Method for producing botulinum toxin by using solubilizing partner and GP41.1 intein

Abstract

The invention relates to a method for producing botulinum toxin by utilizing a solubilizing partner and GP41.1 intein, in particular to a method for producing the botulinum toxin by utilizing the solubilizing partner and the GP41.1 intein in a recombination manner. The present invention confirms that the receptor binding domain (HC) of the heavy chain of botulinum toxin, which is less soluble, can be expressed in a soluble form in Escherichia coli by adding a solubilizing partner (Soluble Partner). Moreover, the solubilizing partner is usually a protein having a very high molecular weight, but the present invention confirms that the solubilizing partner can be easily removed using the GP41.1 intein.

Description

Technical Field

[0001] The present invention relates to a method for producing botulinum toxin in a recombinant manner by utilizing a solubilizing partner and GP41.1 intein. Background Art

[0002] Botulinum toxin is a neurotoxic protein produced by the bacterium Clostridium botulinum. It specifically targets soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) present in nerve cells, hindering the formation of complexes and inhibiting membrane fusion, thereby blocking the release of neurotransmitters. Botulinum toxin is known to have therapeutic effects on various diseases by inhibiting muscle movement and the sympathetic and parasympathetic nervous systems.

[0003] Specifically, although botulinum toxin is known to be mainly used in cosmetic surgeries such as wrinkle removal, it is also used to treat many neurotransmitter secretion or muscle-related diseases, such as strabismus, blepharospasm, vocal cord disorders, torticollis, myocardial disorders, ulcers and acid reflux disease, loss of appetite, pancreatic diseases, stretch marks, urge urinary incontinence, anal fissures, polio, muscle pain, hip deformity, hyperhidrosis, low back pain, neck pain, chronic headaches, cranial nerve disorders, etc.

[0004] Botulinum neurotoxin (BoNT) is generally divided into two parts: the heavy chain (HC, about 100 kDa) and the light chain (LC, about 50 kDa) that has the enzymatic activity of cleaving the SNARE complex in nerve cells to form proteins. The heavy chain (HC) is further divided into the part that has the function of recognizing and binding to nerve cells (receptor binding domain, RBD or H C ) and a portion that has the function of moving the light chain portion to the neuronal cytoplasm (translocation domain, H N ).

[0005] On the other hand, botulinum toxin is released from bacteria as a single chain. Endogenous proteases then separate the two chains into a heavy chain (HC) and a light chain (LC). These chains reconnect via disulfide bonds, resulting in a total size of approximately 150 kDa. Consequently, the single-chain botulinum toxin released from bacteria requires a process of fragmentation and reconnection, resulting in low yields and inevitably increased production costs. Furthermore, botulinum toxin is highly hazardous, making the costs of production licenses and corresponding safety equipment very high.

[0006] Furthermore, screening for botulinum toxin-producing strains in nature is a very difficult process. It is necessary to explore strains that produce the desired type of toxin, and these strains must also be sufficiently productive. Methods for producing full-length toxins in recombinant Escherichia coli other than Clostridium botulinum are also very difficult. In particular, it is difficult to express a 150 kDa botulinum toxin in a soluble form in E. coli cells. Furthermore, botulinum toxins contain interchain and intrachain disulfide bonds, but there is a problem in that disulfide bonds do not form within E. coli cells. Furthermore, as mentioned above, botulinum toxins must be in a state in which the peptide bonds between the light and heavy chains are accurately cleaved and connected via disulfide bonds. However, E. coli does not contain proteins for cleaving and connecting disulfide bonds as described above, so there is a problem in that a separate additional process is required.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Korean Patent No. 10-2610179 (November 30, 2023) describes a method for producing botulinum toxin using Escherichia coli.

[0010] Patent Document 2: Korean Patent Publication No. 10-2020-0115584 (October 7, 2020) describes a method for producing botulinum toxin using Bacillus as a host. Summary of the Invention

[0011] Technical issues

[0012] The present invention aims to provide a method for producing botulinum toxin safely and conveniently by gene recombination technology.

[0013] Furthermore, the present invention aims to provide a method for expressing botulinum toxin in a soluble form and producing it in high yield.

[0014] Technical Solution

[0015] The present invention provides a method for producing botulinum toxin, characterized in that it comprises: step 1), by respectively making "LC-H N-GP41.1 N ” and “SP-GP41.1 C -H C " expression to produce, the "LC-H N -GP41.1 N " is the light chain (LC) of botulinum toxin, the translocation domain of the heavy chain (H N ) and GP41.1 intein N (GP41.1 N ) botulinum toxin fragments sequentially linked from the 5' end to the 3' end, the "SP-GP41.1 C -H C " is a solubilizing partner (SolublePartner, SP) composed of polypeptides, GP41.1 intein C (GP41.1 C ) and the receptor binding domain of the heavy chain of botulinum toxin (H C ) The botulinum toxin fragments are sequentially connected from the 5' end to the 3' end; Step 2), the LC-H N -GP41.1 N and the SP-GP41.1 C -H C ; Step 3), using protein trans-splicing method to remove the translocation domain (H N ) connected to the "GP41.1 N " and the receptor binding domain (H C ) connected to the "SP-GP41.1 C ", so that when the soluble partner (SP) is removed, the translocation domain (H N ) and the receptor binding domain (H C ) combined; step 4), cutting the translocation domain (H) of the light chain (LC) and the heavy chain N ) between the peptide bond between the cleaved light chain (LC) and the translocation domain (H N ) to induce disulfide bonds between them.

[0016] The present invention provides a method for producing botulinum toxin, characterized in that it comprises: step 1), by respectively making "LC-H N -GP41.1 N ” and “SP-GP41.1 C -H C " expression to produce, the "LC-H N -GP41.1 N " is the light chain (LC) of botulinum toxin, the translocation domain of the heavy chain (H N ) and GP41.1 intein N (GP41.1 N) botulinum toxin fragments sequentially linked from the 5' end to the 3' end, the "SP-GP41.1 C -H C " is a solubilizing partner (SolublePartner, SP) composed of polypeptides, GP41.1 intein C (GP41.1 C ) and the receptor binding domain of the heavy chain of botulinum toxin (H C ) The botulinum toxin fragments are sequentially connected from the 5' end to the 3' end; Step 2), the LC-H N -GP41.1 N and the SP-GP41.1 C -H C ; Step 3), cutting the translocation domain (H) of the light chain (LC) and the heavy chain N ) between the peptide bond between the cleaved light chain (LC) and the translocation domain (H N ) between the induction of a disulfide bond; step 4), using a protein trans-splicing method to remove the translocation domain (H N ) connected to the "GP41.1 N " and the receptor binding domain (H C ) connected to the "SP-GP41.1 C ", so that when the soluble partner (SP) is removed, the translocation domain (H N ) and the receptor binding domain (H C )combined.

[0017] In the method for producing the botulinum toxin of the present invention, preferably, in the GP41.1 intein N (GP41.1 N ) and the solubilization partner are further combined with a purification tag.

[0018] In the method for producing botulinum toxin of the present invention, the botulinum toxin fragment can be produced by expressing it in Escherichia coli (E. coli).

[0019] Effects of the Invention

[0020] The present invention recognizes that the less soluble heavy chain (Hc) can be expressed in a soluble form by the addition of a solubilizing partner.

[0021] Solubilizing partners are typically proteins with very high molecular weights, making their removal difficult. Additional genetic manipulation, such as inserting restriction enzyme recognition sequences, is required. However, the present invention demonstrates that the GP41.1 intein peptide can be used to readily remove such solubilizing partners.

[0022] Furthermore, the present invention confirmed that when the GP41.1 intein is used, in which the sequence characteristic expansion region (scar) without cysteine ​​is retained, even if the solubilization partner and the purification tag are located at positions that interfere with the function of the intein, the intein can still function intact and splice H N and H C .

[0023] In order for botulinum toxin to fully exhibit its activity, it needs to be in the presence of LC and H N The cysteine-mediated disulfide bonds are formed between the two peptides. However, if the sequence-characteristic expanded region containing cysteine ​​is left, other disulfide bonds other than the original disulfide bonds described above may be formed, potentially affecting the activity of the botulinum toxin. Therefore, it is crucial to use an intein that leaves the sequence-characteristic expanded region free of cysteine. The results confirmed that GP41.1 used in the present invention is an intein that meets these conditions.

[0024] Furthermore, the present invention attempts to combine various solubilizing proteins (MBP, GFP, Sumo) with the GP41.1 intein C and the receptor binding domain of the heavy chain of botulinum toxin (H C )-linked proteins were further fused, and the results confirmed that maltose binding protein (MBP) had the best effect in enhancing soluble expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Shown is the expression of the botulinum toxin fragment Fusion partner-R10-GP41.1 in E. coli using various types of solubilization partners (Fusion partner; GFP, SUMO, MBP) C -H C Then confirm whether the result is expressed in a soluble form.

[0026] Figure 2 Part (A) schematically shows the botulinum toxin fragment MBP-R10-GP41.1 C -H C and botulinum toxin fragment LC-H N -GP41.1 N -Structure of R10-H6. Figure 2 Part (B) shows the botulinum toxin fragment MBP-R10-GP41.1 produced in E. coli as confirmed by SDS-PAGE C -H C and botulinum toxin fragment LC-H N -GP41.1 N -Result of R10-H6.

[0027] Figure 3 Part (A) shows the mixing of botulinum toxin fragments MBP-R10-GP41.1 C -H C and botulinum toxin fragment LC-H N -GP41.1 N -R10-H6 to induce protein trans splicing reaction and thereby produce recombinant botulinum toxin (LC-HN, rBoNT) process. Figure 3 Part (B) shows the results of confirming the protein trans splicing reaction by SDS-PAGE.

[0028] Figure 4 Part (A) shows the mixing of MBP-R10-iCL C -H C and botulinum toxin fragment LC-H N -iCL N -R10-H6 was used to induce protein trans splicing, and the results of the reaction were confirmed by SDS-PAGE. Figure 4 Part (B) shows the mixing of MBP-R10-gCL C -H C and botulinum toxin fragment LC-H N -gCL N -R10-H6 was added to induce protein trans splicing, and the results of the reaction were confirmed by SDS-PAGE.

[0029] Figure 5 (A) schematically shows that the protein trans-splicing reaction (PTS) was subjected to affinity chromatography (AC) to remove reaction byproducts (MBP-R10-GP41.1-R10-H6) and non-reactants (LC-H N -GP41.1 N -R10-H6, MBP-R10-GP41.1 C -H C ) process. Figure 5 Part (B) shows the results of SDS-PAGE confirmation of the unbound (UB), elution, and washing fractions obtained through the affinity chromatography process.

[0030] Figure 6 Part (A) schematically illustrates the process of treating recombinant botulinum toxin (LC-HN, rBoNT) with thrombin (thr) to cleave between the light chain (LC) and the heavy chain (HN) of botulinum toxin. Figure 6 Part (B) shows the results of SDS-PAGE comparison of a sample of recombinant botulinum toxin treated with thrombin (reduced, R) and an untreated sample (non-reduced, NR) treated with a reducing agent to confirm whether the recombinant botulinum toxin was accurately cleaved by thrombin treatment. DETAILED DESCRIPTION

[0031] The present invention provides a method for producing botulinum toxin, characterized in that it comprises: step 1), by respectively making "LC-H N -GP41.1 N ” and “SP-GP41.1 C -H C " expression to produce, the "LC-H N -GP41.1 N " is the light chain (LC) of botulinum toxin, the translocation domain of the heavy chain (H N ) and GP41.1 intein N (GP41.1 N ) botulinum toxin fragments sequentially linked from the 5' end to the 3' end, the "SP-GP41.1 C -H C " is a solubilizing partner (SolublePartner, SP) composed of polypeptides, GP41.1 intein C (GP41.1 C ) and the receptor binding domain of the heavy chain of botulinum toxin (H C ) The botulinum toxin fragments are sequentially connected from the 5' end to the 3' end; Step 2), the LC-H N -GP41.1 N and the SP-GP41.1 C -H C ; Step 3), using protein trans-splicing method to remove the translocation domain (H N ) connected to the "GP41.1 N " and the receptor binding domain (H C ) connected to the "SP-GP41.1 C ", so that when the soluble partner (SP) is removed, the translocation domain (H N ) and the receptor binding domain (H C ) combined; step 4), cutting the translocation domain (H) of the light chain (LC) and the heavy chain N) between the peptide bond between the cleaved light chain (LC) and the translocation domain (H N ) to induce disulfide bonds between them.

[0032] The present inventors have developed a method for safely producing botulinum toxin through Korean Patent No. 10-2610179, that is, producing a translocation domain (H domain) comprising a light chain (LC) of botulinum toxin and a heavy chain (H domain) of botulinum toxin. N ) of a botulinum toxin fragment and a receptor binding domain (H C ) of a botulinum toxin fragment, and then using a Cfa intein to bind to the botulinum toxin fragment.

[0033] However, when botulinum toxin is produced as described above, the receptor binding domain (H C ) is likely to be expressed in an insoluble form in Escherichia coli (E. coli), resulting in a low yield during production.

[0034] Furthermore, when Cfa intein is used to bind to a botulinum toxin fragment, a cysteine ​​residue is left behind, and there is a problem that the recombinant botulinum toxin formed by the cysteine ​​residue forms a disulfide bond and forms a dimer. N A cysteine-mediated disulfide bond needs to be formed between the two proteins, but if a sequence characteristic expansion region containing cysteine ​​is left, it may lead to the formation of other forms of disulfide bonds instead of the original form of disulfide bonds as described above, and thus there is a problem that the activity of botulinum toxin may be reduced.

[0035] However, the present invention confirmed that, when GP41.1 in various types of inteins without cysteine ​​residues was used, it was possible to remove the cysteine ​​residues associated with GP41.1. N or GP41.1 C The purification tag and soluble partner are connected and the botulinum toxin fragment is connected.

[0036] Furthermore, the present invention has confirmed that among various solubilization partners, maltose binding protein (MBP) has the best solubilization ability for the receptor binding domain (HBD) of the heavy chain of botulinum toxin. C ) The effect of expressing the soluble form of the protein linked to GP41.1 is particularly good.

[0037] Hereinafter, each step of the method for producing botulinum toxin of the present invention will be described in detail.

[0038] <Step 1: Production of Botulinum Toxin Fragments>

[0039] This step is to make "LC-H N -GP41.1 N ” and “SP-GP41.1 C -H C "Expression to produce the process, the "LC-H N -GP41.1 N " is the light chain (LC) of botulinum toxin, the translocation domain of the heavy chain (H N ) and GP41.1 intein N (GP41.1 N ) botulinum toxin fragments sequentially linked from the 5' end to the 3' end, the "SP-GP41.1 C -H C ” is a solubilizing partner (Soluble Partner, SP) composed of polypeptides, GP41.1 intein C (GP41.1 C ) and the receptor binding domain of the heavy chain of botulinum toxin (H C ) botulinum toxin fragments sequentially linked from the 5' end to the 3' end, characterized in that the botulinum toxin fragments are expressed in a soluble form.

[0040] Generally, techniques are known for producing botulinum toxin fragments by expressing them in a state where the entire sequence is linked or by expressing them in a state where the botulinum toxin light chain (LC) and the botulinum toxin heavy chain (HC) are separated.

[0041] However, in this step, the botulinum toxin is fragmented into the light chain (LC) of the botulinum toxin, the translocation domain (H N ) of a botulinum toxin fragment and a receptor binding domain (H C ) is expressed by a botulinum toxin fragment, characterized in that the botulinum toxin is expressed with higher solubility.

[0042] Furthermore, in this step, the solubilization partner is further linked to the receptor binding domain (H C ) is expressed, characterized in that the receptor binding domain (H C In the following embodiments of the present invention, the receptor binding domain (RBD) of the heavy chain of botulinum toxin can be enhanced by using a solubilizing partner. C ), thereby confirming that botulinum toxin can be produced at a higher yield.

[0043] In this step, the solubilization partner refers to a partner that can enhance soluble expression by being linked to a botulinum toxin fragment, and includes, for example, GFP, SUMO, MBP, etc. On the other hand, according to the following examples, the maltose binding protein (MBP) in the solubilization partner is effective for binding to the GP41.1 intein C and the receptor binding domain (HBD) of the heavy chain of botulinum toxin. C ) linked to a botulinum toxin fragment (GP41.1 C -H C ) showed particularly excellent soluble expression effect.

[0044] On the other hand, when using a solubilizing partner, it is necessary to remove the solubilizing partner separately in order to commercialize the botulinum toxin. However, this is problematic because the solubilizing partner is typically a very high molecular weight protein, requiring additional genetic manipulation such as inserting a restriction enzyme recognition sequence. However, the present invention demonstrates, through the following examples, that the use of an intein allows for easy removal of the solubilizing partner without requiring additional genetic manipulation.

[0045] On the other hand, in the present invention, the intein, which is a self-splicing protein, is a protein composed of intein N (N-terminal intein, intein N) and intein C (C-terminal intein, intein C). Each intein exists separately from each other before meeting in the cell. After meeting, it folds and then acts as an enzyme to splice the proteins located at the side ends of each intein (so-called exteins) to each other, and exhibits a self-elimination mechanism. The present invention confirmed that when using GP41.1, one of the various types of inteins that does not leave a cysteine ​​residue, it is possible to remove the protein associated with GP41.1. N or GP41.1 C The purification tag and solubilizing protein (soluble partner) are connected and the botulinum toxin fragment is connected. The 3' end of intein N and the 5' end of intein C may also contain a linker sequence.

[0046] On the other hand, in this step, preferably, the botulinum toxin fragment, "LC-H N -Intein N" also contains a purification tag at the 3' end. This allows for smoother purification in the following purification process. In this case, the purification tag can be a histidine tag, a lysine tag, etc., but is not limited thereto. Maltose binding protein (MBP) can also be used as a purification tag.

[0047] On the other hand, in this step, expression can be performed using a plasmid in various host cells, and as an example, Escherichia coli (E. coli) can be used.

[0048] <Step 2: Purification of Botulinum Toxin Fragments>

[0049] This step is to purify the botulinum toxin fragment LC-H produced in step 1. N -GP41.1 N and botulinum toxin fragment SP-GP41.1 C -H C process.

[0050] In this step, the purification method can be to further link a purification tag to the botulinum toxin fragment for expression, and then use the purification tag for simple purification, but is not limited thereto. Alternatively, the present invention utilizes maltose binding protein, which can be used as a purification tag due to its ability to bind to maltose.

[0051] <Step 3: Incorporating Botulinum Toxin Fragments Using Protein Trans-Splicing>

[0052] The process of this step is as follows: a protein trans-splicing method using GP41.1 intein is used to remove the translocation domain (H N ) connected to the "GP41.1 N " and the receptor binding domain (H C ) connected to the "SP-GP41.1 C In the state where the soluble partner (SP) is removed, the heavy chain translocation domain (H N ) and the receptor binding domain (H C ) to prepare full-length botulinum toxin (LC-H N -H C ).

[0053] On the other hand, when intein N and intein C meet, they connect to form a complete intein complex. If the intein complex is complete, it will be spliced ​​and fall off, and the extein proteins outside the intein complex will be connected by peptide bonds to form a single protein.

[0054] This step is a process of inducing protein trans-splicing reaction by using the intein as described above. The present invention confirms that although the GP41.1 intein N (GP41.1 N ) and GP41.1 intein C (GP41.1 N) is connected to the inside of the intein, but the protein trans-splicing reaction still occurs intact. When the intein conjugate is removed, the purification tag connected to the C-terminus of the intein N and the solubilization partner connected to the N-terminus of the intein C can be removed together.

[0055] Specifically, in order to perform protein trans-splicing using a split intein, intein N and intein C must first recognize and structurally bind to each other. Therefore, there are limitations: protein tags placed before or after the intein must not hinder their interaction, and subsequent protein tags must not hinder functional splicing. However, the present invention has confirmed that even if the botulinum toxin fragment outside the GP41.1 intein and the purification tag or solubilization tag inside interfere with the binding and function of the GP41.1 intein, the binding force generated by the interaction between intein N and intein C is not disturbed, and no steric hindrance occurs, allowing protein trans-splicing to proceed intact.

[0056] On the other hand, this step has the following characteristics: when the intein conjugate is removed as described above, the purification tag and the solubilization partner are also removed. This characteristic has the advantage that there is no need to separately remove other proteins such as the purification tag and the solubilization partner that are linked to the botulinum toxin via a peptide bond.

[0057] Furthermore, the advantage is that the process of purifying botulinum toxin can be simplified after the protein trans-splicing reaction process. Specifically, when the botulinum toxin fragment "LC-H N -GP41.1 N -H6" and "H6-SP-GP41.1 C -H C "To induce protein trans-splicing reaction, not only full-length botulinum toxin (BoNT) but also non-reactive substances (LC-H N -Intein N-H6, H6-SP-Intein CH C ) and by-products (H6-Intein-SP) and other impurities are mixed together (refer to Figure 3 (A) of the present invention), since the impurities contain a histidine tag (H6) as a purification tag, the impurities can be easily removed by affinity chromatography using the tag. On the other hand, if the solubilization partner (SP) can be used as a purification tag, there is no need to use SP-Intein CH CThe above-mentioned effect can be achieved by using a single purification tag (see Figure 5 (Part (A) of the ).

[0058] On the other hand, in this step, when the intein binder is removed by the protein trans-splicing reaction, some residual sequences of the intein binder (the so-called "sequence characteristic amplification region (scar)") may be left behind. Therefore, in the present invention, the intein preferably uses an intein type that does not leave a cysteine ​​residue, and more preferably uses GP41.1. In the case of using GP41.1, SGYSSS is left as a residual sequence during the protein trans-splicing process, and the residual sequence does not contain cysteine ​​(C, cystein), so it has the advantage of not generating unnecessary disulfide bonds. In addition, the present invention confirms that when using GP41.1 among the various types of inteins that do not leave a cysteine ​​residue, the residues that are related to GP41.1 can be removed. N or GP41.1 C The purification tag and soluble partner are connected and the botulinum toxin fragment is connected.

[0059] Step 4: Translocation domain of the light chain (LC) and heavy chain (H N ) between peptide bonds and induction of disulfide bonds>

[0060] In order for botulinum toxin to fully express its activity, the light chain (LC) and the translocation domain (H N ) need to be connected by disulfide bonds rather than peptide bonds. The process of this step is as follows: cutting the full-length botulinum toxin (LC-H) obtained in step 3 N -H C ) of the light chain (LC) and the translocation domain of the heavy chain (H N ) between the peptide bond between the cleaved light chain (LC) and the translocation domain (H N ) to induce disulfide bonds between the two, thereby preparing a botulinum toxin that exhibits full activity.

[0061] On the other hand, this step can also be performed before performing step 3.

[0062] On the other hand, in this step, the light chain (LC) and the translocation domain (H) of the botulinum toxin are cleaved. N ) can be carried out by protease treatment. That is, when the botulinum toxin fragment LC-H is produced in step 1 N -GP41.1 N When LC and H N In the case of inserting a protease recognition sequence between the two, in this step, the LC and HN The peptide bonds between.

[0063] On the other hand, in this step, the light chain (LC) of botulinum toxin and the translocation domain (H N The formation of disulfide bonds between the two sulfide bonds can be induced by treating with an oxidizing agent. However, in step 1, the formation of disulfide bonds between the two sulfide bonds may be induced by treating with an oxidizing agent. N The disulfide bond is already formed when the botulinum toxin is added to the intein N, and no separate processing is required. N ) exists in the protein, so when expressed in E. coli, disulfide bonds are naturally formed. If there is no separate reducing agent treatment, the disulfide bonds can remain intact, so there is no need for separate treatment.

[0064] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the following examples and includes modifications of equivalent concepts.

[0065] [Example 1: Screening of Soluble Partners Suitable for the Botulinum Toxin Production Method of the Present Invention]

[0066] When the receptor binding domain of the heavy chain of botulinum toxin (H C ), there is a problem that most of the protein is expressed in an insoluble form.

[0067] This example attempts to confirm that when the solubilizing partner is linked to the intein C C ) linked to the receptor binding domain of the heavy chain of botulinum toxin (H C ) to enhance soluble expression. Furthermore, the authors sought to identify which solubilization partner (GFP, SUMO, MBP) exhibited the best effect.

[0068] Preparation of coding solubilization partners (green fluorescent protein (GFP, Green Fluorescent Protein, SEQ ID NO: 1; ubiquitin-like protein modification molecule (SUMO, Small Ubiquitin-like Modifier, SEQ ID NO: 2; maltose binding protein (MBP, Maltose-Binding Protein, SEQ ID NO: 3), linker (R10, SEQ ID NO: 4), GP41.1 C Intein C (SEQ ID NO: 5), the receptor binding domain of the heavy chain of botulinum toxin (H C, SEQ ID NO: 13) were sequentially connected to the plasmid (SP-R10-GP41.1 C -H C ), and then transformed into Escherichia coli (E. coli) BL21 (DE3).

[0069] The transformed E. coli was inoculated into 10 ml of TB medium (Terrific broth), and then cultured at 37°C and 200 rpm for 16 hours. 8 g of glycerol was added to 1 L of TB medium (Terrific broth) to prepare the main culture medium, and then 10 ml of the bacterial culture solution was inoculated and the main culture was carried out at 37°C and 200 rpm for 2 hours to ensure that the OD of the culture solution was 600 IPTG was added to the main culture solution to 1 mM, and the main culture was carried out at 16° C. and 200 rpm for 16 hours.

[0070] The main culture was centrifuged at 4°C, 5210×g for 15 minutes, and the supernatant was discarded. The pellet obtained after discarding the supernatant was resuspended in 150 ml of buffer (50 mM HEPES, 150 mM NaCl, pH 7.4), and then the cells were disrupted using an ultrasonic disruptor (10 kHz, 1 sec on / 2 sec off, 30 min). 1 ml of the disrupted cells was centrifuged at 19,800×g to separate the supernatant and pellet.

[0071] Then, the supernatant obtained by the above process was used as the soluble fraction (S), and the pellet was used as the insoluble fraction (I), and SDS-PAGE ( Figure 1 ).

[0072] like Figure 1 As shown in the figure, when GFP or SUMO was used as a solubilization partner (Soluble Fusion Partner), a larger band was confirmed to appear in the insoluble fraction (Insoluble, I). In contrast, when MBP was used as a solubilization partner (Soluble Fusion Partner), a larger band was confirmed to appear in the soluble fraction (Soluble, S).

[0073] Typically, the receptor binding domain (H C) has very low solubility. This example confirms that it is expressed in a state where solubility is enhanced by solubilizing partners such as GFP, SUMO, and MSP. When MBP is used, it can be confirmed that "GP41.1 C -H C The soluble expression enhancement effect of ” is particularly excellent.

[0074] Example 2: Production of Botulinum Toxin Using a Soluble Partner and Intein

[0075] The method for producing botulinum toxin of the present invention is characterized in that the botulinum toxin is produced as LC-H N -intein N and SP-intein CH C The bipartite fragments are then spliced ​​using protein trans-splicing N With H C The method of producing botulinum toxin is between.

[0076] This example aimed to confirm whether a solubilizing partner (SP) linked to the N-terminus of intein C interferes with the intein action when the protein trans-splicing method described above is performed. Furthermore, it was attempted to confirm whether the effects of using various inteins depend on differences in intein type.

[0077] Regarding inteins, a comparison was made between various types of inteins: the Israel group Cystein-Less (iCL) intein, the Germany group Cystein-Less (gCL) intein, and the GP41.1 intein, which do not retain cysteine ​​residues during protein trans-splicing, as characteristic expansion regions of their sequences. Meanwhile, the Israel group Cystein-Less (iCL) intein retains the residue sequence YIDTDSVYLN, the Germany group Cystein-Less (gCL) intein retains the residue sequence SGDTDS, and the GP41.1 intein retains the residue sequence SGYSSS.

[0078] 2-1. Production and purification of botulinum toxin fragments

[0079] Prepare the coding sequence of botulinum toxin light chain (LC, SEQ ID NO: 11), thrombin recognition sequence (SEQ ID NO: 14), translocation domain of botulinum toxin heavy chain (H N , SEQ ID NO: 12), GP41.1 intein N (GP41.1 intein N, GP41.1 N , SEQ ID NO: 6), linker (R10, SEQ ID NO: 4), and histidine tag (H6 tag, SEQ ID NO: 15) were sequentially connected to the plasmid (LC-H N -GP41.1 N -R10-H6), which was then transformed into Escherichia coli (E. coli) BL21(DE3).

[0080] The transformed E. coli was inoculated into 10 ml of TB medium (Terrific broth), and then cultured at 37°C and 200 rpm for 16 hours. 8 g of glycerol was added to 1 L of TB medium (Terrific broth) to prepare the main culture medium, and then 10 ml of the bacterial culture solution was inoculated and the main culture was carried out at 37°C and 200 rpm for 2 hours to ensure that the OD of the culture solution was 600 IPTG was added to the main culture solution to 1 mM, and the main culture was carried out at 16° C. and 200 rpm for 16 hours.

[0081] The main culture was centrifuged at 4°C and 5210×g for 15 minutes, and then all the supernatant was discarded. The pellet obtained after discarding the supernatant was resuspended in 150 ml of buffer (50 mM HEPES, 150 mM NaCl, pH 7.4), and then the cells were disrupted using an ultrasonic disruptor (10 kHz, 1 sec On / 2 sec Off, 30 min). 1 ml of the disrupted cells was centrifuged at 19800×g to obtain a supernatant, which was then filtered through a 0.22 μm filter to purify the translocation domain (H domain) containing the light chain (LC) of botulinum toxin and the heavy chain of botulinum toxin. N ) of the botulinum toxin fragment (LC-H N -GP41.1 N -R10-H6).

[0082] On the other hand, the botulinum toxin fragment MBP-R10-GP41.1 obtained by the above-mentioned process and Example 1 was purified by cation chromatography. C -H C(97.7 kDa) and botulinum toxin fragment LC-H N -GP41.1 N -R10-H6 (112.0 kDa) confirmed that both botulinum toxin fragments were produced intact ( Figure 2 (B) of the .

[0083] Furthermore, the botulinum toxin fragment (LC-H) was produced and purified using the same method as described above and the method of Example 1. N -iCL N -R10-H6, LC-H N -gCL N -R10-H6, MBP-R10-iCL C -H C , MBP-R10-gCL C -H C ), but using a different type of intein (iCL C : SEQ ID NO: 7, iCL N : SEQ ID NO: 8, gCL C : SEQ ID NO: 9, gCL N : SEQ ID NO: 10).

[0084] On the other hand, this process confirmed that among the Israel group Cystein-Less (iCL) intein, the Germany group Cystein-Less (gCL) intein, and the GP41.1 intein, the expression level of botulinum toxin was higher when the GP41.1 intein was used.

[0085] 2-2. Splicing botulinum toxin fragments using protein trans-splicing

[0086] The botulinum toxin fragments (LC-H N -GP41.1 N -R10-H6, LC-H N -iCL N -R10-H6, LC-H N -gCL N -R10-H6、MBP-R10-GP41.1 C -H C 、MBP-R10-iCL C -H C 、MBP-R10-gCL C-H C ) was added with tris(2-carboxyethyl)phosphine (TCEP) to 2 mM and allowed to stand for 10 minutes.

[0087] Then, the translocation domains containing the light chain (LC) and heavy chain (H) of botulinum toxin were respectively N ) of the botulinum toxin fragment (LC-H N -GP41.1 N -R10-H6, LC-H N -iCL N -R10-H6, LC-H N -gCL N -R10-H6) and the receptor binding domain (H) of the heavy chain of botulinum toxin C ) of the botulinum toxin fragment (MBP-R10-GP41.1 C -H C 、MBP-R10-iCL C -H C 、MBP-R10-gCL C -H C ) were mixed in a 1:1 molar ratio according to the type of intein, and then placed at 20°C and 100 rpm for 2 hours to allow protein trans-splicing reaction to occur ( Figure 3 、 Figure 4 ).

[0088] like Figure 3 As shown in Figure 2, it can be confirmed that the protein trans-splicing process occurs completely to form recombinant botulinum toxin (LC-H N -H C , rBoNT, 149.5 kDa). In contrast, Figure 4 As described above, when the Israel group Cystein-Less (iCL) intein and the Germany group Cystein-Less (gCL) intein were used as inteins, it was confirmed that protein trans-splicing did not occur.

[0089] Thus, in the case of using the GP41.1 intein to splice botulinum toxin fragments, it was confirmed that even if the histidine tag and solubilization partner were located at a position that interfered with the function of the intein, the intein could still function intact, thereby not only connecting the H N With H C , the histidine tag and solubilization partner were also removed.

[0090] 2-3. Production and purification of active botulinum toxin by protease treatment

[0091] First, in the protein trans-splicing reaction obtained in Example 2-2, the buffer (20 mM Tris, 150 mM NaCl, 2.5 mM CaCl2, pH 8.0) was replaced by dialysis to remove tris(2-carboxyethyl)phosphine (TCEP) as a reducing agent.

[0092] Afterwards, the reaction byproduct MBP-R10-GP41.1-R10-H6 and the non-reactant LC-H N -GP41.1 N -R10-H6、MBP-R10-GP41.1 C -H C The terminal MBP or H6 was removed by affinity chromatography (IMAC & MBP) Figure 5 (B) of the .

[0093] Specifically, if Figure 5 As shown in part (B), it was confirmed that the recombinant botulinum toxin (rBoNT) that did not bind to the column was detected in the unbinding fraction, while the MBP-R10-GP41.1-R10-H6 and LC-H N -GP41.1 N -R10-H6、MBP-R10-GP41.1 C -H C It was detected in the elution fraction.

[0094] On the other hand, the recombinant botulinum toxin (rBoNT) purified by the above process can be further purified by anion chromatography, and then 10 units of thrombin are added and reacted at 20°C for 16 hours to remove LC-H in the recombinant botulinum toxin (rBoNT). N peptide bonds between the peptide bonds, thereby producing an active form of recombinant botulinum toxin ( Figure 6 (B) of the .

[0095] like Figure 6 As shown in part (B), the SDS-PAGE results of the non-reduced (NR) sample that was not treated with a reducing agent confirmed that botulinum toxin (rBoNT) was detected, and the SDS-PAGE results of the reduced (R) sample in which the disulfide bonds were broken after treatment with a reducing agent confirmed that the amount of botulinum toxin (rBoNT) was reduced and more light chains (LC) and heavy chains (HC) were detected.

[0096] This confirmed that thrombin treatment accurately cleaves the light chain (LC) and heavy chain (HC), resulting in intact production of the active form of botulinum toxin, with the light chain (LC) and heavy chain (HC) linked by disulfide bonds. Furthermore, it was confirmed that no dimerized botulinum toxin forms due to unnecessary disulfide bonds.

Claims

1. A method for producing botulinum toxin, characterized in that: include: Step 1), by respectively LC-H N -GP41.1 N and SP-GP41.1 C -H C Expression to produce the LC-H N -GP41.1 N The light chain LC of botulinum toxin and the translocation domain H of the heavy chain of botulinum toxin N and GP41.1 intein N (GP41.1 N ) botulinum toxin fragments sequentially linked from the 5' end to the 3' end, the SP-GP41.1 C -H C It is a solubilization partner composed of polypeptides SP, GP41.1 intein C (GP41.1 C ) and the receptor binding domain H of the heavy chain of botulinum toxin C Botulinum toxin fragments are linked sequentially from the 5' end to the 3' end; Step 2), purify the LC-H N -GP41.1 N and the SP-GP41.1 C -H C ; Step 3), using protein trans-splicing method to remove the translocation domain H of the heavy chain N Connected GP41.1 N and the receptor binding domain H of the heavy chain C Connected SP-GP41.1 C , so that in the state where the soluble partner SP is removed, the translocation domain H of the heavy chain N The receptor binding domain H of the heavy chain C Combine; Step 4), cutting the translocation domain H between the light chain LC and the heavy chain N The peptide bond between the cleaved light chain LC and the heavy chain translocation domain H N Induce disulfide bonds between.

2. A method for producing botulinum toxin, characterized in that: include: Step 1), by respectively LC-H N -GP41.1 N and SP-GP41.1 C -H C Expression to produce the LC-H N -GP41.1 N The light chain LC of botulinum toxin and the translocation domain H of the heavy chain of botulinum toxin N and GP41.1 intein N (GP41.1 N ) botulinum toxin fragments sequentially linked from the 5' end to the 3' end, the SP-GP41.1 C -H C It is a solubilization partner composed of polypeptides SP, GP41.1 intein C (GP41.1 C ) and the receptor binding domain H of the heavy chain of botulinum toxin C Botulinum toxin fragments are linked sequentially from the 5' end to the 3' end; Step 2), purify the LC-H N -GP41.1 N and the SP-GP41.1 C -H C ; Step 3), cutting the translocation domain H between the light chain LC and the heavy chain N The peptide bond between the cleaved light chain LC and the heavy chain translocation domain H N Induce disulfide bonds between Step 4), using protein trans-splicing method to remove the translocation domain H of the heavy chain N Connected GP41.1 N and the receptor binding domain H of the heavy chain C Connected SP-GP41.1 C , so that in the state where the soluble partner SP is removed, the translocation domain H of the heavy chain N The receptor binding domain H of the heavy chain C combination.

3. The method for producing botulinum toxin according to claim 1 or 2, characterized in that: In the GP41.1 intein N (GP41.1 N ) is further combined with a purification tag.

4. The method for producing botulinum toxin according to claim 1 or 2, characterized in that: A purification tag is further bound to the solubilization partner.

5. The method for producing botulinum toxin according to claim 1 or 2, characterized in that: The expression is in E. coli.

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