incorporated in this application.
In order to mass-produce recombinant proteins for various purposes such as research, treatment, or other commercial purposes, various vectors and hosts are currently used, and among them, a method for expressing a required protein using an E. coli expression system, and then purifying the protein has been usefully utilized because a large amount of protein can be obtained with less cost and effort.
However, since there is a difference in intracellular environment between mammals and bacteria, when efforts are made to obtain various types of human proteins using an E. coli system, specific expression and purification conditions for each protein need to be established. In particular, amino acid sequences of all proteins produced from E. coli start with methionine (Met), and in contrast, proteins found in the human body or expressed from advanced organisms are degraded by proteases through post-translational modification and activated or subjected to various protein modifications such as glycosylation which attach sugar chains, phosphorylation, acetylation, and carboxylation. Therefore, amino acid sequences of many commercialized proteins do not start with methionine, and accordingly, proteins produced from E. coli are disadvantageous in that the proteins need to be subjected to an additional process such as proteolytic cleavage by enzymatic reactions.
Meanwhile, thymosin beta 4 (TB4) is known to exert efficacy in neovascularization, wound healing, hair growth, and the like, and thus is currently experiencing clinical trials and FDA approval reviews for use as a therapeutic agent for various diseases such as ischemic heart disease, corneal injury, ophthalmic diseases, and epidermolysis bullosa, and is expected to be developed as a therapeutic agent for alopecia.
Since thymosin beta 4 is highly available as a therapeutic agent for various diseases as described above, it is expected that there will be huge demand when thymosin beta 4 is released as a therapeutic peptide in the future, but there are problems in that when the peptide is produced by synthesis, high production costs are required, and when the peptide is expressed from a host (host strain) such as E. coli, additional work such as cleavage by enzymatic reactions after production in order to modify a protein sequence which starts with methionine is required, but research on this is still insufficient.
The present inventors have made intensive efforts to solve the problem in the related art as described above, and as a result, confirmed that when a recombinant vector comprising a disulfide bond isomerase signal peptide was used, a protein whose desired amino acid sequence does not start with methionine could be produced through an E. coli expression system, thereby completing the present invention based on this.
Thus, an object of the present invention is to provide a recombinant vector comprising a base sequence encoding a disulfide bond isomerase signal peptide.
Further, another object of the present invention is to provide a recombinant vector comprising base sequences encoding a disulfide bond isomerase signal peptide and thymosin beta 4.
In addition, still another object of the present invention is to provide a transformant transformed with the recombinant vector.
Furthermore, yet another object of the present invention is to provide a method for producing a thymosin beta 4 protein, the method comprising: transforming E. coli with the recombinant vector, and then culturing the E. coli in a medium.
However, technical problems to be achieved by the present invention are not limited to the aforementioned problems, and other problems that are not mentioned may be clearly understood by those skilled in the art from the following description.
The present invention relates to a recombinant vector comprising a disulfide bond isomerase signal peptide and a use thereof, and the present invention provides a recombinant vector comprising a base sequence encoding a disulfide bond isomerase signal peptide.
Further, the present invention provides a recombinant vector comprising base sequences encoding a disulfide bond isomerase signal peptide and thymosin beta 4.
In addition, the present invention provides a transformant transformed with the recombinant vector.
Furthermore, the present invention provides a method for producing a thymosin beta 4 protein, the method comprising: transforming E. coli with the recombinant vector, and then culturing the E. coli in a medium.
When a recombinant vector comprising a disulfide bond isomerase signal peptide of the present invention is used, it is possible to produce a protein whose amino acid sequence does not start with methionine through an E. coli expression vector, and accordingly, there is an advantage in that the recombinant vector does not need to be subjected to an additional process such as a protease treatment to be commercially used. In particular, although thymosin beta 4 has been highly available as a therapeutic agent for various diseases for a while, thymosin beta 4 has a problem in that when thymosin beta 4 is expressed in a host (host strain), thymosin beta 4 is degraded by intracellular proteases and cannot be produced. The present invention enables the thymosin beta 4 to be produced through an E. coli expression system, and thus to be usefully utilized in the development of a therapeutic agent for alopecia as well as ischemic heart disease, corneal injury, ophthalmic diseases, and epidermolysis bullosa, and further, the E. coli expression system of the present invention can be applied to production of various therapeutic proteins such as a parathyroid hormone, a human growth hormone, and a granulocyte-colony stimulating factor (GCSF) used for the treatment of osteoporosis, and thus is expected to be widely used as a useful technique.
The present inventors confirmed that when a recombinant vector comprising a disulfide bond isomerase signal peptide was used, a protein whose amino acid sequence does not start with methionine could be produced through an E. coli expression system, thereby completing the present invention based on this.
Hereinafter, the present invention will be described in detail.
The present invention provides a recombinant vector comprising a base sequence encoding a disulfide bond isomerase signal peptide.
Further, the present invention provides a recombinant vector comprising a base sequence of SEQ ID NO: 7 in which thymosin beta 4 is linked to the carboxyl terminus of the disulfide bond isomerase signal peptide.
As used herein, the “vector” refers to DNA capable of being proliferated by introducing a target DNA fragment into a host bacterium and the like in a DNA recombination experiment, and is also called a cloning vehicle, wherein vector DNA is cleaved by a restriction enzyme and the like to open the ring, and the target DNA fragment is inserted and linked to the vector for introduction into the host bacterium. As the host bacterium is proliferated, the vector DNA linked to the target DNA fragment is replicated and distributed to each daughter cell hand in hand the division of bacteria to maintain the target DNA fragment from generation to generation, and a plasmid and a phage chromosome are usually used.
As used herein, the “plasmid” is a generic term for a gene which is stably maintained and transmitted to progeny throughout generations in cells, which exists independently of chromosomes and autonomously propagates, but DNA included in mitochondria or chloroplasts of eukaryotic cells is generally called organelle DNA, and is distinguished from the plasmid. The presence of the gene is not always essential for normal cell survival, but in bacterial cells, the gene has functions such as conjugal transfer (F factor), resistance to antibiotics (R factor), and synthesis of antibacterial materials (bacteriocin) (colicin factor). In addition, there are also those that transform plant cells into tumors, such as the Ti plasmids present in soil bacteria, and various forms, such as those which are co-delivered when deliverability factors coexist with each other even if they do not have their own delivery mechanism or those which are not delivered even though they co-exist have been discovered. The plasmid is often used as a vector in tissue conversion DNA experiments (gene manipulation techniques).
As used herein, the “promoter” refers to a genomic region linked to the upstream part of a structural gene, and serves to regulate the structural gene linked thereto to be transcribed into mRNA. The promoter is activated by binding multiple general transcription factors, and includes a TATA box that universally regulates gene expression, a CAAT box region, a region which affects gene expression in response to external stimuli, an enhancer which promotes the expression of almost all genes regardless of position and direction, and the like. Since the proteins required for basic metabolism of an organism have to maintain a constant concentration in cells, the promoters linked to these genes are always activated only by the action of general transcription factors. In contrast, proteins, which have no role in normal times and are required to function only under special circumstances, are linked to a tissue specific promoter or inducible promoter which induces expression of the corresponding structural gene. That is, the tissue specific promoter is activated by the binding of an activated specific transcription factor during the development process of an organism, and the inducible promoter is activated by the binding of a specific transcription factor activated by an external stimulus from the surrounding environmental factors.
As used herein, the “operator” is also called an operator gene, and serves to regulate the expression of a specific gene during the gene regulation of bacteria. The operator is primarily located next to a structural gene and serves to regulate the expression or non-expression of the required gene under specific circumstances.
As used herein, the “recombinant vector” refers to a gene construct including an essential regulatory element operably linked to express a gene insert as a vector capable of expressing a target protein or a target RNA in a host cell, and refers to plasmids, viruses or other vehicles known in the art, in which gene base sequences encoding a promoter and a target protein operably linked to the promoter can be inserted or introduced.
In addition, the present invention provides a transformant transformed with a recombinant vector prepared by the present invention.
As used herein, the “transformation” means that the trait of an individual or a cell is genetically modified by DNA, which is a genetic material given from the outside.
Another aspect of the present invention provides a method for producing a transformed protein, the method comprising: a) constructing a recombinant vector of the present invention; b) transforming E. coli with the recombinant vector; and c) culturing the E. coli in a medium.
In an exemplary embodiment of the present invention, a plasmid was constructed using a pGE vector and a reference DNA sequence in which thymosin beta 4 (TB4) is linked to the carboxyl terminus (C-term) of a disulfide bond isomerase signal peptide (DsbC-SP) which is a signal peptide (see Example 1).
In another exemplary embodiment of the present invention, transformed E. coli strains BL21 and DH5a were constructed using the constructed plasmid (see Example 2), and the growth of the transformed E. coli strain BL21 was confirmed (see Example 3).
In still another exemplary embodiment of the present invention, it was confirmed that TB4 was expressed in the transformed E. coli strain (see Example 4).
Hereinafter, preferred examples for helping the understanding of the present invention will be suggested. However, the following Examples are provided only to more easily understand the present invention, and the contents of the present invention are not limited by the following Examples.
In order to synthesize a plasmid which allows thymosin beta (TB4) to be expressed, by using a pGE vector and a reference DNA sequence in which TB4 was linked to the carboxyl terminus (C-term) of a disulfide bond isomerase signal peptide (DsbC-SP) which is a signal peptide, a plasmid in which the DNA sequence was inserted into a vector was synthesized.
The base sequences for plasmid synthesis are shown in the following Table 1.
As a result of confirming that the DNA sequence of the synthesized plasmid and the reference DNA sequence were aligned and matched (Http://Multalin.toulouse.inrafr/), the two sequences were 100% identical, as illustrated in
Further, a vector map as illustrated in
A transformed E. coli strain was constructed by performing the following experiment using a plasmid constructed by the method in Example 1.
<2-1> Preparation of LB Medium
In order to prepare a Luria Bertani media broth (LB broth), 800 mL of tertiary water was put into a 2 L beaker, 25.03 g of an LB broth was measured, the LB broth was put into the 2 L beaker, and then the resulting mixture was stirred until it was completely dissolved. And then, the final volume was adjusted to 1 L by adding tertiary water thereto, the resulting product was transferred to a 2 L bottle, and the bottle was sterilized under the conditions of 121° C. and 30 minutes.
2-2. Preparation of LB Agar Plate Supplemented with Kanamycin
In order to prepare an LB agar plate, 300 mL of tertiary water was put into a 2 L beaker, 19.98 g of LB agar was put into the 2 L beaker, and the resulting mixture was stirred until it was completely dissolved. And then, the final volume was adjusted to 500 mL by adding tertiary water thereto, the resulting product was transferred to a 2 L bottle, and the bottle was sterilized under the conditions of 121° C. and 30 minutes. And then, the bottle was taken out from the autoclave when the temperature of the autoclave was 40 to 50° C. and transferred to a clean bench, and 500 μL of kanamycin (stock concentration of 50 mg/mL) was added thereto until the concentration became 50 μg/mL. About 20 mL of the LB agar solution was dispensed into a petri dish, the petri dish was dried in a clean bench until the LB agar of the petri dish was solidified, and then the fully dried petri dish was wrapped in plastic wrap and stored in a refrigerator (2 to 8° C.) for up to 1 month.
2-3. Construction of Transformed E. coli Strain for Protein Expression
First, in order to construct a transformed E. coli strain BL21 (BL21/DsbC-SP TB4) for the expression of a target protein using the plasmid (DsbC-SP TB4) synthesized by the method in Example 1, transformation was performed according to the user protocol TB009 (Novagen), and the transformed strain was spread on an LB agar plate supplemented with 50 μg/mL kanamycin and cultured in an incubator set at 37° C. overnight (14 hours).
It was confirmed that in the transformed E. coli BL21, colonies were formed in the LB agar plate supplemented with kanamycin (50 μg/mL), and a single colony of BL21/DsbC-SP TB4 was collected by loop and needle. The LB agar plate supplemented with kanamycin (50 μg/mL) was sealed with Parafilm and stored in a refrigerator (2 to 8° C.) for up to 1 month.
And then, the collected BL21/DsbC-SP TB4 was inoculated into a 14 mL round-bottom tube containing 4 mL of an LB broth, cultured in a shaking incubator set at 37° C. and 200 rpm overnight (14 hours), 200 μL of a culture solution was inoculated into a 250 ml flask containing 100 ml of the LB broth, and then the culture was terminated when OD600=approximately 0.2 by measuring the OD600 value at intervals of 30 minutes. The E. coli strain was recovered by centrifugation at 4,000 rpm for 15 minutes, and the volume at which OD600=1.0 was calculated according to the following calculation equation.
(Volume at which OD600=1.0)=(Volume at the end of culture)×(OD600 value at the end time point of culture)
The pellet was resuspended by putting the LB broth containing sterilized glycerol so as to have a volume of 15% at a volume calculated by the above equation, and then 200 μL each of the suspension was aliquoted into sterilized microtubes, and the microtubes were stored in a deep freezer (−80° C. to −70° C.) or an LN2 tank (−196° C.).
And then, as a result of constructing a transformed E. coli strain DH5a (DH5α/DsbC-SP TB4) in order to mass-produce plasmid DNA using a DsbC-SP TB4 plasmid by the same method, a plate in which colonies were formed was obtained.
The DH5α/DsbC-SP TB4 constructed as described above was subjected to the same process for the single colony of the BL21/DsbC-SP TB4 and stored in a deep freezer (−80° C. to −70° C.) or an LN2 tank (−196° C.).
In order to confirm the growth of the E. coli BL21 constructed by the method in Example 2, 20 ml of the LB Broth and 20 μl of the kanamycin stock (50 mg/ml) were placed in a 50 mL conical tube. After the DsbC-SP TB4 1 vial stored in the deep freezer was taken out, the vial was thawed in a water bath set at 37° C. for 2 to 3 minutes. The thawed cells were inoculated into the 50 ml conical tube containing the LB broth and kanamycin, and cultured in a shaking incubator at 37° C. and 200 rpm for 3 hours. And then, 2.5 ml of the culture solution was inoculated into a baffled Erlenmeyer flask containing 250 ml of the LB broth, and 1 mL of the culture solution was sampled (sampling) every hour after the inoculation. The OD600 value of the extracted culture solution was measured by a UV spectrophotometer, and when the measured OD600 value was 0.8 or higher, the culture solution was diluted and measured again.
As a result, as illustrated in
4-1. Confirmation of Expression of DsbC-SP TB4
In order to confirm the expression of a thymosin beta 4 (TB4) protein in the E. coli strain constructed by the method in Example 2, the DsbC-SP TB4 cell stock was thawed in 20 mL of an LB medium supplemented with kanamycin (50 μg/mL). And then, after the cell stock was cultured in a shaking incubator set at 37° C. and 200 rpm for 3 hours (a primary seed culture solution), the primary seed culture solution was diluted 1/10,000 in 20 mL of an LB medium containing kanamycin (50 μg/mL), and then cultured overnight (a secondary seed culture solution). 2.5 mL of the secondary seed culture solution was inoculated into 250 mL of LB and cultured again in a shaking incubator set at 37° C. and 200 rpm, and the OD600 value was measured every 1 hour.
When the measured OD600 value reached 0.6, IPTG was added thereto for induction so as to become 1.0 mM, and the culture was terminated at 3 hours after the induction. Cells were recovered by centrifuging 10 mL of the culture solution at 4,000 rpm for 30 minutes, and the pellet was crushed with B-PER according to User Guide: B-PER Bacterial Protein Extraction Reagent, and then centrifuged at 15,000 rpm for 10 minutes to isolate only the supernatant. After a sample for SDS-PAGE was produced by mixing the isolated supernatant with a 4× Laemmli sample buffer at a ratio of 1:3 and loaded onto a gel, the sample was subjected to electrophoresis at 200 V for 30 minutes, Coomassie-stained for 30 minutes, and then destained. Thereafter, a target protein band was analyzed using Gel-doc.
When the expression of the protein was induced, the growth degree of BL21/DsbC-SP TB4 is as illustrated in
4-2. N-Term Sequencing of DsbC-SP TB4
The DsbC-SP TB4 cell stock was thawed in 20 mL of an LB medium supplemented with kanamycin (50 μg/mL), and cultured in a shaking incubator set at 37° C. and 200 rpm for 3 hours (a primary seed culture solution). And then, the primary seed culture solution was diluted 1/10,000 in 20 mL of an LB medium supplemented with kanamycin (50 μg/mL), and then cultured overnight (a secondary seed culture solution). 2.5 mL of the secondary seed culture solution was inoculated into 250 mL of LB and cultured again in a shaking incubator set at 37° C. and 200 rpm, and the OD600 value was measured every 1 hour.
When the measured OD600 value reached 0.6, IPTG was added thereto for induction so as to become 1.0 mM, and the culture was terminated at 3 hours after the induction. Cells were recovered by centrifuging 10 mL of the culture solution at 4,000 rpm for 30 minutes, and the pellet was crushed with B-PER according to User Guide: B-PER Bacterial Protein Extraction Reagent, and then centrifuged at 15,000 rpm for 10 minutes to isolate only the supernatant. After a sample for SDS-PAGE was produced by mixing the isolated supernatant with a 4× Laemmli sample buffer at a ratio of 1:3 and loaded onto a gel, the sample was subjected to electrophoresis at 200 V for 30 minutes. The sample was transferred to a PVDF membrane, and a band of a transformed target protein was confirmed by staining the membrane with Ponceau.
As a result, as illustrated in
Further, as a result of analyzing the sequence with respect to 10 amino acids of the protein N-term corresponding to the expected target band size (5 to 6 kDa) confirmed above, as shown in the following Table 3, the sequence matched that of WT (wild type)_TB4.
Meanwhile, the amino acid sequence of WT_TB4 is as shown in the following Table 4, and in particular, the part expressed in bold type refers to the sequence of 10 N-term amino acids.
SDKPDMAEIE
KFDKSKLKKTETQEKNPLP
From the above, it can be seen that the E. coli strain constructed by the method of Example 2 is a strain which is transformed with a plasmid having a sequence of WT_TB4 and expresses WT_TB4.
For reference, the base sequence and amino acid sequence of the disulfide bond isomerase signal peptide of the present invention are shown in the following Table 5, but are not limited thereto.
The above-described description of the present invention is provided for illustrative purposes, and those skilled in the art to which the present invention pertains will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are only exemplary in all aspects and are not restrictive.
When a recombinant vector comprising a disulfide bond isomerase signal peptide is used, it is possible to produce a protein whose amino acid sequence does not start with methionine through an E. coli expression system, and accordingly, there is an advantage in that the recombinant vector does not need additional processes such as a protease treatment for commercial availability. In particular, although thymosin beta 4 has been highly available as a therapeutic agent for various diseases for a while, thymosin beta 4 has a problem in that when thymosin beta 4 is expressed in a host (host strain), thymosin beta 4 is degraded by intracellular proteases and cannot be produced. The present invention enables the thymosin beta 4 to be produced through an E. coli expression system, and thus to be usefully utilized in the development of a therapeutic agent for alopecia as well as ischemic heart disease, corneal injury, ophthalmic diseases, and epidermolysis bullosa, and further, the E. coli expression system of the present invention can be applied to production of various therapeutic proteins such as a parathyroid hormone, a human growth hormone, and a granulocyte-colony stimulating factor (GCSF) used for the treatment of osteoporosis, and thus is expected to be widely used as a useful technique.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0010055 | Jan 2018 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/001055 | 1/25/2019 | WO | 00 |
