A soluble expression method, expression vector and expression engineering bacteria of Japanese leech unsteady enzyme

Abstract

The application provides a soluble expression method, an expression vector and an expression engineering bacterium of Japanese medical leech destabilizing enzyme, and belongs to the field of prokaryotic expression technology. The application provides a novel soluble prokaryotic expression method of destabilizing enzyme. Through accurate design of vector construction elements and selection of expression hosts, the soluble efficient expression of the destabilizing enzyme in supernatant is realized, the cumbersome steps of inclusion body renaturation and the risk of folding errors are avoided, the natural conformation and biological activity of the target enzyme are ensured, the subsequent purification process is greatly simplified, the preparation efficiency is improved, a feasible technical scheme is provided for large-scale preparation and practical application of the destabilizing enzyme, and the application has important industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of prokaryotic expression technology, specifically relating to a soluble expression method, expression vector, and engineered expression bacteria for the unstable enzyme of Hirudo medicinalis. Background Technology

[0002] Due to their structural characteristics, unstable enzymes are readily expressed as inclusion bodies in prokaryotic expression systems, posing a key challenge to their efficient preparation and application. While existing technologies attempt to restore the physicochemical properties of proteins expressed in inclusion body form through refolding, the refolding process struggles to precisely induce the protein to form its natural, correct spatial folding conformation. This often results in significant loss or even complete inactivation of the target enzyme's biological activity. Furthermore, the cumbersome refolding procedure and poor process controllability further increase preparation costs, severely limiting the industrial application of unstable enzymes in fields such as biocatalysis and biomedicine. Summary of the Invention

[0003] This invention provides a soluble expression method, expression vector, and engineered bacteria for unstable enzymes from Hirudo medicinalis, which can solve the problems of complex revival and easy inactivation of unstable enzymes after expression in inclusion body form.

[0004] This invention provides a prokaryotic expression method for a Japanese medicinal leech destabilizing enzyme, comprising the following steps: constructing a fusion protein by combining the Japanese medicinal leech destabilizing enzyme with other components; codon-optimized and ligating the coding gene of the fusion protein into a prokaryotic expression vector to construct a recombinant expression vector; transforming E. coli competent cells with the recombinant expression vector and inducing expression with IPTG; collecting and lysing the cells, with the supernatant containing the fusion protein; the other components include a chaperone molecule and a TEV restriction site.

[0005] In one specific embodiment of the present invention, the amino acid sequence of the Japanese medicinal leech destabilizing enzyme is shown in SEQ ID No. 1.

[0006] In one specific embodiment of the present invention, the amino acid sequence of the chaperone molecule is shown in SEQ ID No. 2; and the amino acid sequence of the TEV restriction site is shown in SEQ ID No. 3.

[0007] In one specific embodiment of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID No. 4.

[0008] In one specific embodiment of the present invention, the prokaryotic expression vector includes the pET-15b vector.

[0009] In one specific embodiment of the present invention, the nucleotide sequence inserted into the pET-15b vector is shown in SEQ ID No. 5.

[0010] The present invention also provides a recombinant expression vector for expressing the destabilizing enzyme of Hirudo medicinalis based on supernatant, comprising using pET-15b vector as the base vector and inserting the nucleotide sequence shown in SEQ ID No. 5.

[0011] The present invention also provides a recombinant Escherichia coli expressing Hirudo medicinalis destabilizing enzyme in supernatant, comprising transforming the above recombinant expression vector into BL21(DE3) competent cells.

[0012] The present invention also provides a method for producing Japanese medicinal leech destabilizing enzyme, comprising inducing the expression of the above-mentioned recombinant Escherichia coli using IPTG, collecting the bacterial cells and cleaving them, purifying the protein components in the supernatant to obtain a purified fusion protein; treating the purified fusion protein with rTEV protease, and purifying it again to obtain Japanese medicinal leech destabilizing enzyme.

[0013] In one specific embodiment of the present invention, the purification includes purification using a Ni column.

[0014] Beneficial effects: This invention provides a novel method for the soluble prokaryotic expression of unstable enzymes. By precisely designing vector construction elements and selecting expression hosts, the method achieves efficient and soluble expression of unstable enzymes in supernatants. This avoids the cumbersome steps and folding error risks associated with inclusion body refolding, ensuring the native conformation and biological activity of the target enzyme. It significantly simplifies subsequent purification processes and improves preparation efficiency, providing a practical technical solution for the large-scale preparation and application of unstable enzymes, and has significant industrial application value. Attached Figure Description

[0015] Figure 1 This image shows the expression, purification, and restriction enzyme digestion of the target protein supernatant.

[0016] Figure 2 The graph shows the expression, denaturation, purification, and refolding of the target protein inclusion bodies. Detailed Implementation

[0017] This invention provides a prokaryotic expression method for a Japanese medicinal leech destabilizing enzyme, comprising the following steps: constructing a fusion protein by combining the Japanese medicinal leech destabilizing enzyme with other components; codon-optimized and ligating the coding gene of the fusion protein into a prokaryotic expression vector to construct a recombinant expression vector; transforming E. coli competent cells with the recombinant expression vector and inducing expression with IPTG; collecting and lysing the cells, with the supernatant containing the fusion protein; the other components include a chaperone molecule and a TEV restriction site.

[0018] The prokaryotic expression method of the present invention can solublely express the Japanese medicinal leech destabilizing enzyme by IPTG induction. In one embodiment, the amino acid sequence of the Japanese medicinal leech destabilizing enzyme is shown in SEQ ID No. 1: MMNYAIFALLVALYVIDIAQCTVPSNCLRCICQVEGCDNEIGKCGMDMGSLSCPYQIKEVYWIDCGRPDGDYQRCAKNKACSERCVHAYMARYALSCTGGRPPTCQDYAKIHNGGPNGCNSASNHYWDNVNRCLA

[0019] To ensure the soluble expression of the *Hirudo medicinalis* destabilizing enzyme, this invention modifies the *Hirudo medicinalis* destabilizing enzyme by fusing it with other elements, including a chaperone molecule and a TEV restriction site. In one embodiment, the chaperone molecule is slyD, and the amino acid sequence of slyD is shown in SEQ ID No. 2: KVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLISGLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVVDGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDHDGCCGGHGHDHGHEHGGEGCCGGKGNGGCGCH; the amino acid sequence of the TEV restriction site is shown in SEQ ID No. 3: ENLYFQG.

[0020] The structure of the fusion protein described in this invention, in one embodiment, is a chaperone molecule (slyD)-TEV restriction site-destabilizing enzyme-taa (stop codon). The fusion protein is inserted into the expression vector with NdeI and BamHI treatment. Therefore, NdeI and BamHI restriction sites are added to both ends of the fusion protein, resulting in the amino acid sequence inserted into the prokaryotic expression vector as shown in SEQ ID No. 4: NdeI+ MKVAKDLVVSLAYQVRTEDGVLVDESPVSAPLDYLHGHGSLIS GLETALEGHEVGDKFDVAVGANDAYGQYDENLVQRVPKDVFMGVDELQVGMRFLAETDQGPVPVEITAVEDDHVVV DGNHMLAGQNLKFNVEVVAIREATEEELAHGHVHGAHDHHHDHDGCCGGHGHDHGHEHGGEGCCGGKGNGGCGCH + ENLYFQG + TVPSNCLRCICQVEGCDNEIGKCGMDMGSLSCGPYQIKEVYWIDCGRPDGDYQRCAKNKACSERCVH AYMARYALSCTGGRPPTCQDYAKIHNGGPNGCNSASNHYWDNVNRCLA + taa (stop codon) + BamH I

[0021] The construction principles are as shown above. Single lines represent chaperone molecules, double lines represent the restriction sites of rTEV enzymes, and dashed lines represent the gene sequence of leech thrombolytic enzymes.

[0022] After codon optimization, the nucleotide sequence inserted into the prokaryotic expression vector is shown in SEQ ID No. 5: ATGATGAACTACGCTATCTTTGCTCTCTTAGTGGCACTTTATGTCATCGACATTGCGCAATGCACTGTCCCATCCAACTGCCTGAGATGCATTTGCCAGGTAGAGGGATGCGACAATGAAATTGGAAAGTGCGGCATGGACATGGGAAGTCTGAGTTGTGGTCCTTACCAGATCAAGGAGGTCTACTGGATCGACTGTGGCAGACC AGATGGAGATTACCAGCGATGTGCTAAGAACAAAGCATGTTCTGAAAGGTGCGTCCATGCTTACATGGCCAGATACGCCCTCAGCTGTACGGGTGGACGCCCACCGACCTGCCAAGACTATGCCAAAATCCACAACGGCGGACCGAACGGATGCAACAGTGCAAGTAACCACTACTGGGATAACGTCAATAGATGTTTGGCCTGA.

[0023] The prokaryotic expression vector of the present invention includes the pET-15b vector.

[0024] The present invention also provides a recombinant expression vector for expressing the destabilizing enzyme of Hirudo medicinalis based on supernatant, comprising using pET-15b vector as the base vector and inserting the nucleotide sequence shown in SEQ ID No. 5.

[0025] In the recombinant expression vector of this invention, the chaperone molecule can promote extracellular secretion and soluble expression. Regarding the TEV restriction site: because the addition of (slyD) in constructing this prokaryotic expression system resulted in the recombinant protein being twice the size of the original protein, which might affect subsequent protein function verification, a recombinant rTEV enzyme was added to specifically recognize the seven-amino acid sequence "EXXYXQ↓(G / S)" and cleave it between aminoamide and glycine / serine to separate the protein tag from the target sequence; "taa" serves only as a stop codon in the prokaryotic expression system.

[0026] The present invention also provides a recombinant Escherichia coli expressing Hirudo medicinalis destabilizing enzyme in supernatant, comprising transforming the above recombinant expression vector into BL21(DE3) competent cells.

[0027] This invention uses BL21(DE3) competent cells as the expression host, and the recombinant expression vector is transformed into the host. This invention does not have a special limitation on the transformation method, and conventional transformation methods in the field can be used. In one embodiment, the heat shock method is used.

[0028] The present invention also provides a method for producing Japanese medicinal leech destabilizing enzyme, comprising inducing the above-mentioned recombinant Escherichia coli to express using IPTG, collecting the bacterial cells and cleaving them, purifying the protein components in the supernatant, and obtaining the purified fusion protein; The purified fusion protein was treated with rTEV protease and then purified again to obtain Japanese medicinal leech destabilizing enzyme.

[0029] In this invention, when the recombinant Escherichia coli is cultured in LB liquid medium containing ampicillin sodium on a shaker until OD reaches zero... 600 When the concentration of IPTG is 0.6~0.8, the expression of the target protein is induced using IPTG. The final concentration of IPTG during induction is 0.5 mM. The culture temperature is then adjusted to 16℃, and the culture is continued for 12~24 h to induce the expression of the recombinant protein. After the induction culture is completed, the bacterial culture is transferred to a centrifuge tube and centrifuged at 4℃ and 8000 rpm for 10 minutes. The supernatant is discarded and the bacterial pellet is collected. The bacterial pellet is resuspended in pre-cooled PBS buffer and centrifuged again at 4℃ and 8000 rpm for 10 minutes. The supernatant is discarded. This step is repeated 2-3 times to thoroughly remove residual components of the culture medium. An appropriate amount of RIPA lysis buffer containing protease inhibitors is added to the washed bacterial pellet. After gently mixing by pipetting, the centrifuge tube is placed on ice and ultrasonically disrupted using an ultrasonic homogenizer. The ultrasonic parameters are set as follows: power 200W, working time 5 seconds, interval 10 seconds, total duration 15-20 minutes, until the bacterial culture changes from turbid to clear. After ultrasonic disruption, the bacterial culture was centrifuged at 4°C and 12,000 rpm for 20 minutes to separate the supernatant and precipitate. The supernatant was purified using a Ni column. The purified protein was then subjected to enzymatic digestion with recombinant rTEV protease at a specific concentration to remove its chaperone molecules. The purified protein was then purified again by Ni column filtration to obtain the pure target protein.

[0030] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, of a soluble expression method, expression vector, and engineered expression bacteria for the expression of Hirudo medicinalis destabilizing enzyme provided by the present invention, should not be construed as limiting the scope of protection of the present invention.

[0031] Example 1: Construction of a prokaryotic expression system for pet-15b After optimizing rare codons according to the prokaryotic system, the pET-15b vector was constructed and expressed using N-His tag fusion.

[0032] Construction method: Nde I + chaperone molecule (slyD) + TEV restriction site + destabilizing enzyme + taa (stop codon) + BamHI, i.e., the amino acid sequence is shown in SEQ ID No. 4 and the nucleotide sequence is shown in SEQ ID No. 5. After double digestion of the pET-15b vector with Nde I and BamHI, it was ligated with the fragment shown in SEQ ID No. 5 to construct the recombinant plasmid.

[0033] In recombinant protein expression experiments, introducing the constructed recombinant plasmid into BL21(DE3) competent cells is a crucial initial step. First, BL21(DE3) competent cells stored at -80℃ were removed from the freezer and slowly thawed on ice. Then, an appropriate amount of the constructed plasmid was added, gently mixed, and incubated on ice for 30 minutes. Next, heat shock was performed by rapidly placing the centrifuge tubes in a 42℃ water bath for 90 seconds, followed immediately by cooling on ice for 2-3 minutes to promote plasmid entry into the cells. 800 μL of antibiotic-free LB broth was added to the centrifuge tubes, and the cells were activated and cultured at 37℃ and 200 rpm for 1 hour to allow the cells to return to normal growth and express the resistance gene. After activation, an appropriate amount of the bacterial culture was spread onto a medium containing 50 μg / mL of antibiotic-free LB broth. -1 On LB agar plates containing ampicillin sodium, after the bacterial culture is completely absorbed by the medium, the plates are inverted and incubated at 37°C. Once single colonies have grown on the plates, a single colony with regular morphology and clear edges is picked using a sterile inoculation loop and inoculated into 5 mL of a container containing 50 μg / mL ampicillin sodium. -1 Seed culture was obtained by overnight incubation of ampicillin sodium in LB broth centrifuge tubes at 37°C and 220 rpm for 16 hours using a shaker. The seed culture was then inoculated into fresh LB broth containing ampicillin sodium at a ratio of 1:100 and incubated at 37°C and 220 rpm using a shaker. The OD of the bacterial culture was measured every hour using a spectrophotometer. 600 Value, when OD 600 When the value reaches 0.6-0.8, it indicates that the cells are in the mid-logarithmic growth phase. At this time, IPTG (isopropyl-β-D-thiogalactoside) is added to the culture medium to a final concentration of 0.5 mmol / L, and the culture temperature is adjusted to 16℃. Culture is continued for 14 hours to induce recombinant protein expression. After induction culture, the bacterial culture is transferred to a centrifuge tube and centrifuged at 4℃ and 8000 rpm for 10 minutes. The supernatant is discarded, and the bacterial pellet is collected. The bacterial pellet is resuspended in pre-cooled PBS buffer and centrifuged again at 4℃ and 8000 rpm for 10 minutes. The supernatant is discarded. This step is repeated 3 times to thoroughly remove residual components of the culture medium.

[0034] Add an appropriate amount of RIPA lysis buffer containing protease inhibitors to the washed bacterial pellet, gently mix by pipetting, place the centrifuge tube on ice, and sonicate using an ultrasonic homogenizer. The ultrasonic parameters are set as follows: power 200W, 5 seconds operation, 10-second interval, total duration 20 minutes, until the bacterial solution changes from turbid to clear. After ultrasonic homogenization, centrifuge the bacterial solution at 4°C and 12000 rpm for 20 minutes to separate the supernatant and pellet.

[0035] Take an appropriate amount of supernatant and precipitate sample, add 2×SDS-PAGE loading buffer, and heat in a boiling water bath at 100℃ for 5 minutes to denature the protein. Then perform SDS-PAGE electrophoresis. After Coomassie brilliant blue staining and destaining, observe the position and concentration of the protein bands to verify the expression of the recombinant protein. Subsequently, purify the protein using a Ni column. The purified protein is then digested with recombinant rTEV protease at its specific concentration to remove chaperone molecules. The purified protein is then filtered again through a Ni column to obtain the pure target protein. 1L of LB can express approximately 1.5mg of the purified target protein. Figure 1 ).

[0036] Comparative Example 1: Construction of a prokaryotic expression system for pCZN1: After optimizing rare codons according to the prokaryotic system, the pCZN1 vector was constructed. The N-His tag fusion expression construction method was: Nde I + destabilizing enzyme + taa (stop codon) + Xba I (SEQ ID No. 6). The pCZN1 vector was digested with Nde I and Xba I and then ligated with the fragment shown in SEQ ID No. 6: TVPSNCLRCICQVEGCDNEIGKCGMDMGSLSCGPYQIKEVYWIDCGRPDGDYQRCAKNKACSERCVHAYMARYALSCTGGRPPTCQDYAKIHNGGPNGCNSASNHYWDNVNRCLA to construct the recombinant plasmid.

[0037] First, recombinant plasmids were constructed. Then, the validated recombinant plasmids were transformed into Arctic-Express competent cells. After treatment with ice bath for 30 minutes, heat shock at 42°C for 90 seconds, and ice bath for 2 minutes, the cells were added to TB liquid culture medium and incubated at 37°C on a shaker for 1 hour. Finally, the plasmids were plated with a solution containing 50 μg / mL... -1 Transformants were obtained by incubating ampicillin-containing TB plates inverted at 37°C overnight. Protein expression was then induced, and single colonies were picked and inoculated onto plates containing 50 μg / mL ampicillin. -1 Ampicillin was cultured overnight at 37°C and 220 rpm in TB medium, and then transferred to fresh TB medium at a 1:100 dilution the next day. The culture was then incubated until OD (October Expected). 600When the concentration reaches 0.6~0.8, uninduced samples are collected, and IPTG is added to the remaining culture to a final concentration of 0.2mM. The culture is induced at 15℃ and 220rpm for 16 hours. The bacterial cells are collected, ultrasonically disrupted, and the supernatant and precipitate are separated.

[0038] Protein expression was detected using 15% SDS-PAGE gel electrophoresis. Each sample was mixed with loading buffer, boiled to denature, and then loaded. Electrophoresis was performed at 80V for 30 minutes, then adjusted to 180V. After staining and destaining, the bands were observed to confirm protein expression and distribution. If the protein existed in the precipitate as inclusion bodies, the precipitate was resuspended in pre-chilled RIPA lysis buffer containing PMSF, sonicated on ice for 20 minutes, collected by centrifugation, washed three times with inclusion body washing buffer, resuspended in inclusion body dissolving buffer, sonicated to dissolve, and the supernatant was collected and stored. Finally, protein purification was performed using a Ni-NTA-Sefinose affinity chromatography column. After column equilibration, the sample was loaded, and the target protein fraction was specifically collected by imidazole gradient elution to complete the purification.

[0039] The purified protein solution was placed in a dialysis bag and dialyzed overnight in PBS solution at 4°C. The medium was changed every 3 hours for a total of 3 times. After the last medium change, the bag was dialyzed for 16 hours at 4°C. 1L of TB medium yields 8.5mg of the target protein. Figure 2 ).

[0040] Experimental example: Table 1. Isopeptidase activity of proteins under different expression methods Inclusion body WP Shangqing WP Isopeptidase activity 0.37±0.02 d 1.28±0.02 b

[0041] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A prokaryotic expression method for an unstable enzyme from *Hirudo medicinalis*, characterized in that, Includes the following steps: After constructing a fusion protein by combining the Japanese medicinal leech destabilizing enzyme with other components, the coding gene of the fusion protein was codon-optimized and ligated into a prokaryotic expression vector to construct a recombinant expression vector. The recombinant expression vector was used to transform competent E. coli cells, and expression was induced by IPTG. After collecting the cells, they were lysed, and the supernatant contained the fusion protein. The other components include chaperone molecules and TEV restriction sites.

2. The prokaryotic expression method according to claim 1, characterized in that, The amino acid sequence of the Japanese medicinal leech destabilizing enzyme is shown in SEQ ID No.

1.

3. The prokaryotic expression method according to claim 1, characterized in that, The amino acid sequence of the chaperone molecule is shown in SEQ ID No. 2; the amino acid sequence of the TEV restriction site is shown in SEQ ID No.

3.

4. The prokaryotic expression method according to claim 3, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID No.

4.

5. The prokaryotic expression method according to claim 1, characterized in that, The prokaryotic expression vector includes the pET-15b vector.

6. The prokaryotic expression method according to claim 5, characterized in that, The nucleotide sequence inserted into the pET-15b vector is shown in SEQ ID No.

5.

7. A recombinant expression vector based on supernatant expression of destabilizing enzymes from *Hirudo medicinalis*, characterized in that, This includes inserting the nucleotide sequence shown in SEQ ID No. 5 into the pET-15b vector as the base vector.

8. A recombinant *Escherichia coli* strain expressing *Hirudo medicinalis* destabilizing enzymes in its supernatant, characterized in that... This includes transforming the recombinant expression vector of claim 7 into BL21(DE3) competent cells.

9. A method for producing destabilizing enzymes from Japanese medicinal leeches, characterized in that, The process includes inducing the expression of the recombinant Escherichia coli according to claim 8 using IPTG, collecting the bacterial cells and cleaving them, purifying the protein components in the supernatant to obtain a purified fusion protein; treating the purified fusion protein with rTEV protease, and purifying it again to obtain Hirudo medicinalis destabilizing enzyme.

10. The production method according to claim 9, characterized in that, The purification process includes purification using a Ni column.

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