Urokinase affinity chromatography filler

Abstract

The invention discloses a urokinase affinity chromatography filler, which is characterized in that the amino acid sequence of plasminogen is subjected to mutation design, so that urokinase loses corresponding catalytic activity to a plasminogen mutant, and the plasminogen mutant on the affinity chromatography filler does not generate peptide fragment shedding due to the activity of the urokinase. The affinity of the plasminogen mutant and urokinase is superior to that of wild plasminogen. The initial static loading capacity of the affinity chromatography filler prepared from the plasminogen mutant to urokinase is superior to that of the affinity chromatography filler prepared from wild plasminogen, and the initial static loading capacity is 30 mg / mg or above. The acid resistance of the affinity chromatography filler prepared from the plasminogen mutant is superior to that of the affinity chromatography filler prepared from wild plasminogen. And the residual static loading capacity is at least 80% of the initial static loading capacity after being soaked in a 0.1 M acetic acid solution at 22 + / -2 DEG C for 24 hours.

Description

Technical Field

[0001] The invention relates to the field of purification and separation, and more specifically to a urokinase affinity chromatography filler. Background Art

[0002] Urokinase (UK) is an enzyme protein isolated from healthy human urine or obtained through human kidney tissue culture. Its function is to catalyze the cleavage of the Arg560-Val561 peptide bond in plasminogen to generate plasmin. Plasmin can not only degrade fibrin in thrombus, but also decompose fibrinogen, coagulation factor V, coagulation factor VIII, etc. in the blood, thus having the function of dissolving thrombus. Therefore, urokinase is often used in clinical thrombolytic therapy to prevent and control myocardial infarction, hypertension, arteriosclerosis and other diseases.

[0003] The preparation methods of urokinase mainly include ion exchange chromatography, gel molecular sieve method, affinity chromatography and p-aminobenzamidine-Sepharose affinity chromatography. Affinity chromatography is based on the highly specific binding of the target protein to a specific ligand, which can greatly improve the purity of urokinase. Compared with traditional chromatography methods, affinity chromatography can effectively and selectively remove impurity proteins and retain the target protein. And usually only a few steps are required, which is simple and efficient to operate. The use of affinity chromatography columns can significantly shorten the purification time, thereby improving work efficiency.

[0004] Affinity chromatography has high specificity and can retain urokinase to the maximum extent, thereby increasing the yield of the final product. And it is usually carried out under mild conditions (such as appropriate pH and salt concentration), which can reduce the loss of urokinase activity and maintain its function. Affinity chromatography has received much attention due to its specific adsorption, but currently no affinity chromatography filler with strong affinity and multiple reuse has been found. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide an affinity chromatography filler that can improve the activity, yield and purification multiple of urokinase.

[0006] To achieve the above invention purpose, the technical scheme adopted is: a urokinase affinity chromatography filler, the ligand of the affinity chromatography filler is agarose, and the ligand is a plasminogen mutant; the plasminogen mutant is based on the wild-type plasminogen described in SEQ ID NO: 1, and the following mutation set is mutated: L100Q+G121S+K196C+N251C. The sequence of the wild-type plasminogen comes from the NCBI database: GenBank: CAB46014.1.

[0007] Preferably, the plasminogen mutant further comprises the following mutations: S598N+K729C.

[0008] Preferably, the plasminogen mutant further comprises the following mutations: S598N+V730P.

[0009] Preferably, the plasminogen mutant further comprises the following mutations: K729C+V730P.

[0010] Preferably, the plasminogen mutant further comprises the following mutations: S598N+K729C+V730P.

[0011] Preferably, the dissociation constant of the plasminogen mutant with urokinase is no more than 15 nM.

[0012] Preferably, the initial static loading of the affinity chromatography medium is at least 50 mg / mL.

[0013] Preferably, the affinity chromatography filler has a residual static loading of at least 80% of the initial static loading after being immersed in a 0.1 M acetic acid solution at 22±2° C. for 24 hours.

[0014] Preferably, the ligand is a cross-linked 4% agarose matrix.

[0015] Compared with the prior art, the advantages of the present invention are: urokinase has obvious catalytic activity to wild-type plasminogen, and loses the corresponding catalytic activity to the modified plasminogen mutants. The plasminogen mutants on the affinity chromatography filler will not produce peptide shedding due to the activity of urokinase. The dissociation constants of the plasminogen mutants and urokinase are all less than half of the wild type, so the affinity of the five plasminogen mutants to urokinase is better than the affinity between the wild-type plasminogen and urokinase. The initial static loading of the affinity chromatography filler prepared from the plasminogen mutants on urokinase is better than that of the affinity chromatography filler prepared from the wild-type plasminogen. And the initial static loading is more than 30 mg / mg. The acid resistance of the affinity chromatography filler prepared from the plasminogen mutants is better than that of the affinity chromatography filler prepared from the wild-type plasminogen. And the remaining static loading after soaking in 0.1M acetic acid solution at 22±2°C for 24 hours is at least 80% of the initial static loading. DETAILED DESCRIPTION

[0016] The term "recombinant gene" refers to a DNA or RNA that can express the plasminogen of the present invention. Usually, the recombinant gene is initially synthesized in vitro by the solid phase phosphoramidite trisaccharide method or the TdT biosynthesis method or other suitable techniques known in the art. With the template sequence, it can be amplified by PCR or other suitable techniques known in the art. With the recombinant strain, it can be further amplified on a large scale by culturing the strain. In certain embodiments, the recombinant gene may also include residual sequences of restriction sites, other accessory elements, such as control elements (such as promoters, etc.), markers (such as fluorescent markers, etc.), and other sequences that do not affect the expression of the target gene.

[0017] The term "cloning scar" refers to a promoter sequence that initiates transcription of messenger ribonucleotides (mRNA), followed by a ribosome-binding site (RBS) that attracts the translation machinery, followed by a signal peptide sequence that facilitates protein transport to the periplasm. The mature protein is usually cloned after the signal peptide, from which it is cleaved by a signal peptidase as it passes through the membrane. However, when cloning constructs after the signal peptide, restriction endonucleases usually require specific sequences to cut the DNA, which leaves a cloning scar after the signal peptide sequence.

[0018] The term "signal peptide" refers to a short peptide (usually 16-30 amino acids long) present at the N-terminus of most newly synthesized proteins that are destined to enter the secretory pathway. It may also be called a signal sequence, targeting signal, localization signal, localization sequence, transit peptide, leader sequence or leader peptide. The signal peptide is usually cleaved from the protein by a signal peptidase.

[0019] The term "promoter" refers to a region of DNA that initiates transcription (writes to mRNA) of a specific gene. A promoter is usually located near the transcription start site of a gene, on the same strand and upstream of the DNA (toward the 5' region of the sense strand). A promoter can be inducible, which means that the expression of a gene operably linked to the promoter can be turned on by the presence of an inducer substance. Alternatively, a promoter can be constitutive, i.e., it is not regulated by any inducer substance.

[0020] The abbreviation "RBS" refers to ribosome-binding site, or the binding site of the ribosome. This is the sequence of nucleotides upstream of the start codon of the mRNA transcript that is responsible for recruiting the ribosome during the initiation of protein translation.

[0021] The term "expression" refers to the process of transcribing DNA into messenger RNA (mRNA) and then translating it into protein. In order to achieve the smooth expression and screening of plasminogen, the above-mentioned signal peptide, promoter, and RBS may be introduced into the recombinant gene, so some corresponding peptides may remain on the expressed plasminogen protein. This part of the peptide will not affect the function of plasminogen, so even if the above-mentioned peptides are added to the product, as long as the amino acid sequence of the main part is the same as the sequence of the present invention, the product is still not an infringing product.

[0022] The term "expression vector" has the ability to incorporate and express heterologous polynucleic acid fragments in a host cell. Many prokaryotic and eukaryotic expression vectors are commercially available. Selecting an appropriate expression vector is within the knowledge of the technician.

[0023] The term "chassis cell" refers to a suitable host vector for expressing the DNA of the present invention. The host may include any organism capable of containing and expressing the nucleic acid or gene disclosed herein, but is not limited thereto. Chassis cells may be prokaryotes or eukaryotes, unicellular or multicellular, including mammalian cells, plant cells, fungi, and the like. According to the prior art, those skilled in the art can achieve heterologous expression of the recombinant DNA of the present invention in different disclosed chassis cells by adjusting parameters by a limited number of experiments. Chassis cells may be selected from at least one of Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae, Hansen yeast, Candida, Rhodotorula, Bacillus, Escherichia, Salmonella, Clostridium, Streptomyces, Staphylococcus, Neisseria, and Shigella. The present invention only lists the types of chassis cells and does not constitute a limitation on the types of chassis cells. The chassis cells are preferably Escherichia coli, and suitable Escherichia coli strains (including many other strains) include BL21 (DE3), C600, DH5αF′, 1113101, JM83, JM101, JM103, JM105, JM107, JM109, JM110, MC1061, MC4100, MM294, NM522, NM554, TGI, χ1776, XL1-Blue and Y1089+, etc. The above Escherichia coli strains are all commercially available strains.

[0024] Plasminogen is the catalytic substrate of urokinase, so the affinity chromatography filler obtained by coupling plasminogen as a ligand has a specific adsorption effect on urokinase and can be used for the separation and purification of urokinase. However, urokinase can cleave specific peptide bonds of plasminogen (such as Arg561-Val562 bonds), decomposing it into plasmin with a smaller molecular weight, which will lead to a decrease in the loading capacity of the affinity chromatography filler and a decrease in the purity of the obtained urokinase.

[0025] The present invention will be further described below in conjunction with specific embodiments.

[0026] Embodiment 1:

[0027] The target sequence is: The plasminogen mutant uses the wild-type plasminogen described in SEQ ID NO: 1 as the parent, and the following mutation set is mutated: L100Q+G121S+K196C+N251C.

[0028] Construction of recombinant cells:

[0029] The nucleotide sequences of the target genes were synthesized by Beijing Qingke Biotechnology Co., Ltd. These nucleotide sequences were inserted into expression vectors, specifically, into plasmid pUC18 to obtain the corresponding plasmids. The synthesized plasmids were then transferred into chassis cells (E. coli BL21 (DE3)), thereby constructing an E. coli strain containing the recombinant plasmid. There are many other plasmids and chassis cells available in the prior art, and only a specific solution is provided here.

[0030] Expression and purification of plasminogen:

[0031] Spread BL21 (DE3) containing the expression plasmid pUC18 on a solid LB medium containing ampicillin and culture at 37°C overnight. Pick a single colony and inoculate it into 5mL LB medium containing ampicillin at 37°C and 250rmp to OD 0.7. Transfer the bacterial solution to a new LB medium and culture it at 37°C and 220rmp for 3h, then add IPTG to a final concentration of 0.2mM / L, and induce it at 16°C and 220rmp overnight. Collect the induced bacteria for SDS-PAGE analysis to detect the induction results. There are many other methods available in the prior art for inducing the expression of recombinant bacteria, and only a specific scheme is provided here.

[0032] Take the induced bacteria and resuspend them with equilibrium buffer, and ultrasonically disrupt them on ice. The disruption conditions are: power 600W, ultrasonic 2s, pause 4s, and ultrasonic 30min. After disruption, centrifuge at 4°C and 12000rmp for 10min, and take the supernatant. Ammonium sulfate is gradually added to the supernatant until the protein is precipitated, and the crude protein is obtained by centrifugation. After dissolving the crude protein with a neutral or slightly acidic buffer solution, it is purified with an anion exchange resin (such as DEAE-cellulose or Q-Sepharose, etc.). Then use a gel filtration column (such as SephadexG-100 or Sephacryl S-200) for purification. Finally, use an ultrafiltration membrane to concentrate plasminogen and remove salt and impurities at the same time. Take a sample for SDS-PAGE electrophoresis verification. There are many other methods for purifying plasminogen available in the prior art, and only a specific scheme is provided here.

[0033] Coupling method: The agarose matrix Sepharose4Fast Flow purchased from Cytiva was washed with coupling buffer (0.1M boric acid buffer). Then, 10 mg of plasminogen was added per milliliter of filler, and the coupling was carried out overnight at 30°C and 150rpm. After the coupling was completed, the medium was washed with coupling buffer, and 3 volumes of blocking solution (PBS buffer, pH7.5) were added, and blocked at 37°C for 3 hours. After the blocking solution was drained, the resin was washed with 3 volumes of deionized water. Then, it was washed twice with 0.1M glycine buffer (pH 3.3), deionized water, PBS buffer (pH 7.5) and deionized water in sequence to obtain affinity chromatography filler. Finally, an equal volume of preservation solution was added and the medium was stored at 4°C. There are many choices of ligands in the prior art (such as Agarose Beads or Agarose for Affinity Chromatography from Sigma-Aldrich), and the present invention only selects one of them.

[0034] The loading capacity and acid resistance of plasminogen are closely related to the coupling method. The present invention uses a common coupling method to couple the homemade ligand, which is mainly used to compare the initial static loading capacity of the initial plasminogen ligand and the mutant ligand and the residual static loading capacity after acid treatment. Although the absolute values ​​of the initial static loading capacity and the residual static loading capacity after acid treatment may be different when using other coupling methods, the change rules are basically the same.

[0035] Embodiment 2:

[0036] The difference from Example 1 is that the plasminogen mutant is based on the wild-type plasminogen described in SEQ ID NO: 1, and the following mutation set is mutated: L100Q+G121S+K196C+N251C+S598N+K729C.

[0037] Embodiment 3:

[0038] The difference from Example 1 is that the plasminogen mutant is based on the wild-type plasminogen described in SEQ ID NO: 1 as the parent, and the following mutation set is mutated: L100Q+G121S+K196C+N251C+S598N+V730P.

[0039] Embodiment 4:

[0040] The difference from Example 1 is that the plasminogen mutant is based on the wild-type plasminogen described in SEQ ID NO: 1 as the parent, and the following mutation set is mutated: L100Q+G121S+K196C+N251C+K729C+V730P.

[0041] Embodiment 5:

[0042] The difference from Example 1 is that the plasminogen mutant is based on the wild-type plasminogen described in SEQ ID NO: 1, and the following mutation set is mutated: L100Q+G121S+K196C+N251C+S598N+K729C+V730P.

[0043] Comparative Example:

[0044] The difference from Example 1 is that the amino acid sequence of plasminogen is the wild-type sequence described in SEQ ID NO:1.

[0045] Method for determining the activity of urokinase on plasminogen: fibrinolysis method

[0046] 1. Develop a standard curve of plasmin to fibrin: Prepare plasmin solutions of different concentrations and add them to fibrin gels. The content of fibrin in the fibrin gel is 1%. Incubate for 30 minutes at 22±2℃. Record the size of the dissolution ring on different gels and develop a standard curve between the concentration of plasmin solution and the size of the dissolution ring.

[0047] 2. Prepare samples: add 2% of pure wild-type plasminogen powder (plasminogen protein prepared in the comparative example), 2% of pure plasminogen S1 powder (plasminogen protein prepared in Example 1), 2% of pure plasminogen S2 powder (plasminogen protein prepared in Example 2), 2% of pure plasminogen S3 powder (plasminogen protein prepared in Example 3), 2% of pure plasminogen S4 powder (plasminogen protein prepared in Example 4), and 2% of pure plasminogen S5 powder (plasminogen protein prepared in Example 5) to multiple portions of the same PBS buffer (pH 7.5), and mix them evenly to obtain a wild-type plasminogen solution, a plasminogen S1 solution, a plasminogen S2 solution, a plasminogen S3 solution, a plasminogen S4 solution, and a plasminogen S5 solution, respectively;

[0048] 3. Add 1% pure fibrin powder to the above solutions and make them into gels;

[0049] 4. Add 1% urokinase solution to the surface of the gel and incubate at 22±2℃ for 30min;

[0050] 5. Record the size of the dissolution ring on the gel, calculate the concentration of plasmin based on the standard curve determined in step 1, and calculate the activity of urokinase based on the concentration of plasmin. The results are shown in Table 1. 1U of urokinase activity is defined as the amount of urokinase required to convert 1 mol of plasminogen into plasmin at 22±2℃ within 30 minutes.

[0051] Table 1 Activity of urokinase on plasminogen

[0052]

[0053]

[0054] As shown in Table 1, urokinase has obvious catalytic activity for wild-type plasminogen, but loses the corresponding catalytic activity for the five modified plasminogen mutants. The plasminogen mutants on the affinity chromatography medium will not produce peptide shedding due to the activity of urokinase.

[0055] Method for determining the affinity of plasminogen for urokinase:

[0056] The affinity of plasminogen for urokinase is usually quantified by the dissociation constant (K_D). K_D represents the strength of the protein-ligand interaction, and the smaller the K_D value, the stronger the affinity. The K_D value is determined by surface plasmon resonance.

[0057] 1. Experimental preparation: Plasminogen: As the analyte, plasminogen can be fixed on the surface of the SPR sensor chip. The samples are plasminogen proteins prepared in the comparative example and Examples 1 to 5. Urokinase: As the target molecule, urokinase, as the substance to be tested, will interact with the plasminogen fixed on the chip. Sensor chip: Use Biacore TM Sensor chip, used to fix one molecule and detect the binding of another molecule to it. Flow cell buffer: PBS buffer, used to maintain physiological conditions and control the pH and ionic strength in the flow cell. SPR instrument: Biacore. Configure the SPR instrument and adjust parameters such as temperature and flow rate to ensure that the binding analysis with the target molecule is carried out under appropriate conditions. Determine the flow rate required for the reaction, usually 30-100L / min, to ensure stable flow conditions;

[0058] 2. Immobilization of plasminogen: Immobilize plasminogen on the chip surface through EDC / NHS coupling reaction, and the concentration of plasminogen is 50g / mL;

[0059] 3. Flow of urokinase: Add different concentrations of urokinase solution to the flow cell to measure the binding of urokinase to plasminogen. The concentration of urokinase ranges from 1 to 100 nM. By measuring the change of SPR signal (i.e., the change of reflection angle), the binding process of urokinase to plasminogen is monitored in real time.

[0060] 4. Determine the binding kinetics: At a certain flow rate, the urokinase solution is continuously injected into the flow cell to bind to the plasminogen fixed on the chip surface. This process will cause a change in the surface refractive index, and the SPR device records this change in real time, usually as an increased signal. Binding and dissociation phase: Monitor the process of urokinase binding to plasminogen and record the process of urokinase dissociating from the surface. The signal will increase rapidly after binding and gradually recover after dissociation.

[0061] 5. Calculation of affinity constant (K_D): By recording the binding data at different urokinase concentrations, a concentration-response curve was drawn, and the binding constant (K_on) and dissociation constant (K_off) were calculated using the first-order kinetic model.

[0062] The affinity constant (K_D) can be calculated using the formula K_D = K_off / K_on. These data can be fitted and analyzed using the software that comes with the SPR instrument to directly provide the calculation results of K_D.

[0063] 6. Blank control: Use a chip without plasminogen as a control to ensure that any signal changes in the experiment are related to the binding of plasminogen.

[0064] Nonspecific binding: Use blank mobile phase or buffer without urokinase as a control to eliminate the influence of nonspecific binding.

[0065] The determination results of K_D of different plasminogens and urokinase are shown in Table 2.

[0066] Table 2 K_D values ​​of different plasminogens and urokinase

[0067] Plasminogen K_D value with urokinase (nM) Wild-type plasminogen 30.6 Plasminogen S1 14.0 Plasminogen S2 13.5 Plasminogen S3 13.2 Plasminogen S4 12.7 Plasminogen S5 12.1

[0068] As shown in Table 2, the dissociation constants of the five plasminogen mutants with urokinase are all less than half of that of the wild type, so the affinity of the five plasminogen mutants with urokinase is better than the affinity between the wild type plasminogen and urokinase.

[0069] Method for determining the static loading capacity of affinity chromatography media: UV absorption method

[0070] 1. Determine the specific absorption coefficient: prepare urokinase solutions of different concentrations, and then measure the absorbance of the corresponding solutions at a wavelength of 280nm to calculate the specific absorption coefficient;

[0071] 2. Equilibrium chromatography column: The affinity chromatography fillers prepared in the comparative example and Examples 1 to 5 were equilibrated with PBS buffer (pH 7.5) respectively;

[0072] 3. Prepare washing solution: dissolve 2 g urokinase in 150 mL PBS buffer (pH 7.5), and dilute to 100 mL with PBS buffer (pH 7.5) to obtain urokinase solution;

[0073] 4. Soaking and adsorption: Take 10 mL of urokinase solution, then add 5 mg of the affinity chromatography filler prepared in the comparative example, stir thoroughly and filter to obtain a filtrate. Repeat the above steps with the affinity chromatography fillers prepared in Examples 1 to 5 to obtain filtrates respectively;

[0074] 5. Calculate the static loading capacity of the affinity chromatography filler: Measure the absorbance of the filtrate at a wavelength of 280 nm, calculate the concentration of the filtrate based on the measured specific absorption coefficient, and thus calculate the initial static loading capacity of the affinity chromatography filler. The measurement results are shown in Table 3.

[0075] Table 3 Initial static loading of different affinity chromatography media

[0076] Affinity chromatography media Initial static load (mg / mg) Comparative Example 18.3 Example 1 32.6 Example 2 33.1 Example 3 32.7 Example 4 35.9 Example 5 36.4

[0077] As shown in Table 3, the initial static loading of urokinase by the affinity chromatography fillers prepared from the five plasminogen mutants is better than that by the affinity chromatography fillers prepared from the wild-type plasminogen, and the initial static loading is all above 30 mg / mg.

[0078] The affinity chromatography fillers prepared in the comparative example and Examples 1 to 5 were respectively immersed in 0.1 M acetic acid solution at 22±2°C for 24 hours, and then the urokinase loading capacity of the affinity chromatography fillers after immersion was determined according to the above-mentioned method for determining the static loading capacity of the affinity chromatography fillers. The measurement results are shown in Table 4.

[0079] Table 4 Remaining static load after immersion in 0.1M acetic acid solution at 22±2℃ for 24h

[0080]

[0081]

[0082] As shown in Table 4, the acid resistance of affinity chromatography fillers prepared from the five plasminogen mutants is better than that of affinity chromatography fillers prepared from wild-type plasminogen, and the remaining static loading after soaking in 0.1M acetic acid solution at 22±2°C for 24h is at least 80% of the initial static loading.

[0083] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary researchers in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A urokinase affinity chromatography filler, characterized in that: The ligand of the affinity chromatography filler is agarose, and the ligand is a plasminogen mutant; the plasminogen mutant is based on the wild-type plasminogen described in SEQ ID NO: 1, and the following mutation set is mutated: L100Q+G121S+K196C+N251 C.

2. The urokinase affinity chromatography filler according to claim 1, characterized in that: The plasminogen mutant also includes the following mutations: S598N+K729C.

3. The urokinase affinity chromatography filler according to claim 1, characterized in that: The plasminogen mutant also includes the following mutations: S598N+V730P.

4. The urokinase affinity chromatography filler according to claim 1, characterized in that: The plasminogen mutant also includes the following mutations: K729C+V730P.

5. The urokinase affinity chromatography filler according to claim 1, characterized in that: The plasminogen mutant also includes the following mutations: S598N+K729C+V730P.

6. A urokinase affinity chromatography filler according to any one of claims 1 to 5, characterized in that: The dissociation constant of the plasminogen mutant with urokinase is less than 15 nM.

7. A urokinase affinity chromatography filler according to any one of claims 1 to 5, characterized in that: The initial static loading of the affinity chromatography medium is at least 30 mg / mg.

8. A urokinase affinity chromatography filler according to any one of claims 1 to 5, characterized in that: After the affinity chromatography filler is immersed in a 0.1 M acetic acid solution at 22±2° C. for 24 hours, the remaining static loading capacity is at least 80% of the initial static loading capacity.

9. A urokinase affinity chromatography filler according to any one of claims 1 to 5, characterized in that: The ligand is a cross-linked 4% agarose matrix.

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