Method for producing botulinum toxin using soluble partner and intein

Abstract

The present invention relates to a method for producing botulinum toxin in a recombinant manner by using a soluble partner and intein. In the present invention, it was confirmed that HC having low solubility can be expressed in a soluble form by attaching a soluble partner. It was also confirmed that a soluble partner is generally a protein having a very high molecular weight, and when intein is used in the present invention, the soluble partner can be easily removed.

Description

Method for producing botulinum toxin using a water-soluble partner and intein

[0001] The present invention relates to a method for producing botulinum toxin by recombinant means using a soluble partner and an intein.

[0002] Botulinum toxin is a neurotoxic protein produced by strains of the bacterium Clostridium botulinum. It specifically targets SNAREs present in nerve cells, inhibiting complex formation and membrane fusion, thereby blocking the release of neurotransmitters. This suppression of muscle movement and the sympathetic and parasympathetic nervous systems is known to be effective in treating various diseases.

[0003] Specifically, botulinum toxin is known to be used primarily for cosmetic purposes such as wrinkle removal, but it is also used to treat many neurotransmitter secretion or muscle-related diseases such as strabismus, blepharospasm, vocal cord disorders, torticollis, heart disorders, ulcers and acid reflux disease, loss of appetite, pancreatic diseases, stretch marks, urgency incontinence, hemorrhoids, polio, muscle pain, hip deformities, hyperhidrosis, back pain, neck pain, chronic headaches, and cranial nerve disorders.

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

[0005] Meanwhile, botulinum toxin, once released from bacteria as a single chain, is cleaved by endogenous proteases into two chains—a heavy chain (HC) and a light chain (LC). These two chains are then reconnected via disulfide bonds, resulting in a total size of approximately 150 kDa. Therefore, single-chain botulinum toxin released from bacteria requires a process of breaking and reconnecting, which inevitably lowers yield and increases production costs. Furthermore, because botulinum toxin is highly hazardous, the costs of obtaining production permits and the associated safety equipment are extremely high.

[0006] Furthermore, screening for strains producing botulinum toxin in nature is a very difficult process. It is necessary to identify strains that produce the desired type of toxin and ensure sufficient productivity. Producing full-length toxin in recombinant Escherichia coli, rather than Clostridium botulinum, is also very difficult. In particular, it is difficult to express botulinum toxin, which has a size of 150 kDa, in a soluble form within E. coli cells. Furthermore, botulinum toxin contains interchain and intrachain disulfide bonds, which do not form within E. coli cells. Furthermore, as described above, botulinum toxin requires precise cleavage of the peptide bond between the light and heavy chains and their disulfide bonds. However, E. coli lacks a protein that cleaves the peptide bonds and reconnects the disulfide bonds, necessitating a separate, additional process.

[0007] The present invention aims to provide a method for safely and easily producing botulinum toxin through genetic recombination technology.

[0008] In addition, the present invention aims to provide a method for producing botulinum toxin in a high yield by expressing it in a water-soluble form.

[0009] The present invention relates to 1) a light chain (LC) of botulinum toxin, a translocation domain (H) of a heavy chain of botulinum toxin N ), 'LC-HN-Intein N', a botulinum toxin fragment in which Intein N is sequentially linked from the N-terminus to the C-terminus, and a soluble partner (SP) composed of a polypeptide, Intein C, and a receptor binding domain (H) of the botulinum toxin heavy chain. C ) are produced by expressing 'SP-Intein C-HC', which are botulinum toxin fragments sequentially linked from the N-terminus to the C-terminus; 2) The above LC-H N -Intein N and the above SP-Intein CH C 3) Using the protein trans-splicing method, the translocation domain (H) of the heavy chain is purified respectively. N ) and the receptor binding domain (H) of the heavy chain C ) by removing the 'SP-Intein C' linked to the heavy chain, the translocation domain (H) of the heavy chain is removed, with the soluble partner (SP) removed. N ) and the receptor binding domain of the heavy chain (H C ) and 4) the translocation domains (H) of the light chain (LC) and heavy chain N ) cleaves the peptide bond between the cleaved light chain (LC) and the translocation domain (H) of the heavy chain. N ) is provided. The present invention provides a method for producing botulinum toxin, characterized in that it comprises inducing a disulfide bond between the two.

[0010] In addition, the present invention provides 1) a light chain (LC) of botulinum toxin, a translocation domain (H) of a heavy chain of botulinum toxin N), 'LC-HN-Intein N', a botulinum toxin fragment in which Intein N is sequentially linked from the N-terminus to the C-terminus, and a soluble partner (SP) composed of a polypeptide, Intein C, and a receptor binding domain (H) of the botulinum toxin heavy chain. C ) are produced by expressing 'SP-Intein C-HC', which are botulinum toxin fragments sequentially linked from the N-terminus to the C-terminus; 2) The above LC-H N -Intein N and the above SP-Intein CH C 3) The light chain (LC) and heavy chain translocation domains (H) are purified respectively. N ) cleaves the peptide bond between the cleaved light chain (LC) and the translocation domain (H) of the heavy chain. N ) to induce a disulfide bond between the heavy chains; 4) using the protein trans-splicing method, the translocation domain (H) of the heavy chain N ) and the receptor binding domain (H) of the heavy chain C ) by removing the 'SP-Intein C' linked to the heavy chain, the translocation domain (H) of the heavy chain is removed, with the soluble partner (SP) removed. N ) and the receptor binding domain of the heavy chain (H C ) is combined; and a method for producing botulinum toxin is provided.

[0011] In the above method for producing botulinum toxin of the present invention, it is preferable that a purification tag is further combined with the intein N.

[0012] In the above method for producing botulinum toxin of the present invention, it is preferable that a purification tag is further combined with the water-soluble partner.

[0013] In the method for producing the botulinum toxin of the present invention, the botulinum toxin fragment may be expressed and produced in E. coli.

[0014] In the present invention, it was confirmed that Hc with low water solubility can be expressed in a water-soluble form by attaching a water-soluble partner.

[0015] Soluble partners are typically very large proteins, making their removal challenging. Additional genetic manipulation, such as the insertion of restriction enzyme recognition sequences, is required. However, the present invention demonstrates that the use of inteins facilitates their removal.

[0016] In addition, the present invention provides a receptor binding domain (H) of an intein and a botulinum toxin heavy chain. C ) was further fused with various solubilized proteins (MBP, GFP, Sumo) to the linked protein, and it was confirmed that maltose binding protein (MBP) had the best effect in increasing solubility expression.

[0017] Figure 1 shows the botulinum toxin fragment Fusion partner-R10-GP41.1 using various types of soluble partners (Fusion partner; GFP, SUMO, MBP) in E. coli. C -H C After expressing it, the results of confirming whether it is expressed in a water-soluble manner are shown.

[0018] In Figure 2, (A) is a botulinum toxin fragment MBP-R10-GP41.1 C -H C and botulinum toxin fragment LC-H N -GP41.1 N - Schematic representation of the structure of R10-H6. (B) in Fig. 2 shows the botulinum toxin fragment MBP-R10-GP41.1 produced in E. coli. C -H C and botulinum toxin fragment LC-H N-GP41.1 N -Shows the results of SDS-PAGE analysis of R10-H6.

[0019] In Figure 3 (A) is the botulinum toxin fragment MBP-R10-GP41.1 C -H C and botulinum toxin fragment LC-H N -GP41.1 N -The process of inducing a protein trans-splicing reaction by mixing R10-H6 and thereby producing a recombinant botulinum toxin (LC-HN, rBoNT) is shown. In Fig. 3, (B) shows the results of confirming the protein trans-splicing reaction product by SDS-PAGE.

[0020] In Fig. 4, (A) is MBP-R10-iCL C -H C and botulinum toxin fragment LC-H N -iCL N -R10-H6 was mixed to induce protein trans-splicing reaction, and the results of confirming the reaction product by SDS-PAGE are shown. In Fig. 4, (B) is MBP-R10-gCL. C -H C and botulinum toxin fragment LC-H N -gCL N -R10-H6 was mixed to induce protein trans splicing reaction, and the results of confirming the reaction product by SDS-PAGE are shown.

[0021] In Figure 5, (A) shows the protein trans-splicing reaction product (PTS) subjected to affinity chromatography (AC) to separate the reaction by-product (MBP-R10-GP41.1-R10-H6) and the non-reactant (LC-H N -GP41.1 N -R10-H6, MBP-R10-GP41.1C -H C ) is schematically shown as a process for removing the affinity chromatography. In Fig. 5, (B) shows the results of SDS-PAGE analysis of the pass (UB, unbinding) fraction, elution fraction, and washing fraction obtained through the affinity chromatography process.

[0022] In Fig. 6, (A) schematically shows the process of cleaving a recombinant botulinum toxin (LC-HN, rBoNT) between a botulinum toxin light chain (LC) and a botulinum toxin heavy chain (HN) by treating thrombin (thr). In Fig. 6, (B) shows the results of comparing SDS-PAGE of a sample of thrombin-treated recombinant botulinum toxin treated with a reducing agent (reduced, R) and a sample not treated with a reducing agent (non-reduced, NR) to confirm whether the recombinant botulinum toxin was accurately cleaved by thrombin treatment.

[0023] The present invention relates to 1) a light chain (LC) of botulinum toxin, a translocation domain (H) of a heavy chain of botulinum toxin N ), 'LC-HN-Intein N', a botulinum toxin fragment in which Intein N is sequentially linked from the N-terminus to the C-terminus, and a soluble partner (SP) composed of a polypeptide, Intein C, and a receptor binding domain (H) of the botulinum toxin heavy chain. C ) are produced by expressing 'SP-Intein C-HC', which are botulinum toxin fragments sequentially linked from the N-terminus to the C-terminus; 2) The above LC-H N -Intein N and the above SP-Intein CH C 3) Using the protein trans-splicing method, the translocation domain (H) of the heavy chain is purified respectively. N ) and the receptor binding domain (H) of the heavy chain C) by removing the 'SP-Intein C' linked to the heavy chain, the translocation domain (H) of the heavy chain is removed, with the soluble partner (SP) removed. N ) and the receptor binding domain of the heavy chain (H C ) and 4) the translocation domains (H) of the light chain (LC) and heavy chain N ) cleaves the peptide bond between the cleaved light chain (LC) and the translocation domain (H) of the heavy chain. N ) is provided. The present invention provides a method for producing botulinum toxin, characterized in that it comprises inducing a disulfide bond between the two.

[0024] The present inventors have disclosed a light chain (LC) of botulinum toxin and a translocation domain (H) of a heavy chain of botulinum toxin through Korean Patent No. 10-2610179. N ) and a botulinum toxin fragment comprising the receptor binding domain (H) of the botulinum toxin heavy chain. C ) were produced, and a method for safely producing botulinum toxin was developed by combining the botulinum toxin fragments using inteins.

[0025] However, when producing botulinum toxin as described above, the receptor binding domain (H) of the botulinum toxin heavy chain C ) had a problem in that it was expressed insoluble in E. coli, resulting in low yields during production.

[0026] However, in the present invention, a soluble partner is used to bind the receptor binding domain (H) of the botulinum toxin heavy chain. C ) was confirmed to be able to be expressed in water solubility.

[0027] Furthermore, soluble partners are typically very large-molecular-weight proteins, requiring additional genetic manipulation, such as inserting restriction enzyme recognition sequences, to remove them. However, the present invention has confirmed that using inteins allows for easy removal.

[0028] In addition, in the present invention, among various soluble partners, maltose binding protein (MBP) is used as a receptor binding domain (H) of the botulinum toxin heavy chain. C ) and it was confirmed that the effect of expressing proteins linked to inteins in a water-soluble manner was particularly excellent.

[0029] Hereinafter, the method for producing the botulinum toxin of the present invention will be described in more detail step by step.

[0030]

[0031] < Step 1: Production of botulinum toxin fragments >

[0032] This step is to determine the light chain (LC) of botulinum toxin and the translocation domain (H) of the heavy chain of botulinum toxin. N ), Intein N is sequentially linked from the 5′ end to the 3′ end, and the soluble partner (SP) consisting of a polypeptide, Intein C, and the receptor binding domain of the botulinum toxin heavy chain (H C ) is a process of producing botulinum toxin fragments, 'SP-Intein C-HC', which are sequentially linked from the 5' end to the 3' end, and is characterized by expressing the botulinum toxin fragments in a water-soluble manner.

[0033] In general, technologies are known for producing botulinum toxin by expressing the entire sequence in a connected state or by expressing the botulinum toxin light chain (LC) and botulinum toxin heavy chain (HC) in a split state.

[0034] However, this step involves the botulinum toxin light chain (LC) and the translocation domain (H) of the botulinum toxin heavy chain. N ) and a botulinum toxin fragment comprising the receptor binding domain (H) of the botulinum toxin heavy chain. C ) and expressed by splitting it into botulinum toxin fragments containing botulinum toxin, and is characterized by expressing botulinum toxin with higher water solubility.

[0035] Additionally, this step involves the receptor binding domain (H) of the botulinum toxin heavy chain. C ) by further linking it to a soluble partner, and expressing it by the receptor binding domain (H) of the botulinum toxin heavy chain. C ) is characterized by expressing a botulinum toxin fragment containing a soluble partner with higher water solubility. In the following embodiment of the present invention, the receptor binding domain (H) of the botulinum toxin heavy chain is expressed by using a soluble partner. C ) was able to increase the water-soluble expression ratio, and through this, it was confirmed that botulinum toxin could be produced at a higher yield.

[0036] In this step, the water-soluble partners refer to partners that can increase water-soluble expression by linking to botulinum toxin fragments, such as GFP, SUMO, and MBP. Meanwhile, according to the following example, among the water-soluble partners, maltose binding protein (MBP) binds to intein C and the receptor binding domain (H) of the botulinum toxin heavy chain. C ) linked to the botulinum toxin fragment (Intein CH) C ) has a particularly excellent effect on the expression of water solubility.

[0037] Meanwhile, when using a soluble partner, the soluble partner must be removed separately for commercialization as botulinum toxin. However, since the soluble partner is generally a protein with a very large molecular weight, additional genetic manipulation, such as inserting a restriction enzyme recognition sequence, is required to remove it, which presents a problem. However, the present invention uses intein, and it has been confirmed through the following examples that this can be easily removed without separate genetic manipulation.

[0038] The intein used in the present invention is a self-splicing protein, which is composed of intein N (N-terminal intein, intein N) and intein C (C-terminal intein, intein C). Before each intein meets each other in the cell, it exists separately from each other. After meeting each other, it folds, and thereafter acts as an enzyme to join the proteins (so-called exteins) located at the ends of each intein, and exhibits a mechanism of self-removal.

[0039] Inteins are not limited thereto, but can be Cfa, Npu, Ssp, Rma, Ppu, Cwa, Cra5, Csp8801, Csp0110, Mcht, Maer, Asp, Oli, Aov, Ter-3, Ssp7002, Tvu, Tel, Sel, Aha, GP41.1, etc. derived from DnaE or DnaB of various strains. In addition, the 3'-terminus of intein N and the 5'-terminus of intein C may further include a linker sequence.

[0040] Meanwhile, at this stage, the botulinum toxin fragment, 'LC-H N-Intein N' preferably further includes a purification tag at the 3' end, and the botulinum toxin fragment, 'SP-Intein C-HC' preferably further includes a purification tag at the 5' end. Through this, the purification can be more smoothly performed in the subsequent purification process. At this time, the purification tag is not limited to, but may be a histidine tag, a lysine tag, etc. However, in the case of a water-soluble partner that can be used as both a water-soluble partner and a purification tag, such as maltose binding protein (MBP), a separate purification tag may not be required.

[0041] Meanwhile, expression at this stage may be expressed using a plasmid in various host cells, such as E. coli.

[0042]

[0043] < Step 2: Purification of botulinum toxin fragments >

[0044] This step is to produce the botulinum toxin fragment LC-H through the above step 1. N -Intein N and botulinum toxin fragment, SP-Intein CH C It is a process of refining each.

[0045] The purification method in this step is not limited thereto, but may involve further linking a purification tag to a botulinum toxin fragment, expressing the botulinum toxin fragment, and then easily purifying it using the purification tag. Meanwhile, the maltose-binding protein used in the present invention has the property of binding to maltose, and thus can also be used as a purification tag.

[0046]

[0047] < Step 3: Conjugation of botulinum toxin fragments using protein trans-splicing method >

[0048] This step uses a protein trans-splicing method using inteins to generate the translocation domain (H) of the heavy chain. N ) and the receptor binding domain (H) of the heavy chain C ) is removed at once, and the translocation domain (H) of the heavy chain is removed with the soluble partner (SP) removed. N ) and the receptor binding domain of the heavy chain (H C ) by combining the full-length botulinum toxin (LC-H) N -H C ) is the process of manufacturing.

[0049] Meanwhile, when intein N and intein C meet, they link up and form a complete intein complex. Once the complete intein complex is formed, the intein complex is spliced ​​away and separated, and at this time, the extein proteins on the outside of the intein complex are linked by peptide bonds and become a single protein.

[0050] This step is a process of inducing a protein trans-splicing reaction using the above-described intein. In the present invention, it was confirmed that the protein trans-splicing reaction occurs intact even though a purification tag and a soluble partner are connected to the inside of intein N and intein C, and that when the intein complex is removed, the purification tag connected to the C-terminus of intein N and the soluble partner connected to the N-terminus of intein C can be removed together.

[0051] That is, in order for the protein trans-splicing process to occur using a split intein, the process of intein N and intein C recognizing each other and structurally combining must first be performed, so there is a problem that the protein tags located in front or behind the inteins must not interfere with their interaction and must not inhibit functional joining. However, in the case of the present invention, in the case of GP41.1 intein, there is a fragment of botulinum toxin on the outside and a purification tag or a solubilization tag on the inside, which interferes with binding and function, but it was confirmed that the binding force due to the interaction between intein N and intein C is not interfered with, and steric hindrance does not occur, so that the protein trans-splicing process can be performed intact.

[0052] Meanwhile, the characteristic of this step in which the purification tag and the soluble partner are removed together when the intein complex is removed as described above provides the advantage of not having to separately perform a process for removing other proteins such as the purification tag and the soluble partner linked to the botulinum toxin by peptide bonds.

[0053] In addition, the process of purifying botulinum toxin after the protein trans-splicing reaction process becomes simpler, which is an advantage. Specifically, the botulinum toxin fragment 'LC-H' manufactured by further using a purification tag such as a histidine tag N -Intein N-H6' and 'H6-SP-Intein CH C ', when inducing protein trans-splicing reaction, not only full-length botulinum toxin (BoNT) but also non-reactant (LC-H N -Intein N-H6, H6-SP-Intein CH C) and impurities such as by-products (H6-Intein-SP-H6) are mixed together (see A in Fig. 3). Since the impurities contain a histidine tag (H6), which is a purification tag, they can be easily removed through affinity chromatography using it. Meanwhile, if a water-soluble partner (SP) can serve as a purification tag, SP-Intein CH C The above effect can be achieved without using a separate purification tag, such as in the form (see A in Fig. 5).

[0054] Meanwhile, when the intein complex is removed through the protein trans-splicing reaction in this step, some residual sequences (so-called 'scars') of the intein complex may remain. These residual sequences may vary depending on the type of intein used. Therefore, in the present invention, it is preferable to use intein types that do not leave cysteine ​​residues, and more preferably, it is more preferable to use GP41.1. When GP41.1 is used, SGYSSS is left as the residual sequence during the protein trans-splicing process, and since cysteine ​​(C, cystein) is not included in the residual sequence, there is an advantage in that unnecessary disulfide bonds are not created. In addition, in the present invention, when GP41.1 is used among various types of inteins that do not leave cysteine ​​residues, GP41.1 N or GP41.1 C It was confirmed that the purification tag and soluble partner linked to the botulinum toxin fragment could be removed and linked.

[0055]

[0056] < Step 4: Translocation domains (H) of botulinum toxin light chain (LC) and heavy chain N ) cleavage of peptide bonds and induction of disulfide bonds >

[0057] For botulinum toxin to exhibit full activity, the light chain (LC) and the translocation domain (H) of the heavy chain must be N ) should be linked by a disulfide bond, not a peptide bond. This step is to obtain the full-length botulinum toxin (LC-H) obtained through the above step 3. N -H C ) of the light chain (LC) and the translocation domain (H) of the heavy chain N ) cleaves the peptide bond between the cleaved light chain (LC) and the translocation domain (H) of the heavy chain. N ) is a process for producing botulinum toxin that exhibits full activity by inducing disulfide bonds between them.

[0058] Meanwhile, this step may be performed before performing step 3 above.

[0059] Meanwhile, in this stage, the translocation domains (H) of the light chain (LC) and heavy chain of botulinum toxin N ) can be performed by protease treatment. That is, in step 1 above, the botulinum toxin fragment, LC-H N -When producing Intein N, LC and H N In the case of expression by inserting a protease recognition sequence between, the protease is treated in this step, and LC and H N It is possible to easily cleave the peptide bond between them.

[0060] Meanwhile, in this stage, the translocation domains (H) of the light chain (LC) and heavy chain of botulinum toxin N ) in the case of disulfide bond formation between the botulinum toxin fragments, LC-H, in step 1, may be induced through treatment with an oxidizing agent. N - Since intein N is already formed when it is expressed, a separate processing step may not be necessary. That is, the translocation domain (H) of the light chain (LC) and heavy chain of botulinum toxin N) contains cysteine, and when expressed in E. coli, a disulfide bond is naturally formed. If there is no separate reducing agent treatment, the disulfide bond is maintained intact, so there may be no need for separate treatment.

[0061]

[0062] Hereinafter, the present invention will be described in more detail through the following examples. However, the present invention is not limited to the following examples, and includes variations of equivalent concepts.

[0063]

[0064] [Example 1: Screening for a Soluble Partner Suitable for the Botulinum Toxin Production Method of the Present Invention]

[0065] The receptor binding domain of the botulinum toxin heavy chain (H C ) has the problem that most of them are expressed as insoluble when produced in E. coli.

[0066] In this embodiment, intein C C ) receptor binding domain of botulinum toxin heavy chain linked to C ) to determine whether soluble expression is enhanced. In addition, we aimed to determine which of the various soluble partners (GFP, SUMO, MBP) would exhibit the best effect.

[0067] Soluble partner (GFP, Green Fluorescent Protein, SEQ ID NO: 1; SUMO, Small Ubiquitin-like Modifier, SEQ ID NO: 2; MBP, Maltose-Binding Protein, SEQ ID NO: 3), linker (R10, SEQ ID NO: 4), GP41.1 C Intein C (SEQ ID NO: 5), receptor binding domain of botulinum toxin heavy chain (H C, sequence number 13) were sequentially linked to a plasmid (SP-R10-GP41.1 C -H C ) was manufactured and transformed into E. coli BL21 (DE3).

[0068] The transformed E. coli was inoculated into 10 ml of Terrific broth and cultured at 37°C and 200 rpm for 16 hours. 8 g of glycerol was added to 1 L of Terrific broth to prepare a main culture medium, and 10 ml of the above-mentioned culture solution was inoculated and cultured at 37°C and 200 rpm for 2 hours to determine the OD of the culture solution. 600 This was done to reach 0.5. IPTG was added to the above main culture solution to make it 1 mM, and the main culture was performed at 16°C and 200 rpm for 16 hours.

[0069] The above main culture medium was centrifuged at 4℃, 5210 xg for 15 min, and the supernatant was completely removed. The pellet obtained by removing the supernatant was resuspended in 150 ml of buffer (50 mM HEPES, 150 mM NaCl, pH 7.4), and 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 xg, and separated into the supernatant and the pellet.

[0070] Afterwards, the supernatant obtained through the above process was used as the soluble fraction (S), and the pellet was used as the insoluble fraction (I) and subjected to SDS-PAGE (Fig. 1).

[0071] As shown in Figure 1, when GFP or SUMO is used as a soluble fusion partner, a larger band appears in the insoluble fraction (I). On the other hand, when MBP is used as a soluble fusion partner, a larger band appears in the soluble fraction (S).

[0072] Typically, the receptor binding domain (H) of the botulinum toxin heavy chain of botulinum toxin C ) has very low water solubility, but through this example, it was confirmed that it was expressed in a state where water solubility was increased by water-soluble partners such as GFP, SUMO, and MSP, and 'intein CH C It was confirmed that the effect of enhancing the expression of receptiveness was particularly excellent when MBP was used.

[0073]

[0074] [Example 2: Production of botulinum toxin using a soluble partner]

[0075] The method for producing botulinum toxin of the present invention comprises producing botulinum toxin by LC-H N -intein N and intein CH C After producing it in two parts, H N and H C It is characterized by producing botulinum toxin by splicing proteins using the protein trans-splicing method.

[0076] In this example, we aimed to determine whether a soluble partner (SP) linked to the N-terminus of intein C interferes with the intein's function when performing the protein trans-splicing method described above. Furthermore, we attempted to determine whether the effects differed depending on the type of intein used, using various inteins.

[0077] Among various types of inteins, the Israel group Cystein-Less (iCL) intein, the Germany group Cystein-Less (gCL) intein, and the GP41.1 intein were used for comparison, which do not leave cysteine ​​as a scar during protein trans-splicing. Meanwhile, the Israel group Cystein-Less (iCL) intein leaves the residue sequence YIDTDSVYLN, the Germany group Cystein-Less (gCL) intein leaves the residue sequence SGDTDS, and the GP41.1 intein leaves the residue sequence SGYSSS.

[0078]

[0079] 2-1. Production and purification of botulinum toxin fragments

[0080] 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 N , SEQ ID NO: 6), linker (R10, SEQ ID NO: 4), and histidine tag (H6 tag, SEQ ID NO: 15) were sequentially linked to a plasmid (LC-H N -GP41.1 N -R10-H6) was manufactured and transformed into E. coli BL21 (DE3).

[0081] The transformed E. coli was inoculated into 10 ml of Terrific broth and cultured as a seed at 37°C and 200 rpm for 16 hours. 1 L of Terrific broth was added with 8 g of glycerol to prepare a main culture medium, and 10 ml of the above-mentioned seed culture was inoculated, and the main culture was cultured at 37°C and 200 rpm for 2 hours until the OD600 nm of the culture reached 0.5. 1 mM IPTG was added to the main culture, and the main culture was cultured at 16°C and 200 rpm for 16 hours.

[0082] The above main culture medium was centrifuged at 4°C, 5210 xg for 15 min, and the supernatant was removed. The pellet obtained by removing the supernatant was resuspended in 150 ml buffer (50 mM HEPES, 150 mM NaCl, pH 7.4), and 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 xg to obtain the supernatant, which was then filtered through 0.22 μm to isolate the light chain (LC) of botulinum toxin and the translocated domain (H) of the heavy chain of botulinum toxin. N ) containing botulinum toxin fragment (LC-H) N -GP41.1 N -R10-H6) was purified.

[0083] Meanwhile, the botulinum toxin fragment MBP-R10-GP41.1 obtained through the above process and Example 1 C -H C (97.7 kDa) and botulinum toxin fragment LC-H N -GP41.1 N -R10-H6 (112.0 kDa) was purified using cation chromatography, and it was confirmed that the two botulinum toxin fragments were produced intact (B in Fig. 2).

[0084] In addition, the same process as above and the same method as the process of Example 1 are used to produce and purify, but the type of intein (iCL) C : Sequence number 7, iCL N : Sequence number 8, gCL C : Sequence number 9, gCL N : Sequence number 10) and a botulinum toxin fragment (LC-H) N -iCL N -R10-H6, LC-H N -gCL N -R10-H6, MBP-R10-iCL C -H C , MBP-R10-gCL C -H C) was produced and refined.

[0085] Meanwhile, through this process, it was confirmed that the expression level of botulinum toxin was higher when GP41.1 intein was used among the Israel group Cystein-Less (iCL) intein, the Germany group Cystein-Less (gCL) intein, and the GP41.1 intein.

[0086]

[0087] 2-2. Splicing of botulinum toxin fragments using protein trans-splicing method

[0088] Botulinum toxin fragment (LC-H) prepared through the above process 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 to each of the 2 mM TCEP (tris(2-carboxyethyl)phosphine) and left for 10 minutes.

[0089] Afterwards, the light chain (LC) of botulinum toxin and the translocation domain (H) of the heavy chain of botulinum toxin N ) containing 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 of the botulinum toxin heavy chain (H C ) containing a 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 left at 20°C and 100 rpm for 2 hours to allow protein trans-splicing junction reaction to occur (Figs. 3, 4).

[0090] As shown in Figure 3, the protein trans-splicing process occurs completely, resulting in recombinant botulinum toxin (LC-H N -H C , rBoNT, 149.5 kDa) is produced. On the other hand, looking at Figure 4, it can be confirmed that the protein trans-splicing process does not occur when the Israel group Cystein-Less (iCL) intein and the Germany group Cystein-Less (gCL) intein are used as inteins.

[0091] This allows the intein to function intact when splicing botulinum toxin fragments using inteins, even though the histidine tag and soluble partner are in positions that interfere with the action of the intein, resulting in the H of each botulinum toxin fragment. N Wow H C We confirmed that the histidine tag and the soluble partner can be removed together while connecting them.

[0092]

[0093] 2-3. Production and purification of active botulinum toxin through protease treatment

[0094] First, the protein trans-splicing reaction product obtained through the above Example 2-2 was dialyzed to remove the reducing agent TCEP by replacing the buffer (20 mM Tris, 150 mM NaCl, 2.5 mM CaCl2, pH 8.0).

[0095] Afterwards, the reaction by-product 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 It could be removed through affinity chromatography (IMAC & MBP) using MBP or H6 at the terminal (Fig. 5B).

[0096] Specifically, looking at B of Figure 5, recombinant botulinum toxin (rBoNT) is not bound to the column and is detected in the unbinding fraction, whereas MBP-R10-GP41.1-R10-H6, LC-H N -GP41.1 N -R10-H6, MBP-R10-GP41.1 C -H C It can be confirmed that it is bound to the column and detected in the elution fraction.

[0097] Meanwhile, the recombinant botulinum toxin (rBoNT) purified from the above process was purified again using anion chromatography, and then 10 units of thrombin were added and reacted at 20°C for 16 hours to obtain recombinant botulinum toxin (rBoNT) LC-H. N By removing the peptide bond between them, it was possible to produce an active form of recombinant botulinum toxin (Fig. 6B).

[0098] Looking at B of Figure 6, when looking at the SDS page results of the non-reduced (NR) sample that was not treated with a reducing agent, rBoNT is detected, but when looking at the SDS page results of the reduced (R) sample that was treated with a reducing agent and had its disulfide bonds broken, rBoNT is reduced and LC and HC are more detected.

[0099] Through this, it was confirmed that the active form of botulinum toxin, in which the LC and HC are precisely cleaved by thrombin treatment and the LC and HC are linked by a disulfide bond, was produced intact. Furthermore, it was confirmed that the dimer form of botulinum toxin was not formed by unnecessary disulfide bonds.

Claims

1. 1) Light chain (LC) of botulinum toxin, translocation domain (H) of heavy chain of botulinum toxin N ), 'LC-HN-Intein N', a botulinum toxin fragment in which Intein N is sequentially linked from the N-terminus to the C-terminus, and a soluble partner (SP) composed of a polypeptide, Intein C, and a receptor binding domain (H) of the botulinum toxin heavy chain. C ) is a botulinum toxin fragment sequentially linked from the N-terminus to the C-terminus, 'SP-Intein CH C ' are expressed and produced respectively; 2) The above LC-H N -Intein N and the above SP-Intein CH C Each is purified; 3) Using the protein trans-splicing method, the translocation domain (H) of the heavy chain N ) and the receptor binding domain (H) of the heavy chain C ) by removing the 'SP-Intein C' linked to the heavy chain, the translocation domain (H) of the heavy chain is removed, with the soluble partner (SP) removed. N ) and the receptor binding domain of the heavy chain (H C ) and combine them; 4) The translocation domains (H) of the light chain (LC) and heavy chain N ) cleaves the peptide bond between the cleaved light chain (LC) and the translocation domain (H) of the heavy chain. N ) to induce a disulfide bond between them. A method for producing botulinum toxin, characterized in that it comprises: 2.1) Light chain (LC) of botulinum toxin, translocation domain (H) of heavy chain of botulinum toxin N ), 'LC-HN-Intein N', a botulinum toxin fragment in which Intein N is sequentially linked from the N-terminus to the C-terminus, and a soluble partner (SP) composed of a polypeptide, Intein C, and a receptor binding domain (H) of the botulinum toxin heavy chain. C ) is a botulinum toxin fragment sequentially linked from the N-terminus to the C-terminus, 'SP-Intein CH C ' are expressed and produced respectively; 2) The above LC-H N -Intein N and the above SP-Intein CH C Each is purified; 3) The translocation domains (H) of the light chain (LC) and heavy chain N ) cleaves the peptide bond between the cleaved light chain (LC) and the translocation domain (H) of the heavy chain. N ) induces disulfide bonds between them; 4) Using the protein trans-splicing method, the translocation domain (H) of the heavy chain N ) and the receptor binding domain (H) of the heavy chain C ) by removing the 'SP-Intein C' linked to the heavy chain, the translocation domain (H) of the heavy chain is removed, with the soluble partner (SP) removed. N ) and the receptor binding domain of the heavy chain (H C ) and a method for producing botulinum toxin, characterized in that it comprises combining the following.

3. In paragraph 1 or 2, A method for producing botulinum toxin, characterized in that a purification tag is further combined with the above intein N.

4. In paragraph 1 or 2, A method for producing botulinum toxin, characterized in that the above water-soluble partner is further combined with a purification tag.

5. In paragraph 1 or 2, The above expression is, A method for producing botulinum toxin characterized by being expressed in E. coli.

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