Recombinant aprotinin, its gene and method of production

Abstract

The present application relates to a kind of recombinant aprotinin and its coding gene and preparation method, the preparation method is in the N end of the sequence of bovine aprotinin sequence and adds a sequence consisting of 21 amino acids, the recombinant aprotinin sequence is composed of 79 amino acids, molecular weight 8.812kD, isoelectric point 9.42.And a kind of rapid purification method is established, and the molecular weight of about 8.8kD of recombinant aprotinin can be quickly separated and purified by two-step chromatography, the activity recovery rate of whole purification process is high, specific activity >4.0EPU / mg, about 400mg of recombinant aprotinin pure product can be obtained per liter of fermentation liquor.The whole purification step is simple, the loss of target protein is less, the potency of obtained product is high, and the yield per liter of fermentation liquor is high, suitable for large-scale production application.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a recombinant aprotinin, its encoding gene, and a preparation method thereof. Background Technology

[0002] Aprotinin, also known as bovine trypsin inhibitor, is a non-specific serine protease inhibitor. It is a single-chain basic protein composed of 58 amino acid residues, cross-linked by three disulfide bonds. It has a molecular weight of 6.5 kDa and an isoelectric point of 10.5. It can inhibit the activity of trypsin, chymotrypsin, kallikrein, and other enzymes. Aprotinin can form stable complexes with proteases, thereby blocking the active site of the enzymes.

[0003] Aprotinins are widely used in the biological and medical fields. Currently, genetically engineered aprotinins are commercially available, mostly prepared in E. coli through gene recombination. However, this method often results in inactive aprotininogen inclusion bodies, requiring denaturation and renaturation of the inclusion bodies before enzymatic digestion to remove the precursor portion of the zymogen to obtain the active enzyme. During the denaturation and renaturation processes, disulfide bond mismatches are highly likely to occur, leading to low renaturation yields. Furthermore, the need for enzymatic digestion of the precursor makes the process cumbersome, time-consuming, labor-intensive, and yielding low productivity. Summary of the Invention

[0004] One objective of this invention is to provide a recombinant aprotinin gene and its encoded protein, wherein the base sequence of the recombinant aprotinin gene, from the N-terminus to the C-terminus, comprises: a His-tag gene sequence and an aprotinin gene sequence.

[0005] Preferably, the aprotinin gene sequence is as shown in Seq ID No. 5.

[0006] The recombinant aprotinin gene of this application has a leader peptide sequence added to the N-terminus of the bovine aprotinin sequence. After induction, it can be expressed efficiently. The optimized recombinant aprotinin sequence can also obtain high aprotinin activity without the need for enzyme digestion to remove this His-tag.

[0007] The present invention further provides a recombinant aprotinin encoded by the above-mentioned recombinant aprotinin gene, which consists of 79 amino acids, has a molecular weight of about 8.8 kD, and an isoelectric point of 9.42, as shown in Seq ID No. 6.

[0008] In a second aspect, the present invention provides a method for expressing and isolating an aprotinin in Escherichia coli, the method comprising the following steps: (1) Artificially design and synthesize recombinant aprotinin gene, transduce the aprotinin gene sequence into a vector, transform the constructed recombinant vector into genetically engineered bacteria, and construct recombinant aprotinin engineered bacteria. (2) Culture recombinant aprotinin engineered bacteria and induce the expression of recombinant aprotinin; (3) Collect the bacterial cells and wash them; (4) Disrupt the bacterial cells; (5) Centrifuge to collect inclusion bodies; (6) Transmutation and commutation; (7) Purify the above recombinant aprotinin.

[0009] In one implementation, the recombinant aprotinin gene sequence is shown in Seq ID No. 5.

[0010] Preferably, the vector is plasmid pet28a.

[0011] Preferably, step (1) includes: artificially synthesizing a bovine aprotinin gene sequence, the gene sequence being as shown in Seq ID No. 5, inserting it into the corresponding restriction site of plasmid pet28a or pet28a through the BlpI / NdeI restriction site to construct a recombinant plasmid containing His-tag, preferably pet28a; The constructed recombinant plasmid was introduced into Escherichia coli BL21(DE3) by CaCl2 transformation to construct recombinant aprotinin engineered bacteria.

[0012] Preferably, the engineered bacteria in step (1) is *Escherichia coli* BL21(DE3), and the method of the present invention has no limitation on the type of *E. coli* host. Hosts capable of efficiently expressing recombinant aprotinin are preferred. Transformation methods for converting the aforementioned expression vector into *E. coli* BL21(DE3) are well known in the art, such as electroporation, CaCl2 transformation, etc.

[0013] The method for inducing the host to express recombinant aprotinin in step (1) can be a technique known to those skilled in the art.

[0014] The recombinant aprotinin had a His-tag sequence added to the N-terminus of the bovine aprotinin sequence. In step (6), the aprotinin with this sequence had high activity after renaturation. It was purified and separated by SP chromatography and phenyl hydrophobic chromatography. The SP chromatography equilibration buffer was 20 mM citric acid, pH 3.0. After loading the sample, the UV peak was washed to the baseline with the equilibration buffer and then washed with washing buffer. The washing buffer was 20 mM citric acid, 450 mM sodium chloride, pH 4.0. The washing was performed for 3-5 CV. Then, the elution was performed with elution buffer. The elution buffer was 20 mM citric acid, 1 M sodium chloride, pH 4.0. The elution peak was collected.

[0015] Adjust the pH of the SP elution collection buffer to 7.0 ± 0.2, and perform phenyl hydrophobic chromatography. The phenyl hydrophobic equilibration buffer consisted of 20 mM Tris and 1 M sodium chloride. Collect the flow-through peak of the sample. The phenyl hydrophobic collection buffer was concentrated by 3 kDa ultrafiltration and then lyophilized.

[0016] In a preferred embodiment of the present invention, IPTG is used as an inducer during the expression process to achieve efficient expression of the target protein. A more preferred method is as follows: 1 mL of recombinant engineered bacteria is inoculated into 1 L of LB medium and cultured in a shaker at 37±2℃ for 6-7 h. When OD... 600 The pH was 0.3~3.0. The culture medium was inoculated into a fermenter containing 100L of culture medium. The initial parameters were: fermentation temperature 37±2℃, stirring speed 200±10rpm, pH 7.0±0.2. The stirring speed and aeration rate were controlled to maintain dissolved oxygen above 30%. The culture medium formula was as follows: Fermentation medium: yeast extract 2.5±0.5g / L, dipotassium hydrogen phosphate 10±2g / L, potassium dihydrogen phosphate 2.5±0.5g / L, potassium sulfate 1.125±0.3g / L, ammonium chloride 3.75±0.5g / L, sodium citrate 4.25±0.5g / L, ferric ammonium citrate 0.1875±0.2g / L, and defoamer 0.25±0.2ml / L. Feeding medium: 84±5 g / L magnesium sulfate solution, 10%-37% ammonia solution, 4±1 g / L calcium chloride solution, 50% glucose solution (w / v), trace element solution (manganese sulfate monohydrate 1.0 g / L, zinc sulfate 2.78 g / L, cobalt chloride hexahydrate 2.0 g / L, sodium molybdate dihydrate 2.0 g / L, calcium chloride 3.0 g / L, copper sulfate pentahydrate 1.85 g / L, boric acid 0.5 g / L). Ammonia was used for pH control during fermentation. At 4.0 h of fermentation, residual sugar was measured using a saccharimeter. Once the residual sugar was depleted, the feeding rate of 50% glucose solution (w / v) was adjusted according to cell growth to control the residual sugar concentration at 1-3 mM. When the fermentation broth OD... 600 After reaching 55-65°C, stop adding 50% glucose solution (w / v). Then, add 900 mL of sterilized 84±5 g / L magnesium sulfate solution, 450 mL of 4±1 g / L calcium chloride solution, and 225 mL of trace element solution in sequence. When the sugar concentration is low, the pH is above 7.05, and the dissolved oxygen is above 40%, add IPTG under aseptic conditions to a final concentration of 0.5-1.0 mM. Continue culturing until E. coli growth shows stagnation. Stop adding sugar, cool the fermentation broth in the fermenter to 15±2°C, and collect the cells.

[0017] Preferably, the method for breaking the bacterial cells in step (4) is high-pressure homogenization.

[0018] Preferably, in step (6), denaturation is performed using 8M urea, with a mercaptoethanol concentration of 1 mL / L, pH 10.0 ± 0.2, and denaturation time ≥ 18 h.

[0019] Preferably, the refolding in step (6) is a dilution refolding.

[0020] Preferably, in step (7), the recombinant aprotinin is purified by directly performing chromatography on the recombinant protein whose leader peptide sequence is a His-tag sequence after refolding, to obtain highly active recombinant aprotinin. The chromatography uses ion chromatography, affinity chromatography, or phenyl hydrophobic chromatography, and the chromatographic conditions first involve linear gradient elution.

[0021] Preferably, the recombinant aprotinin obtained after chromatography purification in step (7) is concentrated and lyophilized after 3kD ultrafiltration.

[0022] Advantages of this invention: Compared with the prior art, the method for preparing recombinant aprotinin provided by the present invention has the following advantages: 1) The expression rate and activity of recombinant aprotinin were improved by adding 21 amino acids to the N-terminus; 2) The amino acid sequence added at the N-terminus does not require enzyme digestion for removal, simplifying subsequent purification steps; 4) The recombinant aprotinin has high activity and the preparation process is simple, low-cost and high-yield. Attached Figure Description

[0023] Figure 1 This is a diagram showing the construction and enzyme digestion verification of the His-tag aprotinin recombinant plasmid of this invention.

[0024] Figure 2 This is an SDS-PAGE image of a purified sample of the SOD-aprotinin sequence precursor.

[0025] Figure 3 This is an SDS-PAGE image of a purified sample of the SUMO-aprotinin sequence precursor.

[0026] Figure 4 SDS-PAGE analysis of the SUMO-aprotinin sequence after DEAE chromatography purification.

[0027] Figure 5 This is a comparison of the expression rates of two recombinant vector proteins of His-tag aprotinin.

[0028] Figure 6 This is an SDS-PAGE electrophoresis image of His-tag aprotinin fermentation expression.

[0029] Figure 7 This is an SDS-PAGE electrophoresis analysis of the His-tag aprotinin purification process.

[0030] Figure 8 This is the small molecule gel electrophoresis pattern of the final lyophilized aprotinin powder.

[0031] Figure 9This is the His-tag aprotinin sequence, where the part in red is a His-tag sequence that is inherent in the vector pet28a. Detailed Implementation

[0032] The present invention will be described in detail below with reference to specific embodiments. These specific embodiments are illustrative and do not limit the scope of protection of the present invention in any way.

[0033] Material description: Strains and plasmids: The expression strain BL21 (DE3) and plasmids pSmart-I, pet24a, and pet28a were purchased from Thermo Fisher Scientific.

[0034] Enzymes and reagents: The enzymes used in the molecular biology operations in the examples were all purchased from Thermo, and the corresponding operation steps were performed in strict accordance with the relevant product instructions.

[0035] The synthesis of the nucleotide sequences and the sequencing of the DNA involved in the examples were completed by Hefei Jixiang Biotechnology Co., Ltd.

[0036] All other raw and auxiliary materials whose sources are not specified are commercially available products. Construction of recombinant aprotinin expression vector and engineered bacteria

[0037] 1. Construction of recombinant aprotinin expression vector Comparative Example 1: A DDDDK enterokinase cleavage site was designed based on the leader peptide sequence. The leader peptide sequence can be removed by enterokinase cleavage, and the resulting aprotinin is completely identical to the aprotinin sequence derived from bovine lung. The recombinant protein sequence containing the leader peptide sequence is SOD-DDDDK-bovine aprotinin sequence from the N-terminus to the C-terminus (hereinafter referred to as SOD-aprotinin sequence). Based on this, a recombinant aprotinin gene sequence containing the leader peptide base sequence was artificially synthesized (Seq ID No. 1). NdeI / BlpI cleavage sites were introduced at both ends of the above gene sequence, and the NdeI / BlpI cleavage sites were inserted into the corresponding cleavage sites of plasmid pET24a(+) to construct a recombinant plasmid containing the SOD leader peptide.

[0038] Comparative Example 2: The recombinant aprotinin gene sequence Seq ID No. 3 was synthesized artificially and seamlessly cloned into the corresponding restriction site of plasmid pSmart-I to construct a recombinant plasmid containing a SUMO-tag (the protein expressed by the recombinant vector is bovine aprotinin containing the SUMO protein leader peptide. The SUMO protease can specifically recognize the SUMO protein tag sequence and can remove the SUMO protein tag. After digestion with SUMO enzyme, the recombinant protein can obtain a protein sequence that is completely identical to the bovine lung-derived aprotinin).

[0039] Example 1: A His-tag sequence was added to the N-terminus of the bovine aprotinin gene sequence. BlpI / NdeI restriction sites were introduced at both ends of the bovine aprotinin gene sequence. Based on this, the bovine aprotinin gene sequence was artificially synthesized, as shown in Seq ID No. 5. The sequence was then inserted into the corresponding restriction sites of plasmids pet24a and pet28a via the BlpI / NdeI restriction sites, constructing a His-tag-containing recombinant plasmid.

[0040] 2. Construction of recombinant aprotinin-producing engineered bacteria: The recombinant plasmids constructed in the comparative examples and embodiments above were introduced into *E. coli* BL21(DE3) via CaCl2 transformation to construct recombinant aprotinin-producing engineered bacteria. Sequencing showed that the sequences in the engineered bacteria were consistent with the design. In Example 1, the engineered bacteria containing the two recombinant plasmids were cultured in shake flasks. SDS-PAGE analysis of the fermentation broth showed that the expression level of the pet28a recombinant plasmid engineered bacteria was higher than that of pet24a.

[0041] 3. High-density fermentation with engineered bacteria Taking a 100L fermentation scale as an example: Inoculate 1 mL of recombinant engineered bacteria into 1 L of LB medium and culture on a shaker at 37±2℃ for 6-7 h. When OD 600 The pH was 0.3~3.0. The culture medium was inoculated into a fermenter containing 100L of culture medium. The initial parameters were: fermentation temperature 37±2℃, stirring speed 200±10rpm, pH 7.0±0.2. The stirring speed and aeration rate were controlled to maintain dissolved oxygen above 30%. The culture medium formula was as follows: Fermentation medium: yeast extract 2.5±0.5g / L, dipotassium hydrogen phosphate 10±2g / L, potassium dihydrogen phosphate 2.5±0.5g / L, potassium sulfate 1.125±0.3g / L, ammonium chloride 3.75±0.5g / L, sodium citrate 4.25±0.5g / L, ferric ammonium citrate 0.1875±0.2g / L, and defoamer 0.25±0.2ml / L. Fed culture medium: 84±5 g / L magnesium sulfate solution, 10%-37% ammonia solution, 4±1 g / L calcium chloride solution, 50% glucose solution (w / v), trace element solution (manganese sulfate monohydrate 1.0 g / L, zinc sulfate 2.78 g / L, cobalt chloride hexahydrate 2.0 g / L, sodium molybdate dihydrate 2.0 g / L, calcium chloride 3.0 g / L, copper sulfate pentahydrate 1.85 g / L, boric acid 0.5 g / L). Ammonia was used for pH control during fermentation. At 4.0 h of fermentation, residual sugar was measured using a saccharimeter. Once the residual sugar was depleted, the feeding rate of 50% glucose solution (w / v) was adjusted according to cell growth to control the residual sugar concentration at 1-3 mM. When the fermentation broth OD... 600After reaching 55-65°C, stop adding 50% glucose solution (w / v). Then, add 900 mL of sterilized 84±5 g / L magnesium sulfate solution, 450 mL of 4±1 g / L calcium chloride solution, and 225 mL of trace element solution in sequence. When the sugar concentration is low, the pH is above 7.05, and the dissolved oxygen is above 40%, add IPTG under aseptic conditions to a final concentration of 0.5-1.0 mM. Continue culturing until E. coli growth shows stagnation. Stop adding sugar, cool the fermentation broth in the fermenter to 15±2°C, and collect the cells.

[0042] After being removed from the fermentation tank, the dry weight of the bacterial cells was approximately 60-65 g / L. During the fermentation process, electrophoresis of the bacterial cells at sampling points showed that the expression level reached 20-30% of the total bacterial protein.

[0043] After fermentation, the collected bacterial cells, after being concentrated and washed using hollow fiber, were brought to a final volume of over 70% of the fermentation liquid. Simultaneously, 0.5-1 mol / L disodium edetate solution was added until the final concentration of disodium edetate was 10±5 mM. Then, 2 mol / L sodium chloride solution was added until the final concentration of sodium chloride was 50±10 mM. The mixture was stirred for ≥30 min. Bacterial cell disruption was performed by homogenizing the E. coli cells twice under high pressure to release inclusion bodies within the cells. The homogenization pressure was ≥850 bar.

[0044] 4. Centrifuge to collect inclusion bodies After high-pressure homogenization, the precipitate was centrifuged using a disc centrifuge to collect inclusion bodies. The supernatant was then removed by centrifugation using a tubular centrifuge to obtain solid inclusion bodies.

[0045] 5. Transmutation and renaturation Dilute the inclusion bodies to 3L with water and mix thoroughly to form a suspension. Slowly add the inclusion body suspension to denaturing buffer (6-8M urea, 1-5mL / L mercaptoethanol, pH 10.0±1.0), stir, re-measure the pH, and then adjust the pH to 10.0±1.0. After denaturation, analyze the denaturing solution A. 280 The time for sex reassignment is 18-24 hours.

[0046] Slowly add the denaturing solution to the refolding buffer (30-50 mM glycine, 1-5 mM glutathione, pH 8.0 ± 1.0). After the denaturing solution is completely added to the refolding buffer, readjust the pH to 8.0 ± 1.0. During refolding, maintain stirring and introduce a small flow of air (maintain positive pressure 1.5 ± 0.2 Bar). The refolding time is ≥ 5 h.

[0047] Purification of recombinant aprotinin Experimental Example 1

[0048] To purify the SOD-aprotinin sequence, the precursor was purified using DEAE chromatography with a 0-100% linear gradient elution. Solution A consisted of 20-50 mM Tris, pH 9.0 ± 0.2, and solution B consisted of 20-50 mM Tris, 1 M sodium chloride, pH 9.0 ± 0.2. The linear elution peak was collected, and SDS-PAGE analysis revealed a single-band precursor. Figure 1 As shown in the figure, its amino acid sequence is as indicated by Seq ID No. 2. The activity was measured at 1485 U / mg. However, because the precursor possesses aprotinin activity and inhibits enterokinase, SDS-PAGE after the addition of enterokinase showed that the precursor band was not cleaved. Since the purified precursor aprotinin activity was far lower than commercially available levels and could not be digested to obtain the bovine aprotinin sequence, a high-activity sample could not be obtained from the SOD-aprotinin sequence. Experimental Example 2

[0049] Targeting the SUMO-aprotinin sequence, after renaturation, the sample was eluted by nickel affinity chromatography. The equilibration buffer was 20-50 mM Tris, 150-250 mM NaCl, pH 8.0 ± 0.2, and the elution buffer was 20-50 mM Tris, 150-250 mM NaCl, 250 mM imidazole, pH 8.0. The elution peak was collected to obtain a solution containing the aprotinin precursor, as shown below. Figure 2 As shown, calculations based on the loading and elution buffer A280 indicate that nickel column chromatography can remove approximately 80% of contaminating proteins. Imidazole was removed by ultrafiltration using a 10kD membrane, followed by SUMO enzyme digestion at a ratio of 1:20–1:100, with an optimal ratio ≤1:50. After digestion, DEAE chromatography was performed, followed by linear gradient elution. The linear gradient elution peaks were collected. Solution A consisted of 20–50 mM Tris, pH 8.0 ± 0.5, and solution B consisted of 20–50 mM Tris, 1 M sodium chloride, pH 8.0 ± 0.5. The elution peaks were collected and analyzed by SDS-PAGE.

[0050] The flow-through and elution peak 1 from DEAE chromatography were combined and subjected to SP chromatography with linear gradient elution. The elution peaks were collected. Solution A contained 20-50 mM citric acid at pH 3.0-5.0, and solution B contained 20-50 mM citric acid, 1 M sodium chloride, and pH 3.0-5.0. A single target band with a titer of 2.6-3.54 EPU / mg was obtained. The purified sample of this aprotinin sequence is shown in the following figure. Figure 3 As shown, its amino acid sequence is shown in Seq ID No. 4.

[0051] The SUMO-aprotinin sequence, after refolding, nickel column purification, ultrafiltration concentration and replacement, SUMO protease digestion, DEAE chromatography, and SP chromatography, yields aprotinin with a purity ≥90% (SDS-PAGE) and a potency ≥3.0 EPU / mg. However, using this sequence involves cumbersome procedures, a high SUMO enzyme digestion ratio with incomplete digestion, yielding only 30-50 mg of pure aprotinin per liter of fermentation broth, resulting in low yield. Furthermore, it requires a large amount of SUMO protease, which is expensive, making it uneconomical for large-scale commercial production. Experimental Example 3

[0052] For His-tag aprotinin, the chromatography conditions were explored after refolding, and the optimized chromatography conditions are as follows.

[0053] (1) SP chromatography After refolding, the pH of the refolding solution was adjusted to 3.0±0.2, and the solution was centrifuged using a tubular centrifuge. The supernatant was then filtered through a 0.45μm filter, yielding a specific activity of approximately 500-2500 U / mg. SP chromatography was then performed with an equilibration buffer of 20-50 mM citric acid at pH 3.0-4.0. After sample loading, the sample was washed with the equilibration buffer for 3-5 CV, followed by washing with the washing buffer (20-50 mM citric acid, 200-450 mM sodium chloride, pH 3.0-4.0) for 3-5 CV. Elution was then performed with the elution buffer (20-50 mM citric acid, 1 M sodium chloride, pH 3.0-4.0), and the elution peak was collected. The collected eluent was analyzed by SDS-PAGE, showing a single band with a specific activity of approximately 18000-23000 U / mg. SP chromatography showed A280 recovery rates of approximately 3%-12%, with activity recovery rates of approximately 85.9%-100.61%.

[0054] (2) Phenyl hydrophobic chromatography The collected SP collection solution was adjusted to pH 7.0±0.5 to obtain the HIC loading solution. During loading, the flow-through peak of the phenyl hydrophobic chromatography was collected. The phenyl hydrophobic equilibration solution consisted of 20-50 mM Tris, 1 M sodium chloride, and pH 7.0±0.5. The specific activity of the phenyl hydrophobic collection solution was approximately 21000-26000 U / mg, the A280 recovery rate was approximately 92%-99%, and the activity recovery rate was nearly 100%.

[0055] (3) 3kD ultrafiltration concentration and replacement The HIC collection solution was concentrated to 1.0 ± 0.2 L by ultrafiltration, and then replaced by fed-batch exchange with 10 mM Tris solution at pH 7.0. The final protein concentration was controlled at A. 280 ≥20, with a conductivity of 10-15 mS / cm, yields an ultrafiltration concentrate. The A280 recovery rate of the ultrafiltration concentrate is approximately 65%-90%, and the activity recovery rate is approximately 65%-95%.

[0056] (4) Freeze-drying Lyophilized solution: Dilute ultrafiltration concentrate A 280 =19±1, add 3% mannitol powder (total volume), stir until completely dissolved, and place in a freeze dryer. Pre-freeze at -40℃ for 3 hours, then raise the temperature to -15℃ and vacuum dry for 6 hours (vacuum degree 20 Pa), then raise the temperature to 25℃ and vacuum dry for 30 hours (vacuum degree 20 Pa). Collect the recombinant aprotinin lyophilized powder and send it for testing. Its potency is approximately 4.1-4.2 EPU / mg, and the purity of small molecule gel electrophoresis is >90%. Approximately 430 mg of recombinant aprotinin can be obtained per liter of fermentation broth.

[0057] The amino acid sequence of the above recombinant aprotinin is as shown in Seq ID No. 6 and Figure 9 As shown, where Figure 9 The part marked in red is a sequence (hereinafter referred to as the His-tag sequence) inherent in the vector pet28a, consisting of 21 amino acids. The theoretical molecular weight of the recombinant aprotinin is 8812 kD, and the theoretical isoelectric point is 9.42.

[0058] Construction of recombinant plasmids containing aprotinin sequence Figure 1 As shown, BPT1 is the bovine aprotinin sequence. The bovine aprotinin gene sequence was inserted into the pet28a vector using BlpІ and NdeI to construct a recombinant plasmid, as shown in the figure. Its N-terminus has a histidine tag sequence consisting of 21 amino acids.

[0059] SOD-Aprotinin sequence precursor purified sample SDS-PAGE detection, such as... Figure 2 As shown, 1 is the DEAE loading solution (renaturation solution), 2-4 are the loading flow-through solution, 5-11 are the elution peaks of linear gradient elution, 13 is the marker, and the arrow points to the aprotinin precursor band.

[0060] SDS-PAGE analysis of purified SUMO-aprotinin precursor samples as follows: Figure 3 As shown, 1 is the nickel column loading solution (renaturation solution), 2 is the flow-through solution, 4 is the elution peak, and the arrow points to the SUMO-aprotinin precursor.

[0061] The SDS-PAGE chromatogram of the sample purified by DEAE chromatography after SUMO-aprotinin sequence digestion is shown below. Figure 4 As shown, where: 3 is the marker, 4-8 are the loading solution, elution peak 1, elution peak 2, and elution peak 3 respectively, and 9-15 are the SP chromatography loading solution, flow through, elution peak 1, elution peak 2, elution peak 3, elution peak 4, and elution peak 5 respectively.

[0062] The expression rates of two recombinant vector proteins of His-tag aprotinin were compared, for example... Figure 5As shown, the left figure is the PET24A recombinant vector, and 1-3 are the homogenized liquid, homogenized supernatant and precipitate, respectively; the right figure is the PET28A recombinant vector, and 1-5 are the fermentation broth after induction for 0h, 3h, 5h and 7h, respectively; 6-8 are the homogenized liquid, homogenized supernatant and precipitate, respectively; and 9 is the marker.

[0063] His-tag aprotinin fermentation expression SDS-PAGE electrophoresis image as follows: Figure 6 As shown, 1-6 represent fermentation induction for 0-6 hours, 7 is the marker, and 8 is the fermentation endpoint; His-tag aprotinin purification process and SDS-PAGE electrophoresis analysis are as follows: Figure 7 As shown, 1 is SP loading solution, 2 is marker, 3 is SP elution and collection solution, 4 is phenyl hydrophobic loading solution, 5 is phenyl hydrophobic collection solution, 6 is marker, and 7 is ultrafiltration concentrate.

[0064] The final lyophilized His-tag aprotinin powder small molecule gel electrophoresis pattern is as follows: Figure 8 As shown, 1 is a marker and 2 is pure freeze-dried powder.

[0065] Methods for determining the activity of intermediates in the preparation of recombinant aprotinin The aforementioned recombinant aprotinin intermediates include samples from refolding to ultrafiltration concentration, and the activity assays are all performed according to the activity of inhibiting trypsin.

[0066] Method for determining the potency of recombinant aprotinin lyophilized powder The determination method for the finished product refers to the method for determining the titer of aprotinin in the 2020 edition of the Pharmacopoeia.

[0067] The recombinant aprotinin obtained in the example was tested and found to have a specific activity >4.0 EPU / mg.

[0068] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A recombinant aprotinin gene, characterized in that, The base sequence of the recombinant aprotinin gene, from the N-terminus to the C-terminus, includes: the His-tag gene sequence of the leader peptide and the aprotinin gene sequence.

2. The recombinant aprotinin gene according to claim 1, characterized in that, The aprotinin gene sequence is shown in SeqID No.

5.

3. A recombinant aprotinin encoded by the recombinant aprotinin gene according to claim 1 or 2, characterized in that, It consists of 79 amino acids, has a molecular weight of 8.8 kD, an isoelectric point of 9.42, and its amino acid sequence is shown in Seq ID No.

6.

4. A method for expressing and isolating aprotinin in Escherichia coli, the method comprising the following steps: (1) Artificially synthesize an aprotinin gene, transduce the aprotinin gene sequence into a vector, transform the constructed recombinant vector into engineered bacteria, and construct recombinant aprotinin engineered bacteria, wherein the recombinant aprotinin engineered bacteria includes the His-tag gene sequence of the leader peptide and the aprotinin gene sequence. (2) Cultivate engineered bacteria of recombinant aprotinin and induce the expression of recombinant aprotinin; (3) Collect the bacterial cells and wash them; (4) Disrupt the bacterial cells; (5) Centrifuge to collect inclusion bodies; (6) Transmutation and commutation; (7) Purify recombinant aprotinin.

5. The method according to claim 4, characterized in that, The vector is plasmid pet28a.

6. The method according to claim 4, characterized in that, Step (1) includes: artificially synthesizing the bovine aprotinin gene sequence, the gene sequence is shown in Seq ID No. 5, and inserting it into the corresponding restriction site of plasmid pet28a or pet28a through the BlpI / NdeI restriction site to construct a recombinant plasmid containing His-tag, preferably pet28a.

7. The method according to claim 6, characterized in that, The constructed recombinant plasmid was introduced into Escherichia coli BL21(DE3) by CaCl2 transformation to construct recombinant aprotinin engineered bacteria; Preferably, the engineered bacteria in step (1) is Escherichia coli BL21(DE3).

8. The method according to claim 4, characterized in that, In step (2), the expression of recombinant aprotinin is induced using the inducer IPTG; and / or The specific method for inducing the expression of recombinant protein in step (2) is as follows: 1 mL is added to 1 L of LB medium and cultured in a shaker at 37±2℃ for 6-7 h. When OD 600 The pH was 0.3~3.

0. The culture medium was inoculated into a fermenter containing 100L of culture medium. The initial parameters were: fermentation temperature 37±2℃, stirring speed 200±10rpm, pH 7.0±0.

2. The stirring speed and aeration rate were controlled to maintain dissolved oxygen above 30%. The culture medium formula was as follows: Fermentation medium: yeast extract 2.5±0.5g / L, dipotassium hydrogen phosphate 10±2g / L, potassium dihydrogen phosphate 2.5±0.5g / L, potassium sulfate 1.125±0.3g / L, ammonium chloride 3.75±0.5g / L, sodium citrate 4.25±0.5g / L, ferric ammonium citrate 0.1875±0.2g / L, defoamer 0.25±0.2ml / L; Feeding medium: 84±5 g / L magnesium sulfate solution, 10%-37% ammonia solution, 4±1 g / L calcium chloride solution, 50% glucose solution (w / v), trace element solution including 1.0 g / L manganese sulfate monohydrate, 2.78 g / L zinc sulfate, 2.0 g / L cobalt chloride hexahydrate, 2.0 g / L sodium molybdate dihydrate, 3.0 g / L calcium chloride, 1.85 g / L copper sulfate pentahydrate, and 0.5 g / L boric acid. Ammonia was used for pH control during fermentation. After 4.0 h of fermentation, residual sugar was measured using a saccharimeter. Once the residual sugar was depleted, the feeding rate of 50% glucose solution (w / v) was adjusted according to cell growth to control the residual sugar concentration at 1-3 mM. When the fermentation broth OD... 600 After reaching 55-65°C, stop adding 50% glucose solution (w / v). Then, add 900 mL of sterilized 84±5 g / L magnesium sulfate solution, 450 mL of 4±1 g / L calcium chloride solution, and 225 mL of trace element solution in sequence. When the sugar concentration is low, the pH is above 7.05, and the dissolved oxygen is above 40%, add IPTG under aseptic conditions to a final concentration of 0.5-1.0 mM. Continue culturing until E. coli growth shows stagnation. Stop adding sugar, cool the fermentation broth in the fermenter to 15±2°C, and collect the cells.

9. The method according to claim 4, characterized in that, In step (3), the method used for collecting and washing the bacteria is to concentrate and wash the bacteria using a 500kD hollow fiber. Specifically, the fermentation broth is concentrated using a 500kD hollow fiber to 20%-40% of its volume. An equal volume of purified water and a 2 mol / L sodium chloride solution is added to the concentrated broth, bringing the final sodium chloride concentration to 150 mM. Once the bacterial suspension is concentrated to 20%-40% of the fermentation broth volume, another equal volume of purified water and a 2 mol / L sodium chloride solution is added, bringing the final sodium chloride concentration to 150 mM. This washing process is repeated four times. In step (4), the method for breaking the bacterial cells is high-pressure homogenization, with a homogenization pressure ≥ 850 bar, and homogenization is performed twice.

10. The method according to claim 4, characterized in that, In step (5), disc centrifugation and tube centrifugation are used to collect inclusion bodies; and / or In step (6), denaturation is performed using 6-8M urea. 1-5 mL of mercaptoethanol is added per liter of denaturing solution. Inclusion bodies must be completely dissolved. Denaturation time is ≥18 hours at room temperature. After denaturation, the denaturing solution is slowly added to the refolding solution. The final refolding solution composition is 30-50 mM glycine, 2-5 mM glutathione, and pH 8.0 ± 0.

5. The protein concentration in the refolding solution must be controlled within A... 280 ≤3.0.

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