Method for heterologous expression of lumbrukinase based on escherichia coli

Abstract

The invention belongs to the technical field of biology, and particularly provides a method for heterologous expression of lumbrukinase based on Escherichia coli, which comprises the following steps: firstly connecting a lumbrukinase gene with a vector to construct a recombinant expression vector, then transforming the recombinant expression vector into a competent strain to obtain a recombinant bacterium, then culturing the recombinant bacterium, and inducing expression protein, thereby obtaining the lumbrukinase heterologous expression vector based on Escherichia coli. Purifying to obtain lumbrukinase protein; data prove that the lumbrukinase protein obtained through expression of the method is high in activity and high in thrombolysis capacity, the lumbrukinase obtaining time can be greatly shortened, and the lumbrukinase yield can be greatly increased.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a method for heterologous expression of lumbrokinase based on Escherichia coli. Background Art

[0002] Lumbrokinase has multiple pharmacological effects, including anti-inflammatory, antioxidant, cardiovascular and cerebrovascular protection, and anti-cancer effects, demonstrating superior protection against ischemic stroke. Compared to the FDA-approved t-PA (tissue plasminogen activator), lumbrokinase exhibits thrombolytic activity only in the presence of fibrin, preventing bleeding from excessive thrombolysis during treatment and offering a wider safe dosage range.

[0003] In traditional Chinese medicine, earthworms are used as a traditional Chinese medicine for their antipyretic, calming, diuretic, and antihypertensive properties. The presence of fibrinolytic components in earthworm extracts has long been documented in ancient Chinese medical literature. In 1982, Mihara et al. isolated a group of fibrinolytic enzymes from Lumbricus rubellus and named them lumbrokinases (LK). Lumbrokinases, also known as earthworm fibrinolytic enzymes, are a group of proteases isolated from earthworms with anticoagulant properties. Since the early 1980s, when Mihara et al. first discovered active substances in aqueous extracts of Lumbricus rubellus that directly dissolve fibrin and activate plasminogen, numerous protein fractions with anticoagulant activity have been purified from various earthworm species. Research has revealed that lumbrokinases are a complex class of proteases, characterized by a diverse composition, structure, and enzymatic properties. At least two or more proteases with anticoagulant activity, differing in molecular weight and biochemical characteristics, can be isolated from the same earthworm species. For example, multiple fibrinolytic enzyme fractions have been isolated from both Lumbricus rubellus and Lumbricus sempervirens.

[0004] However, traditional methods for extracting and purifying lumbrokinase from earthworms are complex and time-consuming. The growth cycle of earthworms, the raw material, to meet extraction standards also restricts the production of lumbrokinase. In addition, because lumbrokinase is a protein complex, it is difficult to separate and purify individual lumbrokinase proteins using traditional methods. However, this also results in different protein compositions of the final lumbrokinase products obtained using different extraction and purification methods, which cannot meet uniform standards and the quality of lumbrokinase cannot be guaranteed. Lumbrokinase obtained through traditional extraction and purification processes may also contain other earthworm contaminants, which can cause adverse side effects such as stomach discomfort or vomiting. Summary of the Invention

[0005] To address the aforementioned issues with existing technologies, the present invention provides a method for heterologous expression of lumbrokinase in Escherichia coli. This method produces highly active lumbrokinase protein with strong thrombolytic capacity, significantly shortens the time required to obtain lumbrokinase, and significantly increases lumbrokinase production.

[0006] The technical solution adopted in the present invention is: A method for heterologous expression of lumbrokinase in Escherichia coli comprises the following steps: (1) Connecting the lumbrokinase gene to the vector to construct a recombinant expression vector; (2) transforming the recombinant expression vector into a competent strain to obtain a recombinant bacterium; (3) The recombinant bacteria are cultured and induced to express protein, and lumbrokinase protein is obtained through purification.

[0007] In step (1), the amino acid sequence of the lumbrokinase is (as shown in SEQ ID NO.1): IVGGIEARPYEFPWQVSVRRKSSDSHFCGGSIINDRWVVCAAHCMQGESPALVSLVVGEHDSSAASTVRQTHDVDSIFVNENYDPRTLENDVSVIKTAIAITFDINVGPICAPDPANDY VYRKSQCSGWGTINSGGICCPAVLRYVTLNiTTNAFCDAVYTSDTIYDDMICATDNTGMTDRDSCQGDSGGPLSVKDGSGIFSLVGIVSWGIGCASGYPGVYSRVGFHAGWITDTITNN.

[0008] The nucleotide sequence encoding the lumbrokinase gene is (as shown in SEQ ID NO.2):.

[0009] In step (1), the vector is pET-22b(+).

[0010] The lumbrokinase gene was synthesized by the following method: (a) Combine the pET-22b(+) vector and the target gene using homologous recombination; (b) The lumbrokinase gene was obtained by polymerase chain reaction amplification.

[0011] In step (a), the sequence of the upstream primer is TGGATATCGGAATTAATTCGGATATGATTGTGGGCGGCATTGAAGCGCGC; the sequence of the downstream primer is GTGGTGCTCGAGTGCGGCCGCAAGCTTGTGATGGTGATGGTGATGGCCCTGAAAATACAGGTTTTC.

[0012] In step (b), the polymerase chain reaction system comprises per 50 mL: 35 μL ddH2O, 1 μL target gene template, 1 μL 10 mM dNTPs, 10 μL 5 × TranStart KDPlus Buffer, 1 μL upstream primer, 1 μL downstream primer, and 1 μL KD Plus DNA Polymerase.

[0013] In step (2), the competent strain is Escherichia coli BL21(DE3).

[0014] In step (3), the steps of culturing the recombinant bacteria and inducing protein expression are as follows: After the recombinant bacteria were cultured overnight, a sterile 1.5 mL centrifuge tube was selected and 900 μL of LB liquid culture medium containing 100 μg / mL ampicillin was added to the centrifuge tube; then, a monoclonal colony was selected from the ampicillin-resistant plate and transferred to a conical flask containing 1 L of LB liquid culture medium. 1 mL of 100 mg / mL ampicillin antibiotic was added and cultured in a shaker at 37°C and 180 rpm; when the OD600 value reached between 0.6 and 0.8, the shaker temperature was adjusted to 16°C; after the culture medium and shaker temperature were stabilized at 16°C, 0.2 mM IPTG was added to induce E. coli to begin expressing lumbrokinase protein, and the induction was continued for 20 h.

[0015] In step (3), the specific operation of purification is: The protein produced by the induced expression of the recombinant bacteria was centrifuged at 4000 rpm for 15 min at 4°C, the supernatant was collected, and the supernatant was filtered through a 0.22 μm pore size aqueous filter membrane; then, the supernatant was purified using an AKTA protein purification system to obtain purified lumbrokinase protein.

[0016] The present invention has the following beneficial effects: The present invention provides a method for heterologous expression of lumbrokinase based on Escherichia coli. The method comprises the following steps: first, connecting the lumbrokinase gene with a vector to construct a recombinant expression vector; then, transforming the recombinant expression vector into a competent strain to obtain a recombinant bacterium; then, culturing the recombinant bacterium, inducing protein expression, and purifying the lumbrokinase protein; data show that the lumbrokinase protein expressed by the method of the present invention has high activity and strong thrombolytic ability, and can greatly shorten the time for obtaining lumbrokinase, thereby significantly increasing the yield of lumbrokinase. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0018] Figure 1 The results show the expression of Lumbrokinase protein in E. coli; Figure 2 The results of the protein fiber plate test are shown as Lumbrokinase lanes; Figure 3 Shown is a standard curve graph of urokinase. DETAILED DESCRIPTION

[0019] To make the objectives, technical solutions, and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other implementations obtained by those of ordinary skill in the art without inventive effort are within the scope of protection of the present invention.

[0020] Unless otherwise specified, all reagents involved in the specific embodiments of the present invention are commercially available products and can be purchased through commercial channels.

[0021] Example 1 This embodiment provides a method for heterologously expressing lumbrokinase protein using Escherichia coli, wherein the lumbrokinase protein is lumbrokinase (A8ILN1), comprising the following steps: (1) The Lumbrokinase (A8ILN1) gene was synthesized by gene synthesis technology. The nucleotide sequence encoding the gene is (as shown in SEQ ID NO. 2): ATTGTGGGCGGCATTGAAGCGCGCCCGTATGAATTTCCGTGGCAGGTGAGCGTGCGCCGCAAAAGCAGCGATAGCCATTTTTGCGGCGGCAGCATTATTAACGATCGCTGGGTGGTGTGCGCGGCGCATTGCATGCAGGGCGAAAGCCCGGCGCTGGTGAGCCTGGTGGTGGGCGAACATGATAGCAGCGCGGCGAGCACCGTGCGCCAGACCCATGATGTGGATAGCATTTTTGTGAACGAAAACTATGATCCGCGCACCCTGGAAAACGATGTGAGCGTGATTAAAACCGCGATTGCGATTACCTTTGATATTAACGTGGGCCCGATTTGCGCGCCGGATCCGGCGAACGATTATGTGTATCGCAAAAGCCAGTGCAGCGGCTGGGGCACCATTAACAGCGGCGGCATTTGCTGCCCGGCGGTGCTGCGCTATGTGACCCTGAACATTACCACCAACGCGTTTTGCGATGCGGTGTATACCAGCGATACCATTTATGATGATATGATTTGCGCGACCGATAACACCGGCATGACCGATCGCGATAGCTGCCAGGGCGATAGCGGCGGCCCGCTGAGCGTGAAAGATGGCAGCGGCATTTTTAGCCTGGTGGGCATTGTGAGCTGGGGCATTGGCTGCGCGAGCGGCTATCCGGGCGTGTATAGCCGCGTGGGCTTTCATGCGGGCTGGATTACCGATACCATTACCAACAACACGACC; The amino acid sequence of the lumbrokinase (A8ILN1) is as shown in SEQ ID NO. 1: IVGGIEARPYEFPWQVSVRRKSSDSHFCGGSIINDRWVVCAAHCMQGESPALVSLVVGEHDSSAASTVRQTHDVDSIFVNENYDPRTLENDVSVIKTAIAITFDINVGPICAPDPANDY VYRKSQCSGWGTINSGGICCPAVLRYVTLNiTTNAFCDAVYTSDTIYDDMICATDNTGMTDRDSCQGDSGGPLSVKDGSGIFSLVGIVSWGIGCASGYPGVYSRVGFHAGWITDTITNN; (2) According to the homologous recombination method, primers of the upstream and downstream sequences of the Lumbrokinase (A8ILN1) gene were synthesized as homologous arms for homologous recombination; The sequence of the upstream primer is TGGATATCGGAATTAATTCGGATATGATTGTGGGCGGCATTGAAGCGCGC (as shown in SEQ ID NO. 3); the sequence of the downstream primer is GTGGTGCTCGAGTGCGGCCGCAAGCTTGTGATGGTGATGGTGATGGCCCTGAAAATACAGGTTTTC (as shown in SEQ ID NO. 4).

[0022] The lumbrokinase (A8ILN1) gene was amplified using polymerase chain reaction (PCR). The 50 μL PCR reaction mixture consisted of 35 μL ddH2O, 1 μL target gene template, 1 μL 10 mM dNTPs, 10 μL 5x TranStart KD Plus Buffer, 1 μL upstream primer, 1 μL downstream primer, and 1 μL KD Plus DNA polymerase. The reaction conditions included an initial denaturation step at 94°C for 5 minutes, followed by 32 cycles of denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, and extension at 72°C at 1 kb / min. A final extension step at 72°C for 5 minutes was performed to ensure product integrity. The final reaction was stored at 4°C until subsequent analysis.

[0023] The Lumbrokinase (A8ILN1) gene was inserted into the circular expression vector pET-22b (+) using a seamless cloning kit to construct a linear expression vector. (4) Select competent strains Escherichia coli BL21 (DE3), use chemical transformation to transform the linear expression vector into competent strain. Steps are as follows: ① Take out the competent strain from the -80℃ refrigerator. Escherichia coli BL21 (DE3) test tube, and place it on ice for rapid thawing (about 2 minutes); ② When the substance in the strain test tube becomes an ice-water mixture, take out 30 μL of the competent strain from it Escherichia coli BL21 (DE3) and carefully transfer to a clean centrifuge tube. ④ Add the recombinant DNA product to the tube. During this process, the competent bacteria are relatively sensitive, so be gentle.

[0024] (5) Lumbrokinase protein expression. After the transformed strain was cultured overnight, 5 sterile 1.5 mL centrifuge tubes were selected and 900 μL of LB liquid culture medium containing 100 μg / mL ampicillin was added to each tube. Then, monoclonal colonies were selected from the ampicillin-resistant plate and transferred to a conical flask containing 1 L of LB liquid culture medium. 1 mL of 100 mg / mL ampicillin antibiotic was added to the flask and cultured in a shaker at 37°C and 180 rpm. When the OD600 value reached between 0.6 and 0.8, the shaker temperature was adjusted to 16°C. After the culture medium and shaker temperature were stabilized at 16°C, 0.2 mM IPTG was added to induce E. coli to begin expressing Lumbrokinase protein. The induction was continued for 20 h.

[0025] (6) Lumbrokinase protein purification. The liquid from step (5) was collected and centrifuged at 4000 rpm for 15 min at 4°C. The supernatant was collected and filtered through a 0.22 μm pore size aqueous filter membrane. Next, the lumbrokinase protein was purified using the AKTA protein purification system to obtain the lumbrokinase protein. The kDa of the lumbrokinase (A8ILN1) protein was 25.4.

[0026] Depend on Figure 1 It can be seen that the SDS-PAGE electrophoresis Lumbrokinase lane has an obvious protein "1" near 25kDa compared to the Escherichia coli (E. coli) lane without the Lumbrokinase (A8ILN1) gene introduced, and it is preliminarily judged to be the Lumbrokinase protein.

[0027] Since E. coli does not have thrombolytic activity, Figure 1 The protein in the Lumbrokinase lane was tested with a fiber plate test to verify whether it has thrombolytic activity and the magnitude of its thrombolytic activity. Figure 2 It can be seen that the protein in the Lumbrokinase lane has thrombolytic activity, thus confirming that the heterologous expression of Lumbrokinase protein in E. coli is successful.

[0028] like Figure 3 The figure shows the fitted regression curve generated under different specific activity conditions of urokinase, with the equation being y = 0.3054x - 0.3655 and the fitting degree being 0.9901. Figure 3 The specific activity of Lumbrokinase protein was calculated from the urokinase standard curve to be 10608 U / mL.

[0029] The method for heterologous expression of lumbrokinase in Escherichia coli described in the present invention first connects the lumbrokinase gene with a vector to construct a recombinant expression vector, then transforms the recombinant expression vector into a competent strain to obtain recombinant bacteria, and then cultures the recombinant bacteria, induces protein expression, and purifies to obtain lumbrokinase protein. Data show that the lumbrokinase protein expressed by the method of the present invention has high activity and strong thrombolytic ability, and can greatly shorten the time for obtaining lumbrokinase and significantly increase the yield of lumbrokinase.

Claims

1. A method for heterologous expression of lumbrokinase in Escherichia coli, characterized in that: The steps include: (1) Connecting the lumbrokinase gene to the vector to construct a recombinant expression vector; (2) transforming the recombinant expression vector into a competent strain to obtain a recombinant bacterium; (3) The recombinant bacteria are cultured and induced to express protein, and lumbrokinase protein is obtained through purification.

2. The method for heterologous expression of lumbrokinase in Escherichia coli according to claim 1, characterized in that: In step (1), the amino acid sequence of the lumbrokinase is shown in SEQ ID NO.

1.

3. The method for heterologous expression of lumbrokinase in Escherichia coli according to claim 1, characterized in that: In step (1), the nucleotide sequence encoding the lumbrokinase gene is shown in SEQ ID NO.

2.

4. The method for heterologous expression of lumbrokinase in Escherichia coli according to claim 1, characterized in that: In step (1), the vector is pET-22b(+).

5. The method for heterologous expression of lumbrokinase in Escherichia coli according to claim 3, characterized in that: The lumbrokinase gene was synthesized by the following method: (a) Combine the pET-22b(+) vector and the target gene using homologous recombination; (b) The lumbrokinase gene was obtained by polymerase chain reaction amplification.

6. The method for heterologous expression of lumbrokinase in Escherichia coli according to claim 5, characterized in that: In step (a), the sequence of the upstream primer is shown as SEQ ID NO.3; the sequence of the downstream primer is shown as SEQ ID NO.

4.

7. The method for heterologous expression of lumbrokinase in Escherichia coli according to claim 5, characterized in that: In step (b), the polymerase chain reaction system comprises per 50 mL: 35 μL ddH2O, 1 μL target gene template, 1 μL 10 mM dNTPs, 10 μL 5 × TranStart KD Plus Buffer, 1 μL upstream primer, 1 μL downstream primer, and 1 μL KD Plus DNA Polymerase.

8. The method for heterologous expression of lumbrokinase in Escherichia coli according to claim 1, characterized in that: In step (2), the competent strain is Escherichia coli BL21(DE3).

9. The method for heterologous expression of lumbrokinase in Escherichia coli according to claim 1, characterized in that: In step (3), the steps of culturing the recombinant bacteria and inducing protein expression are as follows: After the recombinant bacteria were cultured overnight, a sterile 1.5 mL centrifuge tube was selected and 900 μL of LB liquid culture medium containing 100 μg / mL ampicillin was added to the centrifuge tube; then, a monoclonal colony was selected from the ampicillin-resistant plate and transferred to a conical flask containing 1 L of LB liquid culture medium. 1 mL of 100 mg / mL ampicillin antibiotic was added to the flask and cultured in a shaker at 37°C and 180 rpm; when the OD600 value reached between 0.6 and 0.8, the shaker temperature was adjusted to 16°C; after the culture medium and shaker temperature were both stable at 16°C, 0.2 mM IPTG was added to induce E. coli to begin expressing lumbrokinase protein, and the induction was continued for 20 hours.

10. The method for heterologous expression of lumbrokinase in Escherichia coli according to claim 1, characterized in that: In step (3), the specific operation of purification is: The protein produced by the induced expression of the recombinant bacteria was centrifuged at 4000 rpm for 15 min at 4°C, the supernatant was collected, and the supernatant was filtered through a 0.22 μm pore size aqueous filter membrane; Then, the AKTA protein purification system is used for purification to obtain the purified lumbrokinase protein.

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