Patent Application
20240132540
The present application requires priority to Application No. 202110217756.1, filed Feb. 26, 2021, which is incorporated herein in its entirety by reference.
The present invention relates to the field of biotechnology and bioengineering, and particularly, to a method for preparing a modified toxin polypeptide.
Polypeptides are a group of compounds formed by the connection of a plurality of amino acids through peptide bonds, generally consisting of 10-100 amino acid molecules, with the same connection manner as proteins and a relative molecular mass of less than 10000. Polypeptides are very common in organisms. Tens of thousands of polypeptides have been found in organisms so far, which are widely involved in regulating the functional activities of various systems, organs, tissues, and cells in the organisms, playing an important role in life activities.
Polypeptide drugs refer to polypeptides with specific therapeutic effects, acquired from chemical synthesis and gene recombination or extracted from animals and plants, and consist of a specific application of polypeptides in the field of pharmaceuticals. Polypeptides have broad and important bioactivities, and can be widely applied to the endocrine system, immune system, digestive system, cardiovascular system, blood vessel system, musculoskeletal system, and the like. As such, the development of polypeptide as drugs has a short history but a fast pace, and has become a hot spot in the market at present. Polypeptides are mainly used for treating serious diseases related to cancers and metabolic disorders, and drugs related to such diseases have fairly important markets all over the world.
Toxin polypeptides occupy a certain share of the polypeptide drugs, including native toxin polypeptides or modified toxin polypeptides produced by genetic recombination. Such toxin polypeptides are well known for their effects in spasm treatment, cosmetology, and antitumor therapies as native molecules or fusion proteins.
Clostridial neurotoxins (CNTs) are known to include seven different serotypes of botulinum neurotoxins (BoNTs) and tetanus toxin (tetX, or tetanus neurotoxin, TeNT), all of which comprise two polypeptide molecules linked by disulfide bonds: a light chain (L) of about 50 KDa and a heavy chain (H) of about 100 KDa. The light chain comprises a protease active region and the heavy chain comprises a translocation domain (N terminus) and a receptor-binding domain (C terminus). All serotypes of botulinum neurotoxins (BoNTs) and tetanus toxins (tetX) produced by Clostridium tetani are Zn2+ proteases. They prevent synaptic exocytosis, inhibit neurotransmitter release, and interrupt nerve signaling by cleaving a protein involved in the formation of SNARE complex that controls cell membrane fusion. Botulinum neurotoxin type A (BoNT/A) has been approved for the treatment of strabismus, blepharospasm, and other diseases in 1989 in the United States. In some applications, the botulinum toxin is injected directly into the muscles to be treated along with an additional bacterial protein in the form of a complex, and the toxin is released from the protein complex at the physiological pH (Eisele et al., 2011, Toxicon 57 (4): 555-65.) to exert the desired pharmacological effect. The FDA has also approved botulinum neurotoxin type B for the treatment of cervical dystonia.
Diphtheria toxin and pseudomonas exotoxin are commonly used to construct toxin fusion protein-based targeted drugs. The native diphtheria toxin consists of 535 amino acids, with two subunits linked by two disulfide bonds connected to a loop of 14 amino acids. The two subunits of diphtheria toxin comprise three protein domains: a protease domain, a receptor-binding domain, and a translocation domain. Once the protease domain enters the cytoplasm, it prolongs the ADP-ribosylation effect of catalytic factor-2, resulting in the inhibition of protein synthesis and cell death. Therefore, it has been reported that the native receptor-binding domain of diphtheria toxin can be replaced with a receptor-binding domain targeting a tumor cell by genetic engineering, and the fusion protein exerts the toxicity of diphtheria toxin through targeting a specific cell (“Targeting Killing Effect of IL-13 Diphtheria Toxin Fusion Protein and DT389-hIL13-13E13K”, Du Juan et al., Journal of Medical Research, 2008 (037) 010, 31-36).
Pseudomonas exotoxin is structurally similar to diphtheria toxin and also has three domains: a protease domain, a receptor-binding domain, and a translocation domain. At present, a series of immune fusion toxins using a pseudomonas toxin as the killing group have been constructed, and interleukins, growth factors, single-chain antibodies, and the like are used as specific ligands (Purification and Renaturation of Recombinant Human Interleukin 2-Pseudomonas Exotoxin (IL2-PE664Glu) Fusion Protein, Hu Zhiming, et al., Journal of First Military Medical University, 1000-2588 (2002) 03-0206-02).
Cholera toxin (CTX) is a toxin polypeptide produced by Vibrio cholerae, which causes serious diarrhea and dehydration in humans. CTX is an oligo-protein with a molecular weight of about 84 KDa, consisting of one A subunit and 5 B subunits surrounding the A subunit. The B subunits are responsible for recognizing and binding the holotoxin to the GM1 ganglioside receptor on the surface of a mammalian cell, and promote the entrance of the A subunit into the cell; the A subunit bears ADP-ribosyl-transferase activity, which down-regulates Gs protein expression and activates AC enzyme, thereby promoting an increase in cAMP level. The A subunit also can ADP-ribosylate the transporter of the outer segment membrane disc of rod cells to inactivate GTPase. Due to the ubiquity of the GM1 ganglioside receptor on eukaryotic cell membranes, CTX is used in a variety of model systems to activate adenylate cyclase (AC). CTX is also a mucosal vaccine adjuvant that induces immune responses of type 2 helper T cells by inhibiting IL-12 production. The fusion protein prepared by utilizing the protease function of the A subunit of the cholera toxin and the targeting recognition and binding functions of the B subunits can be applied to the antitumor treatment field (CN201910673683.X).
Although the role of toxin polypeptides in medical and cosmetic aspects and the role of the fusion protein thereof in antitumor therapies have been recognized, the toxicity of toxin polypeptides makes it necessary to consider environmental reservation and operator protection during the preparation process, and thus the preparation of the toxin polypeptide is usually carried out in a BL-3 environment or in a large-scale isolator, which undoubtedly increases the production cost, and is unfavorable for industrial mass production of such toxins.
In order to overcome the defects in the prior art, the present invention provides a method for preparing a modified toxin polypeptide, specifically as follows:
In a first aspect of the present invention, a method for preparing a modified toxin polypeptide is provided, comprising: step 1), expressing a modified toxin polypeptide precursor; step 2), enriching the toxin polypeptide precursor; and step 3), activating the toxin polypeptide precursor to obtain the modified toxin polypeptide.
Preferably, the step 1) comprises: (1) constructing a nucleic acid molecule encoding the toxin polypeptide precursor; (2) constructing a vector comprising the nucleic acid molecule; (3) transferring the nucleic acid vector into a suitable host cell; and (4) culturing the host cell, and allowing or inducing the host cell to express the toxin polypeptide precursor encoded by the nucleic acid vector.
More preferably, the step 1) further comprises: (5) fermenting a cell capable of expressing the toxin polypeptide precursor.
Even more preferably, the step 1) further comprises: (6) lysing the cell expressing the toxin polypeptide to obtain a cell lysate comprising the toxin polypeptide precursor.
Preferably, the step 2) comprises enriching the toxin polypeptide precursor by conducting a multiplex filtration procedure.
More preferably, the multiplex filtration comprises a crude liquid filtration and a feed liquid circulation filtration.
Even more preferably, the crude liquid filtration step separates the lysate into a feed liquid and a waste residue.
Even more preferably, a material of the crude liquid filtration has a pore size of 0.1-0.65 pm.
Even more preferably, substances that cannot pass through the crude liquid filtration are discharged as the waste residue.
Even more preferably, the waste residue is separated from the material of the crude liquid filtration under the effect of a buffer.
Preferably, the substance entering the crude liquid filtration is switched from the lysate to the buffer when the filtration rate of the crude liquid is lower than 50% of the initial rate.
Even more preferably, the feed liquid circulation filtration step filters the feed liquid multiple times. In the process of multiple filtering, the feed liquid that meets the requirement of the finished liquid collection is collected as the finished liquid, and the feed liquid that meets the requirement of the waste liquid is discharged as the waste liquid.
Even more preferably, the requirement of finished liquid collection is the turbidity of the feed liquid, and when OD600<0.1, the feed liquid is collected as the finished liquid.
Even more preferably, the requirement of waste feed liquid discharge is the turbidity of the feed liquid, and when OD600>0.1, the feed liquid is discharged as the waste feed liquid.
Even more preferably, the requirement of waste feed liquid discharge is that the feed liquid does not meet the requirement of the finished liquid collection after 2 or more cycles of circulation.
Even more preferably, the material of the crude liquid filtration and the material of the feed liquid circulation filtration are selected from a hydrophilic filtration material or a hydrophobic filtration material.
Even more preferably, the hydrophilic filtration material is selected from a cellulose ester, polyethersulfone, polyethylene or the like, or derivatives thereof.
Even more preferably, the hydrophobic filtration material is selected from polyvinylidene fluoride, polypropylene, polytetrafluoroethylene or the like, or derivatives thereof.
More preferably, a lysate separation device is enclosed during operation.
Even more preferably, the lysate separation device does not require an additional isolation measure, for example, an isolator.
Preferably, the step 3) comprises digesting the toxin polypeptide precursor by using a protease to obtain the modified toxin polypeptide.
More preferably, the toxin polypeptide is present in the form of the toxin polypeptide precursor with low toxicity in a cell or a lysate, and the toxin polypeptide with high toxicity is obtained after a protease activation step.
Even more preferably, the low toxicity is relative to the high toxicity after the activation. In a specific embodiment, the activity of the toxin polypeptide after the activation is at least 5000 folds higher than that of the toxin polypeptide precursor, and still further, the activity of the toxin polypeptide after the activation is 6000, 7000, 8000, 9000, 10000, 12000, 15000 or more folds higher than that of the toxin polypeptide precursor.
Preferably, the method further comprises step 4), purifying the toxin polypeptide.
More preferably, the step 4) comprises at least one chromatography procedure. Even more preferably, the step 4) may comprise 2, 3, or more chromatography procedures.
More preferably, the chromatography procedures can be identical or different.
More preferably, the chromatography procedure is affinity chromatography, gel filtration chromatography, and/or ion chromatography.
More preferably, the activation procedure of the step 3) and the purification procedure of step 4) are conducted in an isolator.
Preferably, the toxin polypeptide precursor comprises a second polypeptide fragment, comprising:
More preferably, the metal ion-dependent protease activity domain is a Zn2+-dependent protease activity domain.
More preferably, the first functional amino acid structural region and/or the second functional amino acid structural region are encoded by a natural sequence and/or an artificially synthesized sequence.
Even more preferably, the first functional amino acid structural region of the toxin polypeptide precursor comprises a Zn2+ protease binding domain of the light chain of clostridial neurotoxin.
Even more preferably, a target cell of the receptor-binding domain capable of binding to an antigen on the surface of a human cell in the second functional amino acid structural region refers to a target cell with an SNARE complex. For example, a nerve cell, a pancreatic cell, or other cells with an SNARE complex.
Even more preferably, the target cell of the second functional amino acid structural region refers to a human nerve cell or a pancreatic cell, and the receptor-binding domain is a receptor-binding domain capable of specifically binding to the human nerve cell or the pancreatic cell.
Even more preferably, the receptor-binding domain of the second functional amino acid structural region is a cell surface antigen-binding domain of the heavy chain of clostridial neurotoxin, and the translocation domain of the second functional amino acid structural region that mediates the transfer of the polypeptide across the vesicle membrane is a domain that mediates the transfer of clostridial neurotoxin across the vesicle membrane.
Even more preferably, the clostridial neurotoxin is a botulinum neurotoxin or tetanus toxin.
Even more preferably, the botulinum neurotoxin is selected from any one of serotypes BoNT/A to BoNT/H and derivatives thereof known in the art.
Even more preferably, the clostridial neurotoxin is selected from any one of tetanus toxin or a derivative thereof. More preferably, the first functional amino acid structural region comprises part or all of the light chains of BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H or tetanus toxin.
Even more preferably, the second functional amino acid structural region comprises part or all of the heavy chains of BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H or tetanus toxin.
In one specific embodiment, the first functional amino acid structural region is the light chain of BoNT/A, and the second functional amino acid structural region is the heavy chain of BoNT/A.
Even more preferably, the first functional amino acid structural region and the second functional amino acid structural region may be from different serotypes. The first functional amino acid structural region and the second functional amino acid structural region may be any combination of the above serotypes. For example, the first functional amino acid structural region is derived from the light chain of BoNT/A and the second functional amino acid structural region is derived from the heavy chain of BoNT/B, or the first functional amino acid structural region is derived from the light chain of BoNT/A and the second functional amino acid structural region is derived from the heavy chain of BoNT/C, and the like.
Even more preferably, the second polypeptide fragment has an amino acid sequence comprising a fragment set forth in SEQ ID NO: 7.
Preferably, the toxin polypeptide precursor further comprises a first polypeptide fragment, comprising a tag protein.
More preferably, the first polypeptide fragment further comprises a structural region of a first protease cleavage site.
More preferably, the first polypeptide fragment further comprises a short linker peptide.
In a specific embodiment, the toxin polypeptide precursor comprises:
The term “tag protein” refers to a class of protein molecules that can bind to a specific ligand.
The “tag protein” can increase the solubility of the toxin polypeptide precursor in a host.
The “tag protein” allows the presence of the toxin polypeptide precursor in a host cell in a soluble manner.
More preferably, the tag protein is selected from a tag protein known to those skilled in the art or a tag protein designed by a computer program, and is capable of specifically binding to a known substrate.
Even more preferably, the tag protein is selected from, but not limited to, the following proteins: a glutathione S-transferase (GSTs), C-myc, chitin-binding domain, maltose-binding protein (MBP), SUMO heterologous affinity moiety, monoclonal antibody or protein A, streptavidin-binding protein (SBP), cellulose-binding domain, calmodulin-binding peptide, S tag, Strep tag II, FLA, protein A, protein G, histidine affinity tag (HAT), or polyhistidine.
In a specific embodiment, the tag protein is located at an N terminus of the toxin polypeptide precursor.
In a specific embodiment, the tag protein is a glutathione S-transferase (GSTs), and more preferably, the amino acid sequence of the glutathione S-transferase is set forth in SEQ ID NO: 2.
More preferably, neither the first protease cleavage site nor the second protease cleavage site can be cleaved by a human protease or a protease produced by the host cell expressing the toxin polypeptide precursor.
More preferably, the first protease cleavage site and the second protease cleavage site are identical or different.
The first protease cleavage site and the second protease cleavage site are not recognized and cleaved by the host cell when being expressed or by an endogenous protease of an organism when being used.
More preferably, the activation of the step 3) comprises: digesting the second protease cleavage site in the toxin polypeptide precursor by using a second protease.
Even more preferably, the activation further comprises: digesting the first protease cleavage site in the toxin polypeptide precursor by using a first protease.
In a specific embodiment, the first protease and the second protease are identical.
More preferably, the specific protease is selected from, but not limited to, one of or a combination of two of the following proteases: a non-human enterokinase, a tobacco etch virus protease, a protease derived from Bacillus subtilis, a protease derived from Bacillus amyloliquefaciens, a protease derived from rhinovirus, papain, a homologue of papain of an insect, or a homologue of papain of a crustacean.
In a specific embodiment, the protease that specifically recognizes the first protease and the protease that specifically recognizes the second protease cleavage site are both derived from the proteases of rhinoviruses.
More preferably, the second enzyme cleavage site is embedded into, or partially replaces or completely replaces a natural loop region between a first functional peptide fragment and a second functional peptide fragment. The embedding refers to an embedding between two certain amino acids in the loop region; the partial replacement refers to that the second enzyme cleavage site replaces part of the amino acid sequence of the loop region; the complete replacement refers to that the amino acid sequence of the natural loop region is completely replaced by the second enzyme cleavage site.
More preferably, the first protease cleavage site and the second protease cleavage site are selected from, but not limited to, one of or a combination of two of the following enzyme cleavage sites: DDDDK, EXXYXQS/G, HY, YH, or LEVLFQGP.
In a specific embodiment, the first protease cleavage site and the second protease cleavage site are both LEVLFQGP. More preferably, the short linker peptide has no more than 5 amino acids.
Even more preferably, the short linker peptide enables the first protease cleavage site to be more easily recognized or bound to by a protease thereof.
Even more preferably, the short linker peptide does not affect the function of the toxin polypeptide precursor.
Even more preferably, the short linker peptide retained at the N terminus of the second polypeptide fragment does not affect the function of the second polypeptide fragment after the cleavage of the first protease cleavage site.
Even more preferably, a GS part of the short linker peptide has no more than 5 amino acid residues; more preferably, the short linker peptide is selected from a glycine-serine (GS for short) short peptide, GGS, GGGS, GGGGS, GSGS, GGSGS, GSGGS, GGSGS, GGGSS, and other short linker peptides.
In a specific embodiment, the amino acid sequence of the structural region comprising the first protease cleavage site and the short linker peptide is LEVLFQGPLGS.
In a specific embodiment, the toxin polypeptide precursor sequentially comprises, from the N terminus: the glutathione S-transferase, LEVLFQGPLGS, the light chain of BoNT/A, LEVLFQGP, and the heavy chain of BoNT/A.
More preferably, the toxin polypeptide precursor has an amino acid sequence set forth in SEQ ID NO: 11.
Preferably, the nucleic acid molecule in step (1), constructing a nucleic acid molecule encoding the toxin polypeptide precursor, wherein the nucleic acid molecule is a nucleic acid molecule encoding the toxin polypeptide precursor described above. More preferably, the nucleic acid molecule consists of a nucleotide sequence encoding various parts of the toxin polypeptide precursor.
In a specific embodiment, the sequence encoding the tag protein comprises a sequence set forth in SEQ ID NO: 1.
In a specific embodiment, the nucleotide sequence encoding the structural region comprising the first protease cleavage site comprises a sequence set forth in SEQ ID NO: 3.
In a specific embodiment, the nucleotide sequence encoding the structural region comprising the second protease cleavage site comprises a sequence set forth in SEQ ID NO: 8.
In a specific embodiment, the amino acid sequence of the second polypeptide fragment comprises a sequence set forth in SEQ ID NO: 7.
In a specific embodiment, the nucleotide sequence encoding the second polypeptide fragment comprises a sequence set forth in SEQ ID NO: 6.
In a specific embodiment, the nucleotide sequence encoding the toxin polypeptide precursor is set forth in SEQ ID NO: 10.
Preferably, in step (2), constructing a vector comprising the nucleic acid molecule, the vector comprises the nucleic acid molecule described above or an open reading frame encoding the toxin polypeptide precursor described above.
More preferably, the vector is a plasmid, a phage, a viral vector, or the like.
Preferably, in step (3), transforming the nucleic acid vector into a suitable host cell, the cell is a eukaryotic cell or a prokaryotic cell.
More preferably, the cell is selected from Escherichia coli, a yeast, cyanobacterium or a mammalian cell, an insect cell, a plant cell, or an amphibian cell.
Even more preferably, the cell is Escherichia coli.
Compared with a conventional method for preparing a toxin polypeptide, in the method of the present invention, the toxin polypeptide is expressed as a toxin polypeptide precursor with low toxicity firstly, and activated to become an active toxin polypeptide after enrichment in a large volume, such that the harm of procedures in a large volume to the environment and workers is greatly reduced, and the cost of isolation equipment is reduced. The enrichment procedure of the toxin polypeptide precursor is conducted when a lysate of a large volume is processed, and the yield of a target protein can be improved by using methods of multiplex filtration and graded discharge of the waste. The method is especially suitable for a polypeptide with low toxicity. After the completely enclosed separation procedure of the cell lysate, the obtained waste liquid almost contains no toxin polypeptide precursor with low toxicity, with very little toxicity to the operation environment, and can be directly discharged after simple disinfection treatment.
After further combination with a purification procedure, the content of the toxin polypeptide in the finished liquid can be greater than 90%, and the method provided by the present invention is easier to produce and purify the toxin polypeptide as compared with the prior art due to its higher yield, higher purity, and higher safety, and can be used for preparing a toxin polypeptide of high purity on a large scale for clinical applications.
The present invention is further described with reference to the following specific examples, and the advantages and features of the present invention will be clearer as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It should be appreciated by those skilled in the art that modifications and replacements can be made to the details and form of the technical solutions of the present invention without departing from the spirit and scope of the present invention and that all these modifications and replacements fall within the scope of the present invention.
The process of the method of the present invention is shown in
1. the nucleic acid molecule comprises sequentially from the 5′ end:
There can also be no natural loop region between the first functional amino acid structural region and the second functional amino acid structural region. For example, the KTKSLDKGYNK linker sequence between the light chain and the heavy chain may be removed to reduce non-specific protease cleavage.
The nucleotide sequence encoding a toxin polypeptide precursor is set forth in SEQ ID NO: 10, the toxin polypeptide precursor encoded by the nucleotide sequence has an amino acid sequence set forth in SEQ ID NO: 11.
The genetically optimized GSTs-BoNT/A was artificially synthesized in step (I), and NdeI and NotI enzyme cleavage sites were synthesized and added at the two ends thereof. The GSTs-BoNT/A was digested with the NdeI and NotI at 37° C. (New England Biolabs), purified by using a QIquick gel extraction kit (Qiagen), and inserted into NdeI and NotI sites in a pET28a (Novagen) plasmid vector by using a T4 DNA ligase (NEB).
(III): Transferring the Plasmid Constructed in Step (II) into a Host Cell
1. Preparation of competent cells: A tube of E. coli BL21 DE3 cells (New England Biolabs) was inoculated into a test tube containing 3 mL of LB culture medium and cultured with shaking at 37° C. overnight. On the next day, 0.5 mL of the bacterial culture was inoculated into a 250-mL flask containing 50 mL of LB culture medium and cultured with vigorous shaking (250 rpm) at 37° C. for about 3-5 h. When the OD value of the bacterial colony reached 0.3-0.4 at 600 nm, the flask was transferred to an ice bath and incubated for 10-15 mM. The bacterial culture was poured into a 50-mL centrifuge tube and centrifuged at 1000 g at 4° C. for 10 mM under an aseptic condition. The supernatant was discarded, and the cells were collected. 10 mL of 0.1 M CaCl2 was added into the centrifuge tube, and the mixture was mixed well with shaking to resuspend the bacterial cells. The cells were then subjected to an ice bath for 30 mM, and centrifuged at 1000 g at 4° C. for 10 mM. The supernatant was discarded, and 4 mL of 0.1 M CaCl2 pre-cooled with ice was added to resuspend the collected bacterial cells. The cells were aliquoted at 0.2 mL per tube and stored at 4° C. for later use within 24 h, and the remaining samples were stored in a low-temperature freezer at −70° C.
2. Transfection: An appropriate amount of DNA (ng) and the competent E. coli were mixed, placed in an ice bath for 15 min, heat-shocked at 42° C. for 30 s, placed in the ice bath for 5 mM, shaken at 250 rpm for 1 h in an SOC medium, smeared on a plate with antibiotics, and cultured at 37° C. overnight.
The composition and ratio of the culture medium: 11.8 g/L of tryptone, 23.6 g/L of yeast extract, 9.4 g/L of K2HPO4, 2.2 g/L of KH2PO4, and 4 mL/L of glycerol.
The culture condition: The cells were cultured with shaking at 250 rpm at 37° C. overnight.
Inducing expression and fermentation: The cells were transferred to a fermentor and 1 mM IPTG was added to induce the expression at 25° C. for 5 h when the E. coli grew to a stage in which the OD600=1. The OD600 threshold may be 0.2-1.5, the temperature threshold may be 37° C. to 10° C., and the expression time may be 5-16 h.
Cell lysis: The cells were sheared and degassed by conventional methods, in which the cells were lysed to obtain a cell lysate.
Crude liquid filtration: The cell lysate of Example 1 was subjected to a crude liquid filtration as a crude cell lysate with a filter pore size of 0.12-0.65 μm. The liquid that passed through the crude liquid filtration material entered the next filtration procedure as a feed liquid, and the substance that did not pass through the crude liquid filtration material entered the waste residue discharge flow path as a waste residue under the effect of a buffer.
Feed liquid filtration: The liquid after the crude liquid filtration entered a feed liquid filtration step as the feed liquid. The feed liquid was filtered multiple times (at least 2 times) in a circulation pathway, with a filter pore size of below 0.2 μm, to obtain a finished liquid.
After the completely enclosed separation of the cell lysate, the obtained waste liquid almost contained no toxin polypeptide precursor, with very little toxicity to the operation environment, and could be directly discharged after simple disinfection treatment.
The finished liquid obtained in Example 2 further entered a purification module, and the GSTs-BoNT/A was obtained by a conventional affinity chromatography method.
The method is as follows: A chromatographic column was washed with 20 column volumes of a phosphate buffer. GSTs-BoNT/A was eluted with 10 column volumes of a freshly prepared 10 mM glutathione eluent buffer (0.154 g of reduced glutathione dissolved in 50 mL of 50 mM Tris-HCl (pH 8.0)). The elution of the fusion protein was monitored by absorbance reading at 280 nm.
Determination of the proportion of GSTs-BoNT/A in the total protein level: The purified GSTs-BoNT/A was electrophoretically separated at 200 volts by using 4-12% SDS-PAGE (Biorad) and a major band with a molecular weight of 175 kd was GSTs-BoNT/A.
The GSTs-BoNT/A re-adsorbed on a glutathione purification resin chromatographic column was treated using Genscript 3C enzyme. The enzyme cleavage site between GSTs and BoNT/A was cleaved under the effect of the 3C enzyme. GSTs were separated. The enzyme cleavage site between the light chain and the heavy chain of BoNT/A was also cleaved.
The glutathione purification resin chromatographic column was treated with a phosphate buffer. The GSTs tag protein was retained on the column and was thus removed, while the light chain and the heavy chain of BoNT/A were eluted by the phosphate buffer.
Products before and after the removal of the GSTs tag protein were subjected to a conventional SDS-PAGE experiment shown in
The BoNT/A obtained in Example 4 was further purified by conventional gel filtration chromatography and ion column chromatography to obtain a BoNT/A with a purity of 90% or above.
A product after the removal of GSTs was subjected to a conventional SDS-PAGE experiment shown in
GSS, GSGS, and GGSGS polypeptides were adopted to replace a GS part of a short linker peptide, which demonstrated a similar effect to that of GS. The tag protein can be well exposed and thus completely cleaved.
The products of Example 4 were subjected to a reduction experiment:
A sample was treated by using 100 mM dithiothreitol at 100° C. for 5 min to reduce the sample, and electrophoretically separated at 200 volts by the 4-12% SDS-PAGE (Biorad) to separate the heavy chain and the light chain.
As shown in
The GSTs-BoNT/A obtained in Example 3 had an LD50 of 45-450 ng after intraperitoneal administration in a mice; considering the purity of the injected botulinum toxin protein, the converted LD50 was 22.5-225 ng, with a mid-value of 123.75 ng. The BoNT/A obtained in Example 4 had an LD50 of 0.02-0.05 ng after intraperitoneal administration in mice. Considering the purity of the injected botulinum toxin protein, the converted LD50 was 0.006-0.015 ng, with a mid-value of 0.0105 ng (See Table 1).
The GSTs-BoNT/A had the activity of botulinum toxin, and the median lethal dose (LD50) thereof was approximately 11786 times higher than the LD50 of the BoNT/A protein after intraperitoneal administration in mice, which indicates that the activity of the toxin precursor molecule of GSTs-BoNT/A recombinant protein is approximately 11786 times weaker than that of the final product BoNT/A. The experiment demonstrates that the toxin precursor molecule of the GSTs-BoNT/A recombinant protein has the activity of botulinum toxin, but toxicity much lower than that of the activated BoNT/A. Due to the ultra-high toxicity of botulinum toxin, high safety operation precautions are required in the manufacturing process even before the activation treatment of a precursor molecule.
The examples of the present application demonstrate that the method claimed in the present application is particularly suitable for the preparation of a genetically recombinant toxin polypeptide, which is activated as a toxin molecule with toxicity only by hydrolysis with a specific protease, and is expressed in the form of a mildly toxic precursor in the host cell and present in the form of the mildly toxic precursor in the cell lysate. In this case, the advantage that the enrichment of the polypeptide precursor is conducted in an enclosed system in the method avoids the tedious and costly implementation of isolation facilities, for example, an isolator, when the toxin polypeptide is industrially produced, such that the preparation method is more suitable for the large-scale industrial production of the polypeptide.
The preferred embodiments of the present invention are described in detail above, which, however, are not intended to limit the present invention. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, all of which will fall within the protection scope of the present invention.
In addition, it should be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner where the features do not contradict each other. In order to avoid unnecessary repetition, such combinations will not be illustrated separately.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110217756.1 | Feb 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/077637 | 2/24/2022 | WO |
