CN107177578B - Method for purifying chondrosulphatase ABC - Google Patents

Abstract

The invention discloses a method for purifying chondroitinase ABC. The method comprises obtaining purified chondroitinase ABC from said chondroitinase ABC containing matrix by chromatography using an affinity column on which heparin is immobilized. The method can obtain the high-purity chondroitinase ABC, and has the advantages of simplicity and high recovery rate.

Description

Method for purifying chondrosulphatase ABC

Technical Field

The present invention relates to a method for purifying chondrosulphatase ABC (ChABC), in particular to a method for purifying ChABC from a ChABC-containing matrix.

Background

Chondroitin sulfate is the most abundant glycosaminoglycan in biological systems and consists of a variety of sulfated and non-sulfated carbohydrate derivatives including sulfated glucuronic acid, N-acetylgalactosamine, iduronic acid, and the like. The polymers are typically linked to proteins to form Chondroitin Sulfate Proteoglycans (CSPGs), which play many structural and functional roles in biological systems (e.g., extracellular matrix for cell-cell interactions, directing or inhibiting neural development, synthesizing cartilage, and becoming a structural component). The biosynthesis of these molecules and the maintenance of proper position are critical to the normal function of humans. In fact, many diseases are associated with the deficiency, accumulation and dislocation of CSPG, such as diffuse Lewy body disease, mucopolysaccharidosis type IV-A and type VII, disc herniation, etc.

Chondrosulphatase is a bacterial enzyme that can efficiently degrade chondroitin sulphate. This group of enzymes can be divided into 4 types with different activities and substrate specificities (chondroitinase ABC, chondroitinase AC, chondroitinase B and chondroitinase C). These enzymes, in particular chondrosulphatase ABC, have recently been demonstrated to be potential new biopharmaceuticals for the treatment of many CSPG-related diseases, for example, mcmchon and colleagues have found that chebc can significantly promote functional recovery of the injured spinal cord. Kubota and Naitoh also demonstrated that injection of chondroitinase sulfate into the affected part of a patient with keloids and/or hypertrophic scars could greatly improve symptoms. Takahashi also developed a successful model for treating rabbit herniated disc using chebc. Recent scientific studies have indicated that there are more medical applications to be developed by ChABC, including the treatment of amblyopia, nerve and spinal cord injuries, posterior vitreal detachment and inhibition of tumor metastasis.

The success of ChABC as a biotherapeutic agent depends significantly on the process used to produce and purify high purity ChABC. The existing upstream production techniques of ChABC are based on the fermentation of unmodified Proteus Vulgaris (Proteus Vulgaris) or recombinant expression hosts such as E.coli. By proper optimization, both expression methods can produce a large amount of bacterial liquid or crude enzyme liquid containing ChABC without purification. Because the bacterial liquid or crude enzyme liquid contains a large amount of protein, nucleic acid, endotoxin and other impurities, the development of an efficient downstream purification method is critical to obtaining high-purity ChABC. Furthermore, the amount and nature of impurities from different expression hosts are very different, so a specific downstream purification procedure, individually tailored, would be required to purify ChABC from a particular expression host, especially when only Ion Exchange (IEX) or Hydrophobic Interaction (HIC) chromatography, which is a non-specific interaction, is used. In fact, the development and optimization of IEX/HIC chromatography is time consuming, requiring a large investment of manpower and resources, and the common result is that multiple IEX/HIC chromatography steps are required to obtain the target protein in high purity, at the expense of a greatly reduced recovery and a long purification run.

The use of affinity chromatography in protein purification has significant advantages over IEX/HIC chromatography, such as relatively fast optimization procedures, simple chromatography procedures, high eluent purity, high recovery, etc. In affinity chromatography, molecules or molecular moieties that specifically interact with a target protein are immobilized on a filler. This highly selective interaction allows for efficient extraction of the desired protein and removal of impurities. Furthermore, the use of affinity chromatography as a purification tool for ChABC has never been explored, and therefore the development of new and efficient affinity chromatography methods would have a tremendous contribution to the production of ChABC as a biotherapeutic drug.

However, it is not easy to design affinity chromatography with ChABC. This is because most known molecules that can specifically bind to ChABC are substrates of ChABC, which will be degraded when bound to enzymes. In other words, if these substrate molecules are immobilized on a stationary phase, they will be broken down by the enzyme during purification and thus unable to capture ChABC from the solution. Chondrosulphatase ABC has a broader substrate spectrum than chondrosulphatase AC, chondrosulphatase B and chondrosulphatase C. This makes it more difficult to design affinity chromatography for ChABC than chondroitinase AC, chondroitinase B and chondroitinase C.

In the Yosizawa et al study (1979), a method for purifying chondroitinase B and C was disclosed, comprising separating a crude chondroitinase C fraction and a crude chondroitinase B fraction from a crude enzyme extract of flavobacterium heparinum using a hydroxyapatite column; the crude chondroitinase C fraction and the crude chondroitinase B fraction are then further purified using a phosphocellulose column, and finally, chondroitinase C is separated from hyaluronidase using a dermatan sulfate-coated dermatan sulfate-bound AH-Sepharose 4B column, and chondroitinase B is separated from Δ 4,5 glucuronidase (glycoiduronase) and sulfatase using a dermatan sulfate-coated heparin-bound AH-Sepharose 4B column. The Yosizawa study emphasizes that non-covalent coating of glycosaminoglycan-bound (covalent) AH-Sepharose 4B with one or the other glycosaminoglycan is important for isolation and purification of chondroitinase sulfate.

Therefore, a technology for purifying ChABC by affinity chromatography, which has the advantages of simplicity and high recovery rate, is still needed in the art.

Disclosure of Invention

To solve the problems of the prior art, the inventors found that heparin not only specifically binds to ChABC, but also is not degraded upon binding. Based on this unexpected finding, the present invention provides a method for purifying ChABC from ChABC-containing matrix, which comprises obtaining highly purified ChABC from ChABC-containing matrix by chromatography using an affinity column immobilized with heparin.

According to one aspect of the invention, the method comprises loading a matrix comprising chondroitinase ABC onto an affinity column bound to heparin.

According to one aspect of the present invention, the method for purifying ChABC from ChABC-containing matrix of the present invention comprises the following steps:

pre-equilibrating the affinity column with a pre-equilibration buffer;

loading the sample solution on the pre-equilibrated affinity column;

washing the loaded affinity column with a wash solution to remove non-specifically bound proteins; and

eluting the chondroitinase ABC with an eluent;

wherein the washing solution is a buffer solution with pH of 8-10, and the eluent is a linear gradient solution of NaCl with pH of 6-9.

According to another aspect of the invention, the method for purifying ChABC from ChABC-containing matrix of the invention further comprises removing endotoxin in the ChABC-containing eluate by filtration after said elution step.

According to yet another aspect of the present invention, the method of purifying ChABC from a ChABC containing substrate according to the present invention further comprises adjusting the pH of said chondrosulphatase ABC containing substrate to 7.0, and/or adjusting the conductivity to the range of 1-5 mS/cm. In one embodiment, the method of purifying ChABC from a ChABC containing substrate of the invention further comprises adjusting the conductivity to 1.2, 1.6, 2, 3, 4, 4.5 or 4.8 mS/cm.

According to yet another aspect of the invention, the washing solution is a carbonate buffer, a phosphate buffer, a borate buffer, a Tris-HCl buffer or a Tris acetate buffer.

According to yet another aspect of the invention, the substrate comprises a cell homogenate of bacteria, fungi, insects or animals, an extract containing a surfactant and/or a supernatant collected during fermentation.

According to a further aspect of the invention, the substrate comprises a homogenate or extract from one or more of Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Proteus vulgaris (Proteus vulgaris), Pichia pastoris (Pichia pastoris), and Saccharomyces cerevisiae (Saccharomyces cerevisiae).

According to yet another aspect of the present invention, the heparin is immobilized on a resin for protein purification having a hydroxyl group, an amino group and/or a carboxyl group. Further, the heparin is bound to the resin using a covalent bond.

According to yet another aspect of the present invention, the heparin-immobilized affinity column is not further coated with glycosaminoglycan.

According to yet another aspect of the invention, the resin comprises one or more of agarose, cellulose, dextran, silica polyacrylamide and acrylic acid polymers.

According to yet another aspect of the invention, the pre-equilibration buffer is a pH7.0-7.5 buffer.

The method for purifying ChABC from the ChABC-containing matrix can obtain high-purity chondrosulphatase ABC, and has the advantages of simplicity and high recovery rate.

Drawings

FIG. 1 shows a chromatogram of example 2 of the present invention.

FIG. 2 shows a chromatogram of example 3 of the present invention.

FIG. 3 shows an SDS-PAGE pattern of example 3 of the present invention;

wherein lane (1) is the molecular marker, lane (2) is the matrix before loading on the column, lane (3) overflows, lane (4) is the impurity washed by 60mM Tris-acetate (pH8.0), lane (5) is the impurity washed by 100mM Tris-acetate (pH9.2), lane (6) and (7) eluents.

FIG. 4 shows a chromatogram of example 4 of the present invention.

FIG. 5 shows an SDS-PAGE pattern of example 4 of the present invention;

among these, lane (1) is the molecular marker, lane (2) is the matrix before loading on the column, lane (3) overflows, lane (4) is the impurity washed by 100mM Tris-acetate (pH9.2), lane (5) and (6) eluents.

FIG. 6 shows a chromatogram of example 5 of the present invention.

FIG. 7 shows an SDS-PAGE pattern of example 5 of the present invention;

among these, lane (1) is the molecular marker, lane (2) is the matrix before loading on the column, lane (3) overflows, lane (4) is the impurity washed by 100mM Tris-acetate (pH9.2), lane (5) and (6) eluents.

Detailed Description

The present invention provides a novel and simple purification method of ChABC, which can be applied to various substrates containing ChABC.

The term "substrate" as used herein refers to a mixture of ChABC with extracellular or intracellular material (proteins, DNA, lipids, etc.) from host cells during ChABC production, including but not limited to bacterial, fungal, insect or animal cell homogenates, surfactant-containing extracts, supernatants collected during fermentation, etc.

The current upstream production technology of ChABC is based on the fermentation of unmodified Proteus Vulgaris (Proteus Vulgaris) or recombinant expression hosts such as E.coli. Both expression methods can produce large quantities of substrates containing ChABC. The substrates containing ChABC described in the present invention can be derived from wild type Proteus vulgaris or recombinant expression hosts, including recombinant Escherichia coli, recombinant Bacillus subtilis, recombinant Pichia pastoris, recombinant Saccharomyces cerevisiae, and the like. Recombinant expression hosts can be prepared by transforming host bacteria with recombinant expression plasmids. In transforming a host bacterium with a recombinant expression plasmid, it is necessary to link a gene encoding chondroitinase sulfate to an expression plasmid containing a promoter and Shine-Dalgarno (SD) sequence and capable of achieving a high transcription level in a host cell. The promoter of the vector for the E.coli expression system may be T7, T5, Lac, Trp, araC or any hybrid promoter thereof. The promoter of the vector for Bacillus subtilis may be Spac, SacB, Xyl, PBAD, Pgrac or any hybrid promoter thereof. Furthermore, in addition to the preparation of recombinant expression systems by transforming host bacteria with recombinant expression plasmids, integration of chondroitinase genes into host bacterial chromosomes is also a viable strategy. For example, the integration vector pMG1 was used for Bacillus subtilis MG1P, or the integration vector pSG1112 was used for Bacillus subtilis 1A304(Φ 105MU 331). In one embodiment, the mature chondroitinase expressed by the host cell is set forth in SEQ ID NO 1.

Methods for obtaining ChABC-containing substrates from wild-type Proteus vulgaris or recombinant expression hosts are well known to those skilled in the art. In the case of using wild-type Proteus vulgaris, chondroitinase expression can be induced by adding chondroitin sulfate to the medium by fed-batch fermentation, for example. In the case of using a recombinant expression host, the inducer will depend on the vector and the promoter, for example IPTG as the inducer for the lac promoter and arabinose as the inducer for the pBAD promoter. Depending on the scale of fermentation, the chondroitinase sulfate-containing cells may be collected by centrifugation or cross-flow microfiltration, and the ChABC-containing matrix may be obtained from the collected cells by mechanical disruption, e.g., sonication, liquid homogenization, freeze-thawing, etc., or by chemical disruption, e.g., introduction of surfactants, lysozyme or other lytic enzymes.

The purification method of the present invention is a chromatography method using an affinity column that can selectively capture ChABC. An affinity column is a column on which is immobilized a specific ligand that specifically interacts with ChABC under chromatography. The inventors screened a large number of different types of polysaccharide derivatives, such as glycosaminoglycans or other polysaccharides such as alginates, fucoidans, polygalacturonic acid, pentosan polysulfate, cellulose phosphates, and the like.

Although immobilization of selected polysaccharides on a resin is the most straightforward way to test the applicability of these ligands to the ChABC affinity chromatography, the immobilization chemistry between the ligand and the resin surface, ligand density, spacer groups, etc. can significantly affect the interaction between ChABC and immobilized polysaccharides. In other words, determining the binding between ChABC and the polysaccharide in the solution phase is more appropriate and accurate for revealing the strength of the interaction.

There are several existing methods for quantitatively investigating protein-small molecule interactions, such as differential scanning calorimetry, quartz crystal microbalance, fluorescence anisotropy, etc. However, these methods require complex equipment, expensive and time-consuming protein immobilization or protein labeling. To save time and reduce costs, semi-quantitative evaluation methods, such as enzyme inhibition assays, may be applied. In the present invention, relative binding affinity is assessed by monitoring inhibition of ChABC by selected polysaccharides. The stronger the inhibition observed, the higher the affinity between ChABC and polysaccharide. A more detailed experimental setup is shown in example 1. Heparin was found to almost completely inhibit the activity of ChABC when equal amounts of substrate (chondroitin sulfate C) were applied as determined by enzyme inhibition. Thus, heparin was identified as the most suitable ligand for constructing affinity columns among those selected polysaccharides.

In the present invention, heparin may be immobilized on various resins commonly used for protein purification having hydroxyl, amino, carboxyl groups for ligand immobilization. Heparin may be covalently bound to the resin. As for the resin, the present invention is not particularly specified, and resins to which heparin can be covalently bound may be applied to the present invention, including agarose, cellulose, dextran, silica polyacrylamide, acrylic acid polymer. The heparin or resin may be activated by various activating agents including, but not limited to, cyanogen bromide, EDC/NHS (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide/n-hydroxysuccinimide), sodium periodate, epichlorohydrin, and the like.

In the present invention, the heparin-immobilized affinity column, particularly the affinity column covalently bound to heparin, need not be further coated, e.g., may not be coated with a glycosaminoglycan, although coating is also possible. In one embodiment, the heparin-immobilized affinity column of the present invention, particularly covalently bound heparin, is not further coated with a glycosaminoglycan.

Although heparin can bind and can significantly inhibit ChABC, the affinity between immobilized heparin and ChABC in chromatography is unknown. To demonstrate that ChABC still has affinity for immobilized heparin, purified ChABC was loaded into Affi from Bio-rad

Heparin was gel and washed with various buffers as described in example 2. In fact, a Tris acetate buffer (pH9.2) with a pH value above the isoelectric point (pI ═ 8.5) of ChABC was applied to the column under such conditions whenAt buffer pH values above pI, chebc becomes negatively charged and there should be repulsion between chebc and the negatively charged heparin on the resin. ChABC will elute from the column if it has only non-specific charge interactions with heparin. From the experimental results, however, ChABC was not only successfully captured by the resin, but also not eluted by buffers with pH higher than pI. In other words, ChABC has a specific interaction with heparin, rather than a purely non-specific charge interaction.

In the present invention, a ChABC affinity column has significant advantages over the cation exchange columns most commonly used for ChABC purification, as it has a significantly lower amount of impurities that are non-specifically bound to the resin. Thus, the chromatography can be applied to a variety of substrates with different heterogeneous mass spectra, including but not limited to homogenates or extracts from Escherichia coli (Escherichia coli), Bacillus subtilis (Bacillus subtilis), Pichia vulgaris (Proteus vulgaris), Pichia pastoris (Pichia pastoris), and/or Saccharomyces cerevisiae (Saccharomyces cerevisiae), to obtain highly purified chebc. However, since heparin is a negatively charged polysaccharide, a small amount of positively charged protein can still be non-specifically captured by the heparin-immobilized resin. However, these non-specifically bound proteins can be easily removed by using a high pH buffer (pH 8-10), the pH 8-10 buffer being a carbonate buffer, a phosphate buffer, a borate buffer, a Tris-HCl buffer or a Tris acetate buffer, such as a sodium carbonate buffer, a sodium phosphate buffer, a potassium carbonate buffer, a potassium phosphate buffer, a sodium borate buffer, a potassium borate buffer. Buffers in this pH range can significantly reduce non-specific protein interactions because most of these protein impurities will become negatively charged and thus no longer have charge interactions with the heparin-immobilized resin, whereas chebc with specific interactions will not be affected by high pH buffers. After removal of protein impurities, ChABC was eluted with a linear gradient of NaCl pH 6-9. The eluent may contain ChABC with a purity of up to 98-99% or even above and with low endotoxin levels (examples 3-5). Endotoxin may be further removed by passing the eluate through an endotoxin trap filter. In addition to low endotoxin and protein impurities, another advantage of this process is simplicity and high recovery. Single step chromatography significantly reduces the processing time, thereby avoiding activity loss during processing. In fact, the final yield was in the range of 70-80% (examples 3-5), which is significantly higher than all prior methods. More importantly, affinity chromatography can be applied to different types of matrices produced by different host cells.

Example 1

In the present invention, the identification of molecules with a similar structure to the substrates of ChABC but which are inert to enzymatic degradation is the first key part of the invention. To test the polysaccharides that can actually bind to the active site of chebc, the selected polysaccharides were subjected to a relative enzyme inhibition assay:

mu.L of a substrate solution containing 0.2% chondroitin sulfate C, 0.2% of the selected polysaccharide and 50mM Tris HCl buffer pH8.0 was incubated at 37 ℃ for at least 15 minutes. 10 μ L of ChABC with about 100U/L activity were incubated separately at 37 ℃ for at least 3 minutes. After incubation, the two solutions were gently mixed. The mixture was then incubated at 37 ℃ for 20 minutes to effect enzymatic degradation of chondroitin sulfate. After 20 minutes, the reaction was stopped by heat-inactivating ChABC in a water bath at 100 ℃ for 2 minutes. Blank control was performed separately in the same manner except that the mixture was immediately heat-inactivated without 20 minutes incubation. The absorbance of the heat-inactivated solution at 232nm was then measured and the apparent activity was calculated as shown in formula 1. The determination of the absence of the selected polysaccharide was also performed in the same manner as the reference. The percent inhibition is calculated as shown in equation 2:

wherein

Absorba ═ absorbance difference ═ OD in sample tube_232nmOD in blank tubes_232nm

Extinction coefficient of milligram molecule of unsaturated disaccharide derived from chondroitin sulfate C5.5

Reaction time of 20 min

Vt-measured total volume 0.5mL

Vs ═ volume of enzyme used 0.01mL

DF is dilution factor

1000 converting U/mL to U/L

Wherein

% rA ═ residual percent activity

Activity in the presence of selected polysaccharides

(ref) absorbance measured in the absence of the selected polysaccharide

Of all polysaccharides, alginate and polygalacturonic acid had no significant effect on enzyme activity, while heparin had the strongest inhibitory effect on ChABC. The results of the relative enzyme inhibition assay are shown in table 1.

TABLE 1

Sample (I)	Reference to	Heparin	Polygalacturonic acid	Alginate salts
					Activity (U/L)	121	5.1	117	119
％rA	NA	4.2	97	98

Furthermore, heparin is inert to the enzymatic degradation activity of ChABC, since ChABC alone does not produce any change in absorbance after incubation with heparin (results not shown). Thus, immobilized heparin as a ligand for affinity purification of ChABC is the most suitable choice among the selected polysaccharides.

Example 2

To test whether ChABC has specific interaction with immobilized heparin, Affi filled from Bio-rad was used

A column of heparin gel. The column was pre-equilibrated with 25mM Tris-acetate (pH 7.3) prior to loading the sample onto the column. ChABC was dissolved in 25mM Tris-acetate (pH7.5) and loaded onto the column. The column was washed with (1)25mM Tris-acetate (pH9.2), (2)100mM Tris-acetate (pH9.2), (3)60mM Tris-acetate (pH8), and (4)100mM Tris-acetate (pH 7.5). None of these buffers was able to elute ChABC from the column. While ChABC can be eluted from the column when a linear gradient of 0-0.3M NaCl is applied. The chromatogram is shown in FIG. 1.

Example 3

The gene coding chondroitinase ABC (SEQ ID NO:1) was cloned by PCR using the genome of Proteus vulgaris as a template, the product was ligated to pET3a plasmid by T4DNA ligase, and the recombinant plasmid was introduced into E.coli strain BL21(DE3) by electroporation. The transformed E.coli was inoculated on LB agar plates containing 50. mu.g/ml ampicillin to select successfully transformed colonies. Individual colonies were selected, inoculated into LB medium containing 50. mu.g/ml ampicillin and cultured at 37 ℃ for 18 hours, and the culture was added to LB medium containing 50. mu.g/ml ampicillin at a ratio of 1:100, which was sterile. The medium was further incubated at 37 ℃ to an OD600 of 0.8. Chondrosulphatase production was induced by addition of 1mM IPTG, followed by further culture for 4 hours. Harvested by centrifugation and stored at-80 ℃.

10 g of recombinant E.coli cells were resuspended in 25mM Tris HCl (pH7.0) and lysed by sonication for 5 minutes. Cell debris was removed from the suspension by centrifugation at 17000g for 1 hour. The supernatant was collected and the pH was adjusted to pH7.0 by addition of NaOH. The conductivity of the supernatant was adjusted to 1.2mS/cm by adding di-deionized water.

The supernatants containing ChABC were subjected to 0.22 μm filtration before loading on heparin-immobilized columns pre-equilibrated with pH 7.525 mM Tris-acetate. The column was then washed with (1)100mM Tris-acetate (pH7.5), (2)60mM Tris-acetate (pH8), (3)100mM Tris-acetate (pH9.2), and ChABC on the column was eluted by a linear gradient from 0-0.5M NaCl. The chromatograms and SDS-PAGE are shown in FIGS. 2 and 3.

The degree of purification in the above process is shown in table 2.

TABLE 2

As can be seen, the method of the embodiment can obtain ChABC with the purity of more than 99%, the recovery rate reaches 76% and the endotoxin level is lower than 0.0002 EU/U.

Example 4

Using the genome of Proteus vulgaris as a template, a gene encoding chondroitinase sulfate ABC (sequence SEQ ID NO:1) was cloned by PCR, the product was ligated to pHT43 plasmid by T4DNA ligase, the recombinant plasmid was introduced into Bacillus subtilis strain 168 by electroporation, and the transformed Bacillus subtilis was inoculated on a 2 XTY agar plate containing 5. mu.g/ml chloramphenicol to select successfully transformed colonies. Individual colonies were selected and inoculated into sterile 2 XTY medium containing 5. mu.g/ml chloramphenicol at 37 ℃ for 18 hours. The culture was cultured in the ratio 1:100 were added to sterile LB medium containing 5. mu.g/ml chloramphenicol. The medium was further incubated at 37 ℃ to an OD600 of 0.8. Chondrosulphatase production was induced by addition of 1mM IPTG, followed by further culture for 4 hours. The cells were harvested by centrifugation and stored at-80 ℃.

5g of recombinant Bacillus subtilis cells were resuspended in 25mM Tris-acetate (pH7.0) incubated with 1. mu.g/ml lysozyme for 20 minutes at 30 ℃. Bacterial cells were further lysed by sonication for 5 minutes. Cell debris was removed from the suspension by centrifugation at 17000g for 1 hour. The supernatant was collected and the pH was adjusted to pH7.0 by the introduction of NaOH. The conductivity of the supernatant was adjusted to 1.6mS/cm by introducing double deionized water.

The supernatants containing ChABC were subjected to 0.22 μm filtration before loading on heparin-immobilized columns pre-equilibrated with pH 7.525 mM Tris-acetate. The column was then washed with (1) Tris-acetate pH 7.525mM, (2) Tris acetate pH 860 mM, (3) Tris acetate pH 9100 mM. ChABC on the column was eluted by a linear gradient of 0-0.5M NaCl. The chromatograms are shown in FIGS. 4 and 5. The degree of purification is shown in table 3.

TABLE 3

As can be seen, the method of the embodiment can obtain ChABC with the purity of more than 99%, the recovery rate reaches 74% and the endotoxin level is lower than 0.0002 EU/U.

Example 5

25g of the cell paste of Proteus vulgaris was resuspended in 25mM Tris-acetate (pH7.0) containing 2% Triton X-100, which was preheated at 37 ℃ for 30 minutes. The suspension was continuously stirred at 750rpm and incubated at 37 ℃ for 2 hours. After extraction, the suspension was diluted 2-fold with high purity water at 4-6 ℃. The diluted suspension was then immediately clarified by high speed centrifugation at 17700 g. The supernatant was further clarified by microfiltration through a pore size of 0.2 μm. The pH of the clarified supernatant was adjusted to pH7.0 and the conductivity to 4.8mS/cm, before loading onto a heparin-immobilized column. The heparin immobilization column was pre-equilibrated with pH7.025mM Tris-acetate. After the supernatant was completely loaded onto the column, the column was washed with (1) Tris-acetate pH 7.025 mM, (2) Tris-acetate pH 8.060 mM, and (3) Tris-acetate pH 10100 mM 9.2 to remove impurities including unwanted proteins, DNA, endotoxins. ChABC was eluted with a linear gradient of 0-0.5M NaCl and tested for endotoxin levels below 0.0002 EU/U. The chromatograms for purification are shown in FIGS. 6 and 7. The degree of purification is shown in table 4.

TABLE 4

As can be seen, the method of the embodiment can obtain ChABC with the purity of more than 99%, the recovery rate reaches 81% and the endotoxin level is lower than 0.0002 EU/U.

Various modifications and variations of the present invention are possible to those skilled in the art in light of the above teachings. It is therefore to be understood that the invention may be practiced otherwise than as specifically described without departing from the inventive concepts of the appended claims.

Sequence listing

<110> Aihua Biotechnology Ltd

<120> method for purifying chondroitinase ABC

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Asp Lys Asn Lys Asn Val Glu Thr Thr Leu Phe Gln His Ala Ile Thr

755 760 765

Pro Thr Leu Asn Thr Leu Trp Ile Asn Gly Gln Lys Ile Glu Asn Met

770 775 780

Pro Tyr Gln Thr Thr Leu Gln Gln Gly Asp Trp Leu Ile Asp Ser Asn

785 790 795 800

Gly Asn Gly Tyr Leu Ile Thr Gln Ala Glu Lys Val Asn Val Ser Arg

805 810 815

Gln His Gln Val Ser Ala Glu Asn Lys Asn Arg Gln Pro Thr Glu Gly

820 825 830

Asn Phe Ser Ser Ala Trp Ile Asp His Ser Thr Arg Pro Lys Asp Ala

835 840 845

Ser Tyr Glu Tyr Met Val Phe Leu Asp Ala Thr Pro Glu Lys Met Gly

850 855 860

Glu Met Ala Gln Lys Phe Arg Glu Asn Asn Gly Leu Tyr Gln Val Leu

865 870 875 880

Arg Lys Asp Lys Asp Val His Ile Ile Leu Asp Lys Leu Ser Asn Val

885 890 895

Thr Gly Tyr Ala Phe Tyr Gln Pro Ala Ser Ile Glu Asp Lys Trp Ile

900 905 910

Lys Lys Val Asn Lys Pro Ala Ile Val Met Thr His Arg Gln Lys Asp

915 920 925

Thr Leu Ile Val Ser Ala Val Thr Pro Asp Leu Asn Met Thr Arg Gln

930 935 940

Lys Ala Ala Thr Pro Val Thr Ile Asn Val Thr Ile Asn Gly Lys Trp

945 950 955 960

Gln Ser Ala Asp Lys Asn Ser Glu Val Lys Tyr Gln Val Ser Gly Asp

965 970 975

Asn Thr Glu Leu Thr Phe Thr Ser Tyr Phe Gly Ile Pro Gln Glu Ile

980 985 990

Lys Leu Ser Pro Leu Pro

995

Claims (8)

1. A method for purifying chondroitinase ABC from a chondroitinase ABC-containing matrix comprising obtaining purified chondroitinase ABC from the chondroitinase ABC-containing matrix by chromatography using an affinity column immobilized with heparin, wherein the method comprises:

adjusting the pH of the chondroitinase ABC containing matrix to 7.0 and conductivity to within the range of 1-5mS/cm, 0.22 μm filtering the chondroitinase ABC containing matrix, and loading the chondroitinase ABC containing matrix onto the heparin immobilized affinity column pre-equilibrated with 25mM Tris-acetate pre-equilibration buffer, pH 7.5;

washing the loaded affinity column with a washing solution to remove non-specifically bound proteins, and

eluting the chondroitinase ABC with an eluent;

wherein the washing is performed by one of:

washing with (1)100mM Tris-acetate pH7.5, (2)60mM Tris-acetate pH8, and (3)100mM Tris-acetate pH 9.2;

washing with (1)25mM Tris-acetate pH7.5, (2)60mM Tris-acetate pH8, and (3)100mM Tris-acetate pH 9; or

Washing with (1)25mM Tris-acetate pH7.0, (2)60mM Tris-acetate pH8.0, and (3)100mM Tris-acetate pH 10;

the eluent is 0-0.5M NaCl linear gradient solution with pH of 6-9; and is

The purified chondroitinase ABC has an endotoxin level below 0.0002EU/U, and wherein the chondroitinase ABC is encoded by SEQ ID NO:1 encoding.

2. The method according to claim 1, wherein said method further comprises a step of removing endotoxin in the eluate containing said chondroitinase ABC by filtration after said eluting step.

3. The method according to claim 1 or 2, wherein the substrate comprises a cell homogenate of bacteria and/or a supernatant collected during bacterial fermentation.

4. The method of claim 3, wherein the substrate comprises a material from Escherichia coli (E. coli)Escherichia coli) Bacillus subtilis preparation (B)Bacillus subtilis) And Proteus vulgaris: (Proteus vulgaris) And (c) homogenizing one or more of (a).

5. The method according to claim 1 or 2, wherein the heparin is immobilized on a resin for protein purification having a hydroxyl group, an amino group and/or a carboxyl group.

6. The method of claim 5, wherein the resin comprises one or more of agarose, cellulose, dextran, silica polyacrylamide, and acrylic acid polymers.

7. The method of claim 5, wherein said heparin is covalently bonded to said resin.

8. The method of claim 1, 2 or 7, wherein the heparin-immobilized affinity column is not further coated with a glycosaminoglycan.

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