CN111909920B - Expression and purification method of recombinant human tissue-type plasminogen activator - Google Patents
Expression and purification method of recombinant human tissue-type plasminogen activator Download PDFInfo
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- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
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- C12N9/6459—Plasminogen activators t-plasminogen activator (3.4.21.68), i.e. tPA
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- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21068—Tissue plasminogen activator (3.4.21.68), i.e. tPA
Abstract
The invention provides an expression and purification method of a recombinant human histiotype plasminogen activator, which comprises the following steps: inducing a prokaryotic system to express human tissue-type plasminogen activator protein molecules by a self-induced culture system; purifying the recombinant human histiotype plasminogen activator expressed by a prokaryotic system by using a serine protease affinity chromatographic column; the protein molecules of the human tissue plasminogen activator are renatured by using a dilution dialysis technology. The expression and purification method of the recombinant human histiotype plasminogen activator provided by the invention has the advantages of simple operation, low cost, large protein expression amount and high protein yield.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an expression and purification method of a recombinant human histiotype plasminogen activator.
Background
Tissue-type plasminogen activator (t-PA) or plasminogen activator (t-PA) is a physiological agonist of the fibrinolytic system in vivo, and plays a key role in the balance regulation of fibrinolysis and coagulation in human body. t-PA belongs to serine protease in glycoprotein, is a specific protein of human body, comprises 527 amino acids, wherein 35 cysteine residues, which participate in forming 17 disulfide bonds, can specifically hydrolyze plasminogen, form plasmin and activate a fibrinolytic system, plays an important role in removing insoluble fibrin in blood vessels and ensuring smooth blood circulation, and can effectively prevent and treat thrombotic diseases such as acute myocardial infarction and the like.
The current research on tissue plasminogen activator is more than that of its mutant, such as CN 103159860, CN 107177611, CN103509772, etc., and more than that of eukaryotic expression, such as CN 101717430 and CN 1468862. In the prior art, the problems of difficult cell strain screening, long time, low protein expression amount, high cost, time and labor consumption and the like exist, and the research on the structure and the function of the t-PA is not facilitated. Therefore, a method for purifying t-PA by prokaryotic expression is needed, and the method has the advantages of simple operation, high expression level and low cost.
Chinese patent 201410389379.X discloses a method for constructing, expressing and purifying tissue plasminogen activator. The invention relates to a construction, prokaryotic expression and purification technology of tissue plasminogen activator recombinant plasmid, which is characterized in that a 3C sequence capable of removing a label on a carrier is introduced into the N end on the basis of full-length gene plasmid containing human tPA, an RGDS sequence for improving targeting property is introduced into the C end, PQE30 is used as the carrier to construct a PQE30-rt-PA recombinant plasmid, the plasmid is induced and expressed by a self-induction method, r-tPA protein is purified by using a Ni2+ affinity chromatography column after the ultrasonic method is adopted for bacteria breaking treatment, 3C short peptide on fusion protein is cut by PreScission protease, so that the His-Tag label is removed, and the fusion protein is further purified by using a molecular sieve to obtain high-purity r-tPA protein. However, the purification steps are complicated, which easily causes the problem of low yield of the prepared protein, and further improvement is needed.
Disclosure of Invention
In order to solve the problems, the invention provides an expression and purification method of t-PA containing human tissue plasminogen activator, which can be used for the development of medicaments for thrombolysis treatment of cardiovascular and cerebrovascular diseases such as myocardial infarction.
In one aspect, the invention provides a method for expressing and purifying a recombinant human histotypic plasminogen activator.
The expression purification method comprises the following steps: the human histiotype plasminogen activator protein molecule is expressed by self-induced culture induced prokaryotic system.
The expression purification method further comprises the following steps: the recombinant human histiotype plasminogen activator expressed by a prokaryotic system is purified by a serine protease affinity chromatographic column.
The expression purification method further comprises the following steps: the protein molecules of the human tissue plasminogen activator are renatured by using a dilution dialysis technology.
Further, the prokaryotic system includes but is not limited to pET30a-tPA plasmid.
Further, the self-induction culture system comprises a conventional self-induction culture medium.
Preferably, the self-induction culture medium is a ZYM-5052 self-induction culture medium, and the ZYM-5052 self-induction culture medium is a neutral culture medium, and comprises the following components:
| nitrogen source N-Z-amine (%) | 1-2 |
| Yeast extract (%) | 0.5-1 |
| Na2HPO4(mM) | 20-30 |
| KH2PO4(mM) | 20-30 |
| NH4Cl(mM) | 4-60 |
| Na2SO4(mM) | 3-6 |
| MgSO4(mM) | 1-3 |
| Glycerol (%) | 0.4-0.8 |
| Glucose (%) | 0.04-0.06 |
| Lactose (%) | 0.1-0.4 |
Preferably, the serine protease affinity chromatography column is a Benzamidine Sepharose 4FF affinity chromatography column.
Further, the expression purification method comprises the following steps:
(1) obtaining a gene for coding t-PA protein from a cDNA library, connecting the gene to a prokaryotic expression vector pET30a, and obtaining a recombinant pET30a-tPA plasmid;
(2) transforming the recombinant plasmid into E.coli Origami 2 (DE3) cell, activating Origami 2 strain containing the recombinant plasmid in MDG culture medium, culturing in ZYM-5052 self-induced culture medium, and performing self-induced expression with IPTG to obtain inclusion body containing t-PA protein;
(3) purification was performed under denaturing conditions on a Benzamidine Sepharose 4FF affinity column, renaturation was performed by dilution dialysis techniques and HPLC was performed on a C4 reverse phase column to further obtain correctly folded t-PA protein.
Further, the dilution dialysis technique in step (3) is to calculate the concentration of the primarily purified protein according to the molar extinction coefficient of t-PA molecules, dilute to 0.1-0.3mg/mL with Buffer D (0.1M Tris, 6M Urea, pH7.0), fill into a clean dialysis bag and dialyse at room temperature for renaturation for at least 12 hours.
In another aspect, the invention provides a recombinant human histotypic plasminogen activator.
The recombinant human histiotype plasminogen activator is prepared by the expression and purification method.
In another aspect, the invention provides the use of the expression and purification method in the preparation of recombinant human histotypic plasminogen activator.
In another aspect, the invention provides the application of the expression and purification method in the development of medicaments for thrombolytic treatment of cardiovascular and cerebrovascular diseases such as myocardial infarction.
In another aspect, the invention provides the use of the recombinant human histiotype plasminogen activator in the development of medicaments for thrombolysis treatment of cardiovascular and cerebrovascular diseases such as myocardial infarction.
In another aspect, the invention provides a medical preparation for thrombolytic therapy of cardiovascular and cerebrovascular diseases such as myocardial infarction.
The medical preparation comprises the recombinant human tissue-type plasminogen activator.
The expression and purification method of the recombinant human histiotype plasminogen activator provided by the invention has the following advantages: the prokaryotic expression technology has simple operation, low cost and large protein expression amount, and the high-yield protein induced by IPTG in the prior art can be obtained by a self-induction system; the renaturation technology of dilution dialysis does not need the participation of molecular chaperones, omits the step of removing the molecular chaperones and is beneficial to improving the yield; the T-PA molecule is purified by utilizing the serine protease affinity chromatographic column, the t-PA is not required to be modified, a label is not required to be added, the purification steps can be simplified, and the yield is improved.
Drawings
FIG. 1 shows the elution of correctly folded t-PA protein with a gradient of acetonitrile (25% -65%) over time.
Detailed Description
The present invention will be further illustrated in detail with reference to the following specific examples, which are not intended to limit the present invention but are merely illustrative thereof. The experimental methods used in the following examples are not specifically described, and the materials, reagents and the like used in the following examples are generally commercially available under the usual conditions without specific descriptions.
Example 1 expression and purification method of recombinant human histotypic plasminogen activator
1. The t-PA gene is subjected to PCR amplification by using a cDNA library, and a primer is designed by a general method to connect the t-PA gene to a genetic engineering vector pET30a so as to construct pET30a-tPA plasmid.
2. The constructed pET30a-tPA plasmid was transformed into E.coli DH 5. alpha. competent cells, plated on LB medium plate containing kanamycin, the composition of LB medium is as follows:
| yeast extract (Yeast extract) | 5g/L |
| Tryptone (Tryptone) | 10g/L |
| NaCl | 10g/L |
Adding 200 μ L10M NaOH per liter culture medium to adjust pH to neutral, and sterilizing at high temperature under high pressure. After overnight culture, single colonies were picked and cultured in ZYM-505 liquid medium containing kanamycin, the composition of which is as follows:
| nitrogen source N-Z-amine (%) | 1 |
| Yeast extract (%) | 0.5 |
| Na2HPO4(mM) | 25 |
| KH2PO4(mM) | 25 |
| NH4Cl(mM) | 50 |
| Na2SO4(mM) | 5 |
| MgSO4(mM) | 2 |
| Glycerol (%) | 0.5 |
| Glucose (%) | 0.05 |
Adjusting the culture medium to neutral, and sterilizing at high temperature under high pressure. After the culture is finished, sequencing the quality-improved particles, transforming the plasmid with correct sequencing into E.coli Origami 2 cells, performing activation culture in an MDG culture medium containing kanamycin, and finally preserving the strain at-80 ℃ by 25% of glycerol.
(3) Origimi 2 strain containing pET30a-tPA plasmid was inoculated into MDG medium containing 100mg/L kanamycin and activated for 8-10 hours, and the MDG medium contained the following components:
| Na2HPO4(mM) | 25 |
| KH2PO4(mM) | 25 |
| NH4Cl(mM) | 50 |
| Na2SO4(mM) | 5 |
| MgSO4(mM) | 2 |
| glucose (%) | 0.5 |
| Aspartic acid (%) | 0.25 |
Transferring 1mL of activated bacterial liquid into 250mL of ZYM-5052 self-induced medium (0.2% lactose is added more than ZYM-505 liquid medium) containing 100mg/L kanamycin, and performing self-induced expression for 8-12 h under the culture conditions of 37 ℃ and 220 rpm;
4. collecting thallus at 4 ℃ by using a refrigerated centrifuge, discarding the supernatant, resuspending the thallus by Buffer A (20mM Tris, 150mM NaCl, 2mM EDTA, 0.1% Triton X-100, pH7.0), ultrasonically lysing bacteria in an ice water bath, centrifuging at 17000g at 4 ℃ to collect inclusion bodies, and discarding the supernatant;
5. inclusion bodies were washed sequentially with the following buffers: buffer B1(20mM Tris, 20mM NaCl, 0.5% (v/v) Triton X-100, pH7.0), Buffer B2(20mM Tris, 2M NaCl, pH7.0), Buffer B3(20mM Tris, 150mM NaCl, 2M Urea, pH7.0), discarding the supernatant by centrifugation, Buffer C (8M Urea, 100mM Na)2HPO410mM Tris,1mM beta-mercaptoethanol, pH 8.0) resuspending the inclusion body pellet, placing in a chromatographic cabinet for dissolving overnight;
6. benzamidine Sepharose 4FF affinity chromatography column chromatography with Buffer C (8M Urea, 100mM Na)2HPO410mM Tris,1mM beta-mercaptoethanol, pH 8.0), centrifuging the solubilized inclusion bodies at 17000g for 1h at 4 ℃, retaining the supernatant and discarding the precipitate, filtering the supernatant through a 0.45 μ M filter (ultrasound 3 times if viscous) and loading it onto a chromatography column at a flow rate of 1mL/min, washing off unbound hetero-proteins with about 5 column volumes of Buffer C, and finally eluting with an elution Buffer (8M Urea, 100mM Na) containing 10mM beta-mercaptoethanol2HPO410mM Tris,10mM beta-mercaptoethanol, pH 4.0) to obtain the target protein by linear elution;
7. the C4 column is balanced by ultrapure water containing 0.1% trifluoroacetic acid in advance, t-PA protein with good renaturation and ultrapure water containing 0.1% trifluoroacetic acid are diluted by a ratio of 1:3, then a 0.45 mu m filter membrane is used for filtering and loading the sample on the C4 column, the loading speed is 5mL/min, the ultrapure water containing 0.1% is used for washing away urea and non-combined hybrid protein, finally acetonitrile containing 0.1% trifluoroacetic acid is used for gradient elution of the correctly folded t-PA protein, the gradient change of the acetonitrile concentration (25% -65%) along with time is shown in figure 1, the total flow rate is 5mL/min, the elution peak is approximately positioned at about 50min according to the hydrophobicity of the protein, and the acetonitrile in the eluent is collected and dialyzed to remove the acetonitrile.
8. Calculating the concentration of the primarily purified protein according to the molar extinction coefficient of t-PA molecules, diluting to 0.1-0.3mg/mL by Buffer D (0.1M Tris, 6M Urea, pH7.0), filling into a clean dialysis bag, dialyzing at room temperature for renaturation for at least 12 hours, further purifying and separating the renatured t-PA protein by HPLC through a C4 reverse chromatographic column, and detecting the product.
9. Detecting a high-order structure by circular dichroism spectroscopy: the product was reconstituted with 20mM NaAc, pH 5.0 buffer, measured for concentration and diluted to 10. mu.M. The test sample was added to a cuvette (approximately 600 μ L) and measured using a circular dichroism spectrometer, minus the baseline set with the buffer control. Measurement range: far ultraviolet 260nm to 190nm and near ultraviolet 350nm to 250 nm; taking point intervals: 0.5 nm; scanning speed: 200 nm/min; slit width: 1 nm; measuring the temperature: 25 ℃; the measurement times are as follows: 5 times. And (3) comparing the similarities and differences of the secondary structure and the tertiary structure of the recombinant t-PA protein and the standard product according to the analysis of map data.
The purity of the t-PA protein obtained by the embodiment can reach 95%.
The t-PA protein obtained in the present example was subjected to thrombolysis performance detection by a fibrin plate lysis method, which specifically comprises the following steps:
the recombinant t-PA protein was reconstituted with physiological saline and diluted to approximately 1. mu.g per l mL. Weighing 125mg of agarose, adding 23mL of physiological saline, boiling to swell the agarose, balancing in a water bath at 55-60 ℃, adding 14 mu L of human thrombin solution (100IU/mL) and 280 mu L of human plasminogen solution (0.5mg/mL), shaking uniformly while adding, adding 2.2mL of human fibrinogen solution (6mg/mL), immediately pouring into a plate with the diameter of 8cm after shaking to be turbid, horizontally placing for full solidification, and placing for at least 30 minutes at 4 ℃ for later use. A small hole with the aperture of 2mm is drilled in a fibrin-containing plate, recombinant t-PA protein solution and standard solution are respectively added into the hole, 10 mu L of each hole is formed, each dilution is formed into 2 holes, and the recombinant t-PA protein solution and the standard solution are horizontally placed in a wet box at 37 ℃ for 24 hours. The diameter of the soluble ring is measured longitudinally and transversely, and the average value is taken 2 times each. And performing linear regression on the logarithm of the biological activity of each dilution of the standard solution to the logarithm of the corresponding lysis ring diameter to obtain a linear regression equation, and obtaining the biological activity of the sample to be detected according to the logarithm of the lysis ring diameter of the sample to be detected.
The results show that the thrombolytic activity of t-PA protein obtained in this example can reach 4X 10 relative to the standard5IU/mg。
Claims (2)
1. A method for expressing and purifying a recombinant human histiotype plasminogen activator is characterized by comprising the following steps:
(1) obtaining a gene for coding t-PA protein from a cDNA library, connecting the gene to a prokaryotic expression vector pET30a, and obtaining a recombinant pET30a-tPA plasmid;
(2) transforming the recombinant plasmid into E.coli Origami 2 cells, activating Origami 2 strains containing the recombinant plasmid in an LB MDG culture medium, performing amplification culture in a ZYM-5052 self-induction culture medium, and performing self-induction expression to obtain inclusion bodies containing t-PA protein;
(3) under the denaturation condition, purifying by a Benzamidine Sepharose 4FF affinity chromatographic column, renaturing by using a dilution dialysis technology, and further obtaining correctly folded t-PA protein by an HPLC (high performance liquid chromatography) C4 reverse chromatographic column;
the self-induction culture medium in the step (2) is a ZYM-5052 self-induction culture medium, and the ZYM-5052 self-induction culture medium is a neutral culture medium and consists of the following components:
；
The dilution dialysis technology in the step (3) is to calculate the concentration of the primarily purified protein according to the molar extinction coefficient of t-PA molecules, dilute the protein to 0.1-0.3mg/mL by Buffer D, put the protein into a clean dialysis bag and dialyze and renature the protein for at least 12 hours at room temperature; the Buffer D consists of 0.1M Tris, 6M Urea, pH7.0.
2. Use of the expression purification method of claim 1 for the preparation of recombinant human histotypic plasminogen activator.
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