Expression vector, engineering bacteria and induced expression method of serine protease

Abstract

This invention relates to an expression vector, engineered bacteria, and method for inducing expression of a serine protease. The expression vector contains a nucleic acid sequence encoding a serine protease, and the amino acid sequence of the serine protease includes the sequence shown in SEQ ID NO.1. This invention transforms the expression vector into a host bacterium and utilizes its unique high-efficiency expression system for protein expression. By optimizing culture and induction conditions, high-efficiency expression of the serine protease was successfully induced. This serine protease exhibits highly efficient hydrolytic ability, with an enzyme activity reaching 925 U / mL.

Description

Technical Field

[0001] This invention relates to the field of protease technology, and more particularly to an expression vector, engineered bacteria, and induction expression method for a serine protease. Background Technology

[0002] Proteases can hydrolyze protein peptide chains into soluble short peptides and free amino acids over a wide range of pH and temperature. Based on their function and catalytic properties, proteases can be classified into nine families: aspartic proteases (A), cysteine ​​proteases (C), glutamate proteases (G), metalloproteinases (M), asparaginic acid proteases (N), mixed proteases (P), serine proteases (S), threonine proteases (T), and proteases with unknown functions (U). Serine proteases (EC 3.4.21) are the most representative class of proteases, with over 700 different serine proteases identified to date, making them one of the most abundant enzymes in nature. Approximately one-third of known proteases belong to the serine protease family. Their unique catalytic mechanisms enable them to play a variety of important functions in organisms, participating in blood clotting, food digestion, defense mechanisms, immune responses, and tissue remodeling. In the medical field, serine is mainly used to treat hemostasis and thrombosis, skin diseases, inflammation, and viral infections. In industrial applications, serine proteases are used as detergents to effectively degrade protein components in stains. In the food industry, serine proteases are used to hydrolyze proteins, improving the digestibility and absorption of food or enhancing its taste. In addition, serine proteases are widely used in leather processing, textile industry, and biodegradation.

[0003] Genetically engineered bacteria refer to bacteria into which a target gene is introduced to express and produce the desired protein. This involves using the genetic material of a donor organism or an artificially synthesized gene, which is then cleaved with a suitable vector after in vitro or in vitro restriction enzyme digestion to form a recombinant DNA molecule. This recombinant DNA molecule is then introduced into recipient cells or recipient organisms to construct transgenic organisms. For example, CN112877312A discloses the preparation and application of a recombinant serine protease. The serine protease (PmSpr) gene was cloned using PCR technology, and a recombinant engineered strain BL21 / pET-PmSpr was constructed. The obtained recombinant engineered bacteria were induced at low temperature, and the bacterial cells were finally collected and purified to obtain the recombinant serine protease.

[0004] In summary, providing a novel serine protease expression vector and engineered bacteria, and efficiently expressing serine protease in the host bacteria, has become one of the urgent problems to be solved in this field. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an expression vector, engineered bacteria, and induction expression method for serine proteases. By transferring the recombinant vector into a host bacterium, serine proteases can be expressed efficiently. The invention also provides a method for constructing and expressing the protein.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides an expression vector for a serine protease, the expression vector containing a nucleic acid sequence encoding a serine protease, the amino acid sequence of which includes the sequence shown in SEQ ID NO.1.

[0008] SEQ ID NO.1: MGFFSMVQMVRTHASKLDQPLRETVLHLYKPFKWTPCFLHRFFEGRL KKKKKLRVIIEFKEGAAEAGIQSTKQLMKKSRKTNIKRHFSHIDCCAADLTPAALEELLANGEHIRKIYLDRKVHALLDVATQASHAEEVIRNRTTLT GEGITVAVIDTGIYPHEDLDGRIRDFVDFVKQKTKPYDDNGHGTHCAGDVAGDGAASDGLYKGPAPKANLIGVKVLNKQGAGSLSTIIEGVEWCIQFNE DHPDDPIHIISMSLGGAAQRYDDEQDDPMVRAVNAAWDQGIVVCVAAGNSGPNSQTIASPAVSQKVITVGAYDDRNTPESSDDVVAPFSSRGPTVYGEAKPDILAPGVNIVSLRSPRSFLDKLDKSSRVDEDYTTLSGTSMATPICAGICALLLEHSPDLTPDEVKTLLKENTGKWSGDDPMIYGAGAIDAEKAIKE.

[0009] The serine protease provided in this invention originates from the S8 family of Basillus sp. 8A6. According to the MEROPS database, protease genes of the S8 family are defined as serine proteases. The expression vector provided in this invention can achieve exogenous expression of this serine protease by transformation into host bacteria.

[0010] The low activity of serine proteases limits their widespread application in industry and biomedicine. This invention selects a gene vector for efficiently expressing serine proteases and clones it into Bacillus subtilis using chemical transformation, significantly increasing the expression level and enzyme activity of the serine protease, thus laying a solid foundation for its application in medicine, industry, food, and other fields.

[0011] Preferably, the vector backbone of the expression vector includes a pHT43 vector.

[0012] Preferably, the nucleic acid sequence encoding the serine protease includes the sequence shown in SEQ ID NO.2.

[0013]

[0014] Preferably, the nucleic acid sequence of the expression vector includes the sequence shown in SEQ ID NO.3.

[0015]

[0016] In a second aspect, the present invention provides an engineered bacterium that expresses a serine protease, the engineered bacterium containing the expression vector described in the first aspect.

[0017] Preferably, the host bacteria of the engineered bacteria include Bacillus subtilis WB800N and / or Escherichia coli DH5α.

[0018] Thirdly, the present invention provides a method for preparing engineered bacteria as described in the second aspect, the method comprising converting the expression vector described in the first aspect into a host bacterium.

[0019] Preferably, the method for preparing the engineered bacteria includes the following steps:

[0020] (1) Obtain the nucleic acid fragment encoding the serine protease;

[0021] (2) Connect the nucleic acid fragment described in step (1) to the expression plasmid to obtain an expression vector containing a serine protease encoding gene;

[0022] (3) The expression vector obtained in step (2) is transformed into competent cells, and positive single colonies with correct sequencing results are screened to obtain the engineered bacteria.

[0023] Preferably, the method for obtaining the nucleic acid fragment encoding the serine protease in step (1) includes de novo synthesis of the nucleic acid fragment or PCR amplification from the genome of Basillus sp. 8A6 strain.

[0024] Preferably, the nucleic acid sequence of the primers for PCR amplification includes the sequences shown in SEQ ID NO.4 to SEQ ID NO.5.

[0025] SEQ ID NO. 4: GGATCCATGTTTGGATTTTCTATGGTGCAAATGGTCAG.

[0026] SEQ ID NO. 5: CCCGGGTTAGTGATGGTGATGGTGATGTTCTTTGATTGC.

[0027] Preferably, the expression plasmid in step (2) includes the pHT43 vector.

[0028] Preferably, step (2) specifically includes: digesting the nucleic acid fragment encoding a serine protease with the signal peptide removed and histidine tag added with the expression plasmid using restriction endonucleases BamHI and SmaI, and ligating them using T4 ligase to obtain the expression vector.

[0029] Preferably, the competent cells in step (3) include Bacillus subtilis WB800N and / or Escherichia coli DH5α.

[0030] Fourthly, the present invention provides a method for inducing the expression of a serine protease, the method comprising: inoculating the engineered bacteria described in the second aspect into a culture medium, adding IPTG to induce the expression of the serine protease, centrifuging to collect the supernatant, and obtaining the serine protease.

[0031] Preferably, the culture medium includes LB medium.

[0032] Preferably, the culture temperature of the engineered bacteria is 35-40℃ (e.g., 35℃, 36℃, 37℃, 38℃, 39℃ or 40℃, etc.), and the rotation speed is 150-250 rpm (e.g., 150 rpm, 170 rpm, 190 rpm, 200 rpm, 210 rpm, 230 rpm or 250 rpm, etc.).

[0033] Preferably, after the engineered bacteria are inoculated into the culture medium, they are cultured until the OD reaches a certain level. 600 Add IPTG when the value is between 0.8 and 1 (e.g., it can be 0.8, 0.85, 0.88, 0.9, 0.92, 0.95 or 1, etc.).

[0034] Preferably, after adding IPTG, the final concentration of IPTG is 0.8 to 1.2 mM (e.g., 0.8 mM, 0.9 mM, 1 mM, 1.1 mM, or 1.2 mM).

[0035] Preferably, the induction time for serine protease expression is 12–20 h (e.g., 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, or 20 h, etc.).

[0036] Preferably, the centrifugation speed is 10,000 to 15,000 rpm (e.g., 10,000 rpm, 11,000 rpm, 12,000 rpm, 13,000 rpm, 14,000 rpm, or 15,000 rpm, etc.), and the time is 5 to 15 minutes (e.g., 5 minutes, 7 minutes, 9 minutes, 10 minutes, 11 minutes, 13 minutes, or 15 minutes, etc.).

[0037] Other specific point values ​​within the range of the above values ​​can be selected, and will not be elaborated on here.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] This invention provides an expression vector for a serine protease. Using Bacillus subtilis as the host bacterium, the expression vector is transformed into Bacillus subtilis to obtain a recombinant strain. This invention utilizes the unique high-efficiency expression system of Bacillus subtilis for protein expression. By optimizing culture and induction conditions, high-efficiency expression of the serine protease was successfully induced, significantly improving the expression level and enzyme activity of the serine protease. This serine protease exhibits highly efficient hydrolytic ability, with an enzyme activity reaching 925 U / mL. Detailed Implementation

[0040] To further illustrate the technical means and effects of this invention, the following embodiments are provided for further explanation. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0042] The bacterial strains used in the following examples:

[0043] Bacillus sp. strain 8A6: from BGSC (Bacillus Genetic Stock Center), BGSCID 8A6, classified as Bacillus pumilus.

[0044] Bacillus subtilis WB800N: purchased from Guangzhou Yuexing Biotechnology Co., Ltd.

[0045] Escherichia coli DH5α: purchased from Guangzhou Zhenzhi Biotechnology Co., Ltd.

[0046] Example 1

[0047] This embodiment constructs an expression vector for a serine protease. The preparation process includes the following steps:

[0048] (1) Primer design and PCR amplification

[0049] A partial gene sequence from Basillus sp.8A6 was used, with histidine tags added. Upstream and downstream primers were designed, and PCR amplification was performed using the Basillus sp.8A6 genome as a template. The primer nucleic acid sequences are shown in SEQ ID NO.4 to SEQ ID NO.5, and the nucleic acid sequence encoding the serine protease is shown in SEQ ID NO.2.

[0050] (2) Construction of expression carrier

[0051] The PCR product from step (1) and the pHT43 vector were double-digested with restriction endonucleases BamHI and SmaI, respectively, and ligated with T4 ligase to obtain the expression vector of serine protease. The nucleic acid sequence of the expression vector is shown in SEQ ID NO.3.

[0052] Example 2

[0053] This embodiment provides an engineered bacterium expressing a serine protease. The expression vector provided in Example 1 is transferred into a host bacterium, and the preparation method includes any one of the following two methods:

[0054] Method (I):

[0055] (a) Preparation of stock solution for preparing competent Bacillus subtilis cells

[0056] 10×Spizizen salts stock solution: 15% K2HPO4·3H2O, 6% KH2PO4, 2% (NH4)2SO4, 0.2% MgSO4, 1% sodium citrate, dissolved in distilled water, autoclaved, and stored at room temperature.

[0057] GMI: 1 mL 10×Spizizen salts, 0.1 mL 10% yeast extract, 0.25 mL 20% glucose, 0.2 mL 1% hydrolyzed casein, 0.2 mL 0.25% required amino acids, and add sterile distilled water to a total volume of 10 mL.

[0058] GMII: 1 mL 10×Spizizen salts, 0.05 mL 10% yeast extract, 0.25 mL 20% glucose, 0.04 mL 1% hydrolyzed casein, 0.2 mL 0.25% required amino acids, 0.05 mL 0.1 mol / L CaCl2, 1 mL 25 mmol / L MgCl2, and add sterile distilled water to a total volume of 10 mL.

[0059] (b) Preparation of Bacillus subtilis competent cells

[0060] Bacillus subtilis WB800N was inoculated onto LB agar and cultured overnight at 37°C. A single colony was picked using an inoculation loop and placed in 5 mL of GMII solution, and cultured overnight at 30°C with a shaker at 125 rpm. The next day, 2 mL of the culture was transferred to 18 mL of GMII solution and cultured rapidly at 37°C with a shaker at 250 rpm for 3.5 h. Then, 10 mL of the culture from the previous step was transferred to 90 mL of GMII solution and cultured at 37°C with a shaker at 125 rpm for 90 min. After culturing, the cells were collected by centrifugation at 5000 rpm for 10 min. The cells were gently resuspended in 10 mL of the original culture supernatant. The resuspended cells were the competent cells. After mixing, the cells were aliquoted into centrifuge tubes (0.5 mL / tube) and stored at -80°C.

[0061] (c) Transformation of Bacillus subtilis competent cells with expression vector

[0062] Take 500 μL of Bacillus subtilis competent cells, add 5 μL of DNA, incubate at 37°C for 30 min, then incubate at 37°C and 200 rpm for 90 min on a shaker. Spread the culture onto chloramphenicol-resistant LB medium and incubate overnight at 37°C. Sequencing is used to verify the sequence. A single colony with a correct sequence is the engineered bacteria.

[0063] Method (II):

[0064] The expression vector was used to transform Escherichia coli DH5α competent cells to obtain recombinant strains. The original strains were then extracted and sequenced to verify the sequence. Single colonies with correct sequencing were selected, and the plasmids of these single colonies were extracted and transformed into Bacillus subtilis WB800N competent cells. The transformation steps were performed according to method (I).

[0065] Example 3

[0066] This embodiment describes the induction of serine protease expression in the engineered bacteria obtained in Example 2, including the following steps: positive transformants were inoculated into LB medium and cultured at 37°C and 200 rpm until OD500. 600 After reaching a concentration of 0.8–1, IPTG was added to a final concentration of 1 mM for induction expression. After 16 h of induction expression, the sample was centrifuged at 12,000 rpm for 10 min, and the supernatant was collected, which is the serine protease. The amino acid sequence of the serine protease is shown in SEQ ID NO.1.

[0067] Comparative Example 1

[0068] In this comparative example, the nucleic acid sequence encoding the serine protease in Example 1 was replaced with the nucleic acid sequence shown in SEQ ID NO. 6. The expression vector was obtained according to the preparation process provided in Example 1, and the serine protease was obtained by induction according to Examples 2 and 3. SEQ ID NO. 6 is derived from Basillus sp. 8A6.

[0069]

[0070] Comparative Example 2

[0071] In this comparative example, the nucleic acid sequence encoding the serine protease in Example 1 was replaced with the nucleic acid sequence shown in SEQ ID NO.7. The expression vector was obtained according to the preparation process provided in Example 1, and the serine protease was obtained by induction according to Examples 2 and 3. SEQ ID NO.7 is derived from Basillus sp.8A6.

[0072] SEQ ID NO.7: ATGAAAAAGTCAAAAGCCTTATTACTATTTGTTTCGTGCCTAGTGAT TCTTGCAATTGTATTATGTTGGGGACTATCTGGCGATGTTTCTGAGGATAATAGTAAATCATACAAGACAGAGAAGGAAAAAGACCTGCTATGGGGATATAAAGTTATAGGTCATGAAAAACCTAATAATCATAAGAATTATGTCAAAATTGCTATCCTTGATAGCGGTATTAATAAGTCACATAAAGAGTTTAAAGATTTAACTTTTTATGAGTACAATGCTATTAAACCAAAAAAATCTATCGAAGATGATTATGGTCATGGTACAGCAATTGCCGGAATAATCGCTGCAACTGGTGAAGAAATAAAAGGTATTTCACAGAATCCCATTATTTATGATGTCAAGGTACTAAATAAAAATGGCAAAGGAAAAGTTGAAGATGTTGTCAAAGGGATTAAATGGTGTATTGAGCAAAAGGTAGACATAATTAATATAAGTTTCGGATTTGGAAAAGACTACGATGAATTACGAAATGTTATAAAAATTGCTTTAGATAACAATATAACAATTACAGCTGCTTCTGGAAATACATTAGGTCTAACTGTTGAATACCCTGCAAAATATGAAGGCGTCTTATCGATATCCTCACTTAACGAAAAACTAAAAATTGATCCAATTTCAGCAAAAGGAAAAATTGACTTTTCTGCGCCTGGAGTAAATATTAAAACAACTGATATACACGGAGGATATACAAAGGTAAAAGGTACATCCTTTGCAACTGCGTTTGCAACAGGTGCTATAGGCTGTTTGTATTCTAAAAAATACAAATCTAAAGAGAATGTTCATCAAACACTCAAGAAAAATACAGTTGATCTTGGAAAGCTAGGTTATGACAGAGAGTATGGCAATGGGATGATTATTTGTAACAAGGGAGAGAATTAG。

[0073] Comparative Example 3

[0074] In this comparative example, the nucleic acid sequence encoding the serine protease in Example 1 was replaced with the nucleic acid sequence shown in SEQ ID NO. 8. The expression vector was obtained according to the preparation process provided in Example 1, and the serine protease was obtained by induction according to Examples 2 and 3. SEQ ID NO. 8 is derived from Basillus sp. 8A6.

[0075] SEQ ID NO.8: ATGAAAGGTGAAATTCGCTTAATTCCGTATGAAGTAAAAGCCAATGT 。

[0076] Test Example 1

[0077] This test example measures the enzyme activity of the serine protease induced in Example 3 and Comparative Examples 1-3. The test method is as follows:

[0078] In the experimental group, AZO casein was used as the substrate. 20 μL of 1.5% (w / v) AZO casein (dissolved in 50 mM pH 9 Tris-HCl buffer) was added to 20 μL of crude enzyme solution. The mixture was incubated at 60°C and 500 rpm for 15 min. Then, 100 μL of 0.4 M trichloroacetic acid (TCA) was added, and the mixture was incubated on ice for 15 min to terminate the reaction. The mixture was then centrifuged at 12000 rpm for 5 min, and 100 μL of the supernatant was added to a 96-well plate containing 25 μL of 1.8 M NaOH solution. The absorbance at 405 nm was measured. In the control group, 100 μL of 0.4 M trichloroacetic acid (TCA) was added to 20 μL of 1.5% (w / v) AZO casein substrate, and the mixture was incubated. Then, 20 μL of crude enzyme solution was added, and the mixture was incubated at 60°C and 500 rpm for 15 min. The reaction was then terminated by incubating on ice for 15 min, and the absorbance at 405 nm was measured. An arbitrary unit (U) of protease activity refers to the amount of enzyme required to increase absorbance by 0.01 at 405 nm under the assay conditions.

[0079] The serine proteases induced in Examples 3 and Comparative Examples 1-3 were used to determine their enzyme activity using the method described above. The test results are shown in Table 1.

[0080] Table 1

[0081] Group Enzyme activity (U / mL) Example 3 925 Comparative Example 1 310 Comparative Example 2 690 Comparative Example 3 452

[0082] As shown in Table 1, the serine protease encoding gene provided by this invention can efficiently express serine protease in a specific expression system, with enzyme activity far exceeding that of other serine proteases derived from Basillus sp.8A6, thus achieving efficient exogenous expression of serine protease.

[0083] In summary, this invention provides an expression vector for a serine protease. Using Bacillus subtilis as the host bacterium, the expression vector is transformed into Bacillus subtilis to obtain a recombinant strain. This invention utilizes the unique high-efficiency expression system of Bacillus subtilis for protein expression. By optimizing culture and induction conditions, high-efficiency expression of a serine protease was successfully induced. This serine protease exhibits high hydrolytic capacity, with an enzyme activity reaching 925 U / mL.

[0084] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. An expression vector for a serine protease, characterized in that, The expression vector contains a nucleic acid sequence encoding a serine protease, the amino acid sequence of which includes the sequence shown in SEQ ID NO.

1.

2. The expression vector according to claim 1, characterized in that, The vector backbone of the expression vector includes the pHT43 vector.

3. The expression vector according to claim 1 or 2, characterized in that, The nucleic acid sequence encoding the serine protease includes the sequence shown in SEQ ID NO.2; Preferably, the nucleic acid sequence of the expression vector includes the sequence shown in SEQ ID NO.

3.

4. An engineered bacterium expressing a serine protease, characterized in that, The engineered bacteria contain the expression vector described in any one of claims 1 to 3.

5. The engineered bacteria according to claim 4, characterized in that, The host bacteria of the engineered bacteria include Bacillus subtilis WB800N and / or Escherichia coli DH5α.

6. A method for preparing engineered bacteria as described in claim 4 or 5, characterized in that, The method for preparing the engineered bacteria includes converting the expression vector according to any one of claims 1 to 3 into a host bacterium.

7. The method for preparing engineered bacteria according to claim 6, characterized in that, The method for preparing the engineered bacteria includes the following steps: (1) Obtain the nucleic acid fragment encoding the serine protease; (2) Connect the nucleic acid fragment described in step (1) to the expression plasmid to obtain an expression vector containing a serine protease encoding gene; (3) The expression vector obtained in step (2) is transformed into competent cells, and positive single colonies with correct sequencing results are screened to obtain the engineered bacteria.

8. The method for preparing engineered bacteria according to claim 7, characterized in that, The method for obtaining the nucleic acid fragment encoding the serine protease in step (1) includes de novo synthesis of the nucleic acid fragment or PCR amplification from the genome of Bacillus 8A6 strain; Preferably, the nucleic acid sequence of the primers for PCR amplification includes the sequences shown in SEQ ID NO.4 to SEQ ID NO.5; Preferably, the expression plasmid in step (2) includes the pHT43 vector; Preferably, step (2) specifically includes: digesting the nucleic acid fragment encoding a serine protease with the signal peptide removed and histidine tag added with the expression plasmid using restriction endonucleases BamHI and SmaI, and ligating them using T4 ligase to obtain the expression vector; Preferably, the competent cells in step (3) include Bacillus subtilis WB800N and / or Escherichia coli DH5α.

9. A method for inducing the expression of serine protease, characterized in that, The method for inducing the expression of serine protease includes: inoculating the engineered bacteria of claim 4 or 5 into a culture medium, adding IPTG to induce the expression of serine protease, centrifuging to collect the supernatant, and obtaining the serine protease.

10. The method for inducing the expression of serine protease according to claim 9, characterized in that, The culture medium includes LB medium; Preferably, the culture temperature of the engineered bacteria is 35-40℃, and the rotation speed is 150-250 rpm; Preferably, after the engineered bacteria are inoculated into the culture medium, they are cultured until the OD reaches a certain level. 600 Add IPTG when the concentration is 0.8–1; Preferably, after adding IPTG, the final concentration of IPTG is 0.8–1.2 mM; Preferably, the induction time for serine protease expression is 12–20 h; Preferably, the centrifugation speed is 10,000 to 15,000 rpm and the time is 5 to 15 minutes.