Recombinant plasmid for producing toxic protein

Abstract

The present invention relates to a recombinant plasmid comprising a gene encoding a protein toxic to an organism and a gene encoding an ELP linked thereto. By using the recombinant plasmid, it is possible to produce a fusion protein in an organism in which the toxicity of the protein is mitigated due to fusion with the ELP.

Description

Technology Field

[0001] The present invention provides a recombinant plasmid that mitigates the toxicity of a protein, which was difficult to produce within a living organism due to its toxicity to the organism, thereby enabling its production within the organism. This plasmid can be used universally in fields where protein production is used, for example, in the field of new drug development. Background Technology

[0002] When an organism is used as a host for producing recombinant proteins, if the produced protein is toxic to the organism, the organism may die, stop growing, inhibit protein production, or produce proteins with disrupted structures. To solve this problem, methods have been used so far such as (1) strongly suppressing production until the organism grows to a certain extent, (2) secreting the produced protein outside the cell, or (3) producing another protein that performs detoxification, but there is a need to develop more efficient production methods. The problem to be solved

[0003] The present invention aims to provide a new recombinant plasmid and a method that enable the efficient production of a protein toxic to a living organism within the organism. means of solving the problem

[0004] 1. A recombinant plasmid comprising a gene encoding a protein toxic to an organism and a gene encoding ELP (Elastin-like polypeptides) linked thereto.

[0005] 2. In the above 1, the organism is a recombinant plasmid that is a microorganism.

[0006] 3. In the above 1, the ELP is a recombinant plasmid having a transition temperature lower than the culture temperature of the organism.

[0007] 4. In the above 1, the ELP is a recombinant plasmid comprising 40 to 50 repeating units of the amino acid Val-Pro-Gly-Ile-Gly.

[0008] 5. The recombinant plasmid comprising the sequence of SEQ ID NO. 1 in the above 1.

[0009] 6. A composition for producing a toxic protein in a living organism, comprising a recombinant plasmid of any one of claims 1 to 5 above.

[0010] 7. A non-human organism transformed with a recombinant plasmid of any one of items 1 to 5 above.

[0011] 8. A method for producing a toxic protein in a living organism, comprising the step of culturing a non-human living organism transformed with a recombinant plasmid of any one of 1 to 5 above to obtain a fusion protein of a protein toxic to the living organism and an ELP. Effects of the invention

[0013] By using the recombinant plasmid of the present invention, it is possible to produce a protein toxic to an organism within the organism.

[0014] By using the recombinant plasmid of the present invention, toxic proteins can be produced in living organisms while preserving protein activity without affecting the structure or function of the protein. Brief explanation of the drawing

[0015] Figure 1 is a schematic representation of the pVP65KR-SacB-I48 plasmid used in the present embodiment. Figure 2 is a schematic representation of the addition of the restriction enzyme PmeI site to I48 ELP (plasmid #68937). Figure 3 schematically illustrates the pVP65KR-SacB plasmid used in the present embodiment. Figure 4 is a schematic representation of pVP65KR-SacB with an I48 piece inserted. Figure 5 is a photograph of plates taken to compare the survival of E. coli producing each toxic protein or fusion protein according to culture temperature. The red border indicates the plates where colonies were formed. Specific details for implementing the invention

[0017] The present invention relates to a recombinant plasmid comprising a gene encoding a protein toxic to living organisms and a gene encoding ELP (Elastin-like polypeptides) linked thereto.

[0018] By using a transformant into which the above-mentioned recombinant plasmid has been introduced, a fusion protein of ELP and a protein toxic to an organism (hereinafter referred to as the 'toxic protein') can be produced, and the fusion protein enables the production of the toxic protein within the organism by mitigating the toxicity of the protein to the organism.

[0019] The above organism is not particularly limited as long as it is an organism that can be used as a host for protein production, and includes animals, plants, insects, microorganisms, etc., and the microorganism includes bacteria, yeast, etc., and may be, for example, microorganisms mainly used for protein production, namely Escherichia coli (E. coli), Pseudomonas, cyanobacteria, and Bacillus, etc.

[0020] Toxicity to the aforementioned organism refers to all toxicity that causes disease or pathological symptoms in the organism, inhibits growth or reproduction, or leads to death.

[0021] Proteins possessing the toxicity described above include, for example, EcoRI methylase, EcoRI endonuclease, galactokinase, colicin E3, transcription factor GATA-1, and lysis protein of It could be X174, barnase, Bacillus subtilis SacB protein, E. coli RpsL protein, F-factor CcdB protein, etc.

[0022] The above-mentioned recombinant plasmid refers to a genetic construct recombined to include essential regulatory elements, such as a promoter, so that a target gene can be expressed within a suitable host, and may be in a form integrated into the genome of a host cell or microorganism.

[0023] The above promoter refers to an untranscribed nucleic acid sequence upstream of a coding region that includes a binding site for polymerase and has transcription initiation activity into the mRNA of a promoter-downstream gene.

[0024] In addition, if the above plasmid is a replicable expression plasmid, it may include a replication origin, which is a specific nucleic acid sequence at which replication is initiated.

[0025] Additionally, the plasmid may include a selection marker. The selection marker is intended to select cells or microorganisms transformed by the plasmid, and markers conferring selectable phenotypes, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or the expression of surface proteins, may be used. Since only cells or microorganisms expressing the selection marker survive in an environment treated with a selective agent, the transformed individuals can be selected.

[0026] The above plasmid may be one in which a gene encoding a toxic protein is operatively linked to the promoter, and specifically, may be linked to a downstream region of the promoter.

[0027] The gene encoding the above ELP may be linked to the N-terminus or C-terminus of the gene encoding the above toxic protein.

[0028] Between the gene encoding the ELP and the gene encoding the toxic protein, other genes, such as those encoding a protease cleavage site, an intein, a linker, etc., may be appropriately included by a person skilled in the art to maintain the toxicity mitigation effect by ELP fusion.

[0029] If a gene encoding the protease cleavage site is included, ELP can be removed by treating the generated fusion protein with the protease when a toxic protein with ELP removed is required as the final product. The protease may be, for example, human Rhinovirus (HRV) 3C protease, thrombin, or TEV (Tobacco etch virus) protease.

[0030] Alternatively, a gene coding for intein may be included between the gene coding for the ELP and the gene coding for the toxic protein. For example, after purifying a fusion protein prepared in the form of toxic protein-intein-ELP, if a reducing agent such as DTT is added, the toxic protein upstream of intein can cleave itself without the assistance of other proteases.

[0031] The plasmid of the present invention may use one vector backbone selected from the group consisting of pVP, pQE, pET, pMAL, pGEX, and pGEM as a basic backbone, but is not particularly limited thereto, and any vector backbone known in the art that is capable of recombining the promoter sequence of the present invention is not particularly limited.

[0032] The above ELP is a polypeptide derived from mammalian elastin and synthesized by genetic recombination, a pentapeptide (Val-Pro-Gly-Xaa-Gly)n It consists of repetitions of . Here, Xaa represents a guest residue and can be any amino acid excluding Pro. The above n indicates the number of repetitions.

[0033] ELP can be synthesized in various structures and lengths according to techniques known to those skilled in the art, and can be synthesized, for example, by solution and solid-phase chemical synthesis methods or protein recombination methods.

[0034] ELP has a temperature-sensitive property and has the characteristic of undergoing a phase transition at a specific temperature corresponding to the LCST (lower critical solution temperature), that is, the transition temperature.

[0035] Below the transition temperature, ELP is close to a random coil shape and is well solvated and dissolves well in aqueous solutions; however, when the solution is heated and reaches the transition temperature, ELP becomes insoluble and forms large, micron-sized aggregates visible to the naked eye.

[0036] The above transition is completely reversible, so when the temperature drops back below the ELP transition temperature, the aggregated polypeptide is completely dissolved.

[0037] In the fusion protein of the above ELP and toxic protein, the formation of aggregates by the ELP does not affect the structure or activity of the fused toxic protein.

[0038] In an embodiment of the present invention, it was confirmed that when the temperature is above the transition temperature of the ELP used, that is, when the ELP forms aggregates in the fusion protein of the ELP and the toxic protein, the toxicity of the toxic protein is mitigated, thereby enabling the host organism to survive and the production of the fusion protein within the organism without other treatments for toxicity removal.

[0039] The transition temperature of the above ELP can be varied widely from 0 to 100°C, and polypeptides can be configured in various ways to suit the application. The transition temperature of the ELP may be affected by the pH, concentration, ionic strength, polymer structure, length, etc. of the ELP solution.

[0040] The above ELP may have a transition temperature lower than the culture temperature of the organism, i.e., the host.

[0041] The above culture temperature may be, for example, 25 to 40°C, which is the culture temperature for general bacteria, or 30 to 40°C for E. coli. The above culture temperature may be the optimal culture temperature for the microorganism, and specifically, 36 to 38°C for E. coli.

[0042] If the transition temperature of the above ELP is lower than the culture temperature of the organism, the ELP of the fusion protein aggregates at the culture temperature of the microorganism, allowing the protein to be produced without the death of the organism.

[0043] If the above transition temperature is lower than the optimal culture temperature of the organism, the fusion protein can be produced under temperature conditions where the growth of the organism is smooth, so the protein can be produced efficiently.

[0044] The above ELP may contain 40 to 50 repeating units of the amino acid Val-Pro-Gly-Ile-Gly, specifically 48. At this time, since the transition temperature of the above ELP is 37°C or lower, which is the optimal culture temperature for E. coli, toxic proteins can be efficiently produced in E. coli.

[0045] The above recombinant plasmid may contain the sequence of SEQ ID NO. 1.

[0046] SEQ ID NO. 1 above is the sequence of a plasmid in which ELP I48 is inserted into a plasmid containing the toxic protein SacB, and the I48 fragment was inserted after cleavage with restriction enzymes PmeI and SacI. The I48 is [Val-Pro-Gly-Ile-Gly]48 It means the ELP of the structure.

[0047] A recombinant plasmid consisting of the sequence of SEQ ID NO. 1 is schematically illustrated in FIG. 1 (pVP65KR-SacB-I48), and in the present embodiment, it was confirmed that the toxicity of the toxic protein was effectively mitigated in E. coli transformed using the pVP65KR-SacB-I48 plasmid.

[0049] In addition, the present invention relates to a composition for producing a toxic protein in a living organism, comprising the recombinant plasmid.

[0050] The specific details regarding the above-mentioned recombinant plasmid and the method for enabling the production of toxic proteins within a living organism using it are as described above.

[0051] The composition of the present invention may additionally include an auxiliary substance for producing a target protein with excellent efficiency, and, for specific examples, may further include at least one weak stress treatment substance selected from the group consisting of salt, ethanol, sorbitol, and hydrogen peroxide, but is not necessarily limited thereto.

[0053] In addition, the present invention relates to a non-human organism transformed with the recombinant plasmid.

[0054] The specific details regarding the above-mentioned recombinant plasmid and the method for enabling the production of toxic proteins within a living organism using it are as described above.

[0055] The above-mentioned transformed non-human organism can produce a fusion protein of a toxic ELP with reduced toxicity and a toxic protein.

[0056] The above non-human organism is not particularly limited as long as it is an organism that can be used as a host for protein production, and includes animals, plants, insects, microorganisms, etc. excluding humans, and the microorganism includes bacteria, yeast, etc., and may be, for example, microorganisms mainly used for protein production, namely Escherichia coli (E. coli), Pseudomonas, cyanobacteria, and Bacillus.

[0057] The above transformation refers to the process in which a vector, such as a DNA chain fragment or plasmid containing a type of gene different from that possessed by the original cell, penetrates and binds to the DNA present in the original cell, thereby altering the genetic trait. The above transformation may include the steps of transforming a cell of an organism using a recombinant plasmid and redifferentiating the transformed organism cell from the transformed organism cell.

[0058] The above transformation can be carried out by methods conventional in the art, and, for example, can be introduced through natural introduction, thermal shock, electric shock, etc., but is not particularly limited thereto.

[0060] In addition, the present invention relates to a method for producing a toxic protein in a living organism, comprising the step of culturing a non-human organism transformed with the recombinant plasmid to obtain a fusion protein of a protein toxic to the organism and an ELP.

[0061] The specific details regarding the above-mentioned recombinant plasmid and the method of using it to transform a non-human organism to enable the production of toxic proteins within the organism are as described above.

[0062] The above culture may be carried out at an appropriate culture temperature of the organism, for example, at the optimal culture temperature of the organism. The transition temperature of the ELP of the recombinant plasmid may be lower than the culture temperature of the organism.

[0063] The above method may include the step of cutting the end containing the stop codon of the gene encoding a protein that is toxic to an organism with a first and second restriction enzyme; and the step of obtaining the recombinant plasmid by inserting the gene encoding ELP, which was cut by the first and second restriction enzymes, into the cut location.

[0064] The gene encoding the above ELP can be used by a person skilled in the art using a product available to them, or by manufacturing it if necessary.

[0065] The first and second restriction enzymes mentioned above are not particularly limited as long as they are restriction enzymes usable in a plasmid containing a gene encoding a protein toxic to the organism, and may be used by using a restriction enzyme site within the plasmid or by inserting a restriction enzyme site. The restriction enzymes may be, for example, AsiSI, PmeI, SacI, etc.

[0066] The above method may include a step of separating or purifying the produced protein.

[0067] The above method may include the step of removing ELP from the fusion protein.

[0068] For the above removal, appropriate methods for removing ELP from the fusion protein by those skilled in the art may be used without limitation.

[0069] For example, the above removal may involve treating with a protease to cleave the ELP if a peptide cleavage site exists between the ELP and the toxic protein. The protease may be, for example, human Rhinovirus (HRV) 3C protease, thrombin, or TEV (Tobacco etch virus) protease.

[0070] Alternatively, if the fusion protein is in the form of toxic protein-intein-ELP, the above removal may involve treatment with a reducing agent such as DTT. Upon treatment with a reducing agent such as DTT, the toxic protein upstream of intein can cleave itself without the assistance of other proteases.

[0072] Hereinafter, in order to specifically explain the present invention, it will be described in detail with reference to examples.

[0074] Experimental method

[0075] 1. Production of recombinant plasmids

[0076] 1) The restriction enzyme PmeI site (GTTTAAAC) was added to the 5' position of I48 ELP (plasmid #68937, top of Fig. 2) purchased from Addgene via site-directed mutagenesis (I48 ELP+PmeI, bottom of Fig. 2).

[0077] 2) Cut I48 ELP+PmeI into PmeI and SacI, and separate the I48 fragment.

[0078] 3) The pVP65KR-SacB constructed by the inventors of the present invention (top of FIG. 3) was cut into PmeI and SacI, and an I48 piece was inserted. The completed plasmid was named pVP65KR-SacB-I48 (the middle part of I48 is omitted, and only the ends are shown. 5' end: middle, 3' end: bottom).

[0080] 2. Experiment on the Temperature Dependence of the Effect of I48's Lethality

[0081] 2.1. Temperature-dependent experimental method

[0082] 1) 100 pg of each plasmid was added to XL1-Blue chemically competent cells, and transformation was performed via 42°C heat shock. The proteins produced by each plasmid were barnase, barnase-I48, SacB, and SacB-I48, and pUC19 was a control.

[0083] 2) Barnase and barnase-I48 were cultured on LB+kanamycin plates, and SacB and SacB-I48 were cultured on LB+kanamycin+sucrose plates. Barnase exhibited lethality without additional compounds, but SacB exhibited lethality under 5% sucrose conditions.

[0084] 3) 1 set is cultured at 37℃ and 1 set is cultured at 20℃.

[0086] 2.2. Expected Experimental Results

[0087] 1) At 37°C, due to the aggregation of I48, colony formation is observed on plates producing barnase-I48 and SacB-I48 in addition to pUC19 plates, but not on versions without I48.

[0088] 2) Since I48 cannot aggregate at 20℃, colonies will not form on all plates except pUC.

[0089] 3) If Barnase or SacB inhibits only the growth of E. coli, colony formation will be observed when a plate cultured at 20°C is transferred to 37°C.

[0091] Experimental results

[0092] 1. Lethality Experiment Results

[0093] 1) At 37°C, colony formation was observed only on plates producing barnase-I48 and SacB-I48. Since the formation of insoluble aggregates in the fused state of I48 restricts the movement of toxic enzymes, it is difficult to exhibit efficient lethality.

[0094] 2) At 20℃, barnase-I48 and SacB-I48 also exhibit lethality because they are in a water-soluble state.

[0095] 3) When transferred to 37℃ after incubation at 20℃, no colonies were formed, indicating that the E. coli had already been killed by toxic enzymes during incubation at 20℃.

[0096] 4) Since the versions without I48, barnase and SacB (control), showed lethality regardless of temperature, the lethality of these two genes was confirmed.

[0097] 5) Since colonies were observed on the pUC19 plate (control), there is no problem with the transformation experiment process itself.

[0098] 6) In Fig. 5, the plate where the colony was formed is indicated by a red border.

[0100] 2. Discovery of New Characteristics of I48

[0101] SacB (levansucrase) promotes levan synthesis using sucrose, and since this substance is lethal to E. coli, it is used as a selection marker in the cloning process. Replacing the lethal gene sacB with another gene allows E. coli to grow even in a medium supplemented with 5% sucrose, thereby eliminating self-ligation and facilitating cloning. However, growth of E. coli in sucrose-supplemented media was observed even in samples where SacB was not replaced (negative controls). Hypothesizing that SacB would not be able to play a lethal role if I48 ELP agglutinated at 37°C, another lethal gene, barnase, was inserted into the SacB site.

[0102] Since Barnase is an enzyme that degrades RNA in E. coli, the E. coli dies in the absence of a detoxification protein (barstar). However, pVP65KR-barnase-I48 was successfully produced in XL1-Blue, an E. coli strain lacking barstar. This is thought to be because the agglutination of I48 ELP inhibits the function of lethal genes or toxic proteins.

[0103] Even when a target protein is fused with I48 ELP, I48 ELP does not affect the structure or function of the target protein. Therefore, when a toxic protein with normal structure / function is fused with I48 ELP and this fusion protein is produced at 37°C, the toxicity is sufficiently reduced by the agglutination function of I48 ELP, allowing E. coli to grow.

Claims

Claim 1 A recombinant plasmid for producing a toxic protein in an organism, comprising a gene encoding a protein toxic to the organism and a gene encoding an ELP (Elastin-like polypeptide) linked thereto, wherein the ELP comprises 40 to 50 repeating units of the amino acid Val-Pro-Gly-Ile-Gly. Claim 2 A recombinant plasmid according to claim 1, wherein the organism is a microorganism. Claim 3 Claim 1, wherein the ELP is a recombinant plasmid having a transition temperature lower than the culture temperature of the organism. Claim 4 delete Claim 5 The recombinant plasmid of claim 1 comprising the sequence of sequence no.

1. Claim 6 A composition for producing a toxic protein in a living organism, comprising a recombinant plasmid of any one of claims 1 to 3 and 5. Claim 7 A non-human organism transformed with a recombinant plasmid of any one of claims 1 to 3 and 5. Claim 8 A method for producing a toxic protein in a living organism, comprising the step of culturing a non-human living organism transformed with a recombinant plasmid of any one of claims 1 to 3 and 5 to obtain a fusion protein of a protein toxic to the living organism and an ELP. Claim 9 A method for producing a toxic protein in an organism according to claim 8, further comprising: a step of cutting a terminal containing a stop codon of a gene encoding a protein toxic to an organism with first and second restriction enzymes in a plasmid containing a gene encoding said protein; and a step of obtaining a recombinant plasmid by inserting a gene fragment containing a gene encoding ELP, which was cut by said first and second restriction enzymes, into said cut locations.

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