Recombinant escherichia coli for preparing soluble EC-SOD and application of recombinant escherichia coli

Abstract

The invention provides recombinant escherichia coli for preparing soluble EC-SOD and application of the recombinant escherichia coli, and relates to the technical field of biological engineering. According to the present invention, SOD and KGF2 genes are connected by using a connecting peptide coding sequence containing a protease cleavage site, and are cloned to a pET28a expression vector; the pET28a expression vector is transformed into competent cells of escherichia coli BL21 (DE3), and recombinant escherichia coli is constructed. The obtained recombinant Escherichia coli can solve the problem that an inactive inclusion body is formed when EC-SOD is expressed in Escherichia coli. The method is suitable for preparing soluble EC-SOD.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a recombinant Escherichia coli for preparing soluble EC-SOD and its application. Background Technology

[0002] Human extracellular superoxide dismutase (EC-SOD) is one of the three known SOD (superoxide dismutase) isoenzymes. It is a secreted glycoprotein containing Cu and Zn metal ions. EC-SOD is mainly found in the extracellular matrix and on the cell surface. It is the most abundant SOD isoenzyme in extracellular fluids such as plasma, lymph, synovial fluid, and cerebrospinal fluid, and is also responsible for scavenging extracellular oxygen. 2- EC-SOD is a major enzyme with significant hypoglycemic, hypolipidemic, and hypotensive effects. EC-SOD is present in very small amounts in tissues, making its isolation and purification difficult.

[0003] E. coli expression systems offer numerous advantages, including clear genetic background, high expression levels, rapid propagation, low cost, good stability, strong resistance to contamination, and easy product purification. However, when EC-SOD is expressed in E. coli expression systems, the expression products are all inactive inclusion bodies. Furthermore, because EC-SOD is a four-subunit metalloenzyme, with each subunit containing six sulfhydryl groups, the inclusion body refolding rate is low, denaturation and refolding operations are time-consuming, and the recovery efficiency of active proteins is low.

[0004] Fusion protein expression is an important protein expression strategy to enhance the solubility of target proteins. It involves fusing the coding sequence of a target protein with the coding sequences of other proteins, and then synthesizing the target protein by expressing the gene of the fusion protein. Human keratinocyte growth factor 2 (KGF2), also known as fibroblast growth factor 10 (FGF10), consists of 208 amino acids and is unstable in acid and heat. KGF2 has a wide range of biological functions, promoting epidermal proliferation and development, and has extensive applications in the medical and cosmetic fields. Currently, there are no reports of using KGF2 as a fusion tag to promote the soluble expression of heterologous proteins in *E. coli*. Summary of the Invention

[0005] The purpose of this invention is to improve the soluble expression level of EC-SOD in Escherichia coli by constructing recombinant Escherichia coli.

[0006] On the one hand, the present invention provides a recombinant Escherichia coli for preparing soluble EC-SOD.

[0007] The recombinant Escherichia coli was prepared by inserting the gene encoding KGF2 downstream of the gene encoding SOD in an expression vector, with the SOD and KGF2 genes linked by a linker peptide coding sequence, and then transforming the SOD-KGF2 fusion protein expression vector into competent Escherichia coli cells.

[0008] Furthermore, the host Escherichia coli is Escherichia coli BL21(DE3).

[0009] Furthermore, the expression vector is the expression vector pET28a.

[0010] This invention provides a recombinant *E. coli* strain capable of soluble expression of EC-SOD. The recombinant *E. coli* strain is obtained by linking the SOD gene (with the stop codon TAA removed) and the KGF2 gene (containing the sequence shown in SEQ ID NO. 1) in an expression vector using a linker peptide coding sequence containing a protease cleavage site, and then transforming the vector into competent *E. coli* cells.

[0011] Furthermore, the linker peptide containing the protease cleavage site has a TEV protease cleavage site. Furthermore, the linker peptide containing the protease cleavage site has the nucleic acid sequence shown in SEQ ID NO. 11 and the enzyme cleavage site sequence shown in SEQ ID NO. 12.

[0012] On the other hand, the present invention provides a method for constructing the above-mentioned recombinant Escherichia coli expressing soluble EC-SOD, comprising the following steps:

[0013] (1) The SOD nucleotide sequence derived from superoxide dismutase (SOD) was synthesized according to the codon preference of E. coli, and cloned into the expression vector PET28a to construct the vector pET28a-SOD. The SOD nucleotide sequence is shown in SEQ ID NO.1.

[0014] (2) The amino acid sequence derived from keratinocyte growth factor 2 was synthesized according to the codon preference of E. coli, and the KGF2 nucleotide sequence was cloned into the expression vector pUC19 to construct the vector pUC19-KGF2. The KGF2 nucleotide sequence is shown in SEQ ID NO.2.

[0015] (3) Using the vector pET28a-SOD as a template, the pET28a-SOD vector backbone was amplified using primers P1 (SEQ ID NO.3) and P2 (SEQ ID NO.4), and denoted as fragment 1.

[0016] (4) Using the vector pUC19-KGF2 as a template, a KGF2 fragment with a linker peptide was amplified using primers P3 (SEQ ID NO. 5) and P4 (SEQ ID NO. 6), denoted as fragment 2. This linker peptide contains a protease cleavage site. This linker peptide has a TEV (tobacco mosaic virus) protease cleavage site. Furthermore, this linker peptide has the nucleotide sequence shown in SEQ ID NO. 11 and the enzyme cleavage site sequence shown in SEQ ID NO. 12.

[0017] (5) Fragment 1 and fragment 2 were incubated using the ClonExpress II One Step Cloning Kit to obtain the fusion protein expression vector.

[0018] (6) Transform Escherichia coli BL21(DE3) competent cells with the fusion protein expression vector obtained in step (5) to obtain recombinant E. coli BL21(DE3) / pET28a-SOD-KGF2.

[0019] On the other hand, the present invention provides the application of recombinant Escherichia coli expressing soluble EC-SOD in the preparation of EC-SOD.

[0020] On another front, this invention provides a method for preparing EC-SOD using *E. coli*. The recombinant *E. coli* is cultured, and an inducer is added to induce the expression of a soluble SOD-KGF2 fusion protein. Then, the SOD-KGF2 fusion protein is cleaved by proteases to obtain soluble EC-SOD and soluble KGF2. Compared to the prior art's expression of SOD protein in the form of inactive inclusion bodies, this invention achieves soluble expression of SOD. This method can obtain the SOD-KGF2 fusion protein through soluble expression, and then obtain soluble EC-SOD through protease cleavage, retaining the activity of EC-SOD. Soluble KGF2 can also be obtained simultaneously.

[0021] Furthermore, the *E. coli* is any of the recombinant *E. coli* BL21(DE3) / pET28a-SOD-KGF2 prepared as described above for soluble expression of EC-SOD. The recombinant *E. coli* is inoculated into LB liquid medium to obtain a seed culture; the obtained seed culture is inoculated into LB liquid medium and cultured until the bacterial OD600 reaches 0.6–0.8, and an inducer is added to induce the expression of the SOD-KGF2 fusion protein; the expressed fusion protein is then subjected to protease digestion to obtain EC-SOD and KGF2.

[0022] Furthermore, the inducer can be IPTG (isopropyl-β-D-thiogalactoside), eliminating the need for methanol as an inducer, thus ensuring high safety and minimal harm. In the method described in this invention, the inducer can be added at a final concentration of 0.05-0.5 mM to induce the expression of the recombinant E. coli.

[0023] Furthermore, the protease is a TEV (tobacco mosaic virus) protease. In the method of the present invention, 200 U of TEV protease is added to each 1 mg of the fusion protein and incubated overnight at 4°C.

[0024] The technical solution of this invention has the following advantages:

[0025] This invention induces the expression of SOD-KGF2 fusion protein in Escherichia coli, achieving soluble expression of EC-SOD in Escherichia coli. Furthermore, after protease cleavage, both soluble SOD and KGF2 protein products are obtained simultaneously, reducing production costs. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 Electrophoresis diagram of SOD protein: Lane 1 is the marker; Lane 2 is the cell lysate precipitate induced at 37℃; Lane 3 is the cell lysate supernatant induced at 37℃; Lane 4 is the cell lysate precipitate induced at 28℃; Lane 5 is the cell lysate supernatant induced at 28℃.

[0028] Figure 2 The pET28a-SOD-KGF2 vector spectrum;

[0029] Figure 3 Electrophoresis diagram of SOD-KGF2 fusion protein: Lane 1 is the marker; Lane 2 is the supernatant of cell lysate induced at 37℃; Lane 3 is the precipitate of cell lysate induced at 37℃; Lane 4 is the supernatant of cell lysate induced at 28℃; Lane 5 is the precipitate of cell lysate induced at 28℃; Lane 6 is the supernatant of control cell lysate induced at 28℃ without plasmid.

[0030] Figure 4 Electrophoresis images of the lysed and purified SOD protein and KGF2. Detailed Implementation

[0031] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1: Construction and expression of E. coli BL21(DE3) / pET28a-SOD

[0033] The amino acid sequence derived from superoxide dismutase (SOD) was synthesized according to the codon preference of *E. coli* (SEQ ID NO. 1) and cloned into the expression vector pET28a to obtain the vector pET28a-SOD. The vector pET28a-SOD was transformed into competent cells of *E. coli* strain BL21(DE3). Positive transformants were selected and colony PCR was performed using primers SOD-F (SEQ ID NO. 7) and SOD-R (SEQ ID NO. 8) to verify the transformants. The resulting strain was designated *E. coli* BL21(DE3) / pET28a-SOD and stored at -80℃.

[0034] The engineered strain E. coli BL21(DE3) / pET28a-SOD constructed above was streaked onto LB (Luria-Bertani) solid plates containing 100 μg / mL Kanamycin and incubated upside down at 37°C for 12–16 h. Single colonies were picked and inoculated into LB liquid medium containing 100 μg / mL Kan and cultured at 37°C and 200 rpm for 12 h to obtain a seed culture. The seed culture was then transferred to fresh LB liquid medium containing 100 μg / mL Kan at a 2% v / v inoculation ratio and cultured at 37°C and 200 rpm until the bacterial OD600 reached 0.6–0.8. IPTG (isopropyl-β-D-thiogalactopyranoside) was added to a final concentration of 0.2 mM. Expression was induced for 12 h at 37℃ and 28℃ at 200 rpm. After centrifugation at 4℃ and 8000 rpm for 10 min, the supernatant was discarded, and the wet bacterial pellet was collected. The pellet was resuspended in 10 volumes of 100 mM PBS buffer (pH 7.0, containing 17.03 g Na₂HPO₄ and 10.89 g KH₂PO₄ dissolved in 100 ml deionized water), and the cells were disrupted using an ultrasonic cell disruptor. The cell lysate was collected and centrifuged at 4℃ and 8000 rpm for 10 min. The supernatant and pellet were then subjected to SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) for verification. The 12% SDS-PAGE results for EC-SOD are shown below. Figure 1 As shown, the results indicate that after IPTG induction, EC-SOD mainly exists in the ultrasonically disrupted bacterial precipitate in the form of inclusion bodies.

[0035] Example 2: Construction and expression of engineered bacteria expressing the fusion protein SOD-KGF2

[0036] The amino acid sequence derived from keratinocyte growth factor 2 was synthesized according to the codon preference of E. coli to form the KGF2 nucleotide sequence (SEQ ID NO.2), which was then ligated into the cloning vector pUC19 to construct the pUC19-KGF2 vector.

[0037] Using pET28a-SOD as a template, the pET28a-SOD vector backbone (denoted as fragment 1) was amplified using upstream primer P1 (SEQ ID NO.3) and downstream primer P2 (SEQ ID NO.4). Using pUC19-KGF2 as a template, the KGF2 fragment containing the linker peptide (denoted as fragment 2) was amplified using upstream primer P3 (SEQ ID NO.5) and downstream primer P4 (SEQ ID NO.6). Fragments 1 and 2 have a 20-base homologous arm. Fragments 1 and 2 were incubated using the ClonExpress II One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd.) to insert the KGF2 fragment containing the linker peptide before the stop codon of SOD, constructing the following structure: Figure 2 The expression vector shown was then transformed into *E. coli* BL21(DE3) competent cells. Positive transformants were selected and colony PCR was performed using primers SOD-KGF2-F (SEQ ID NO. 9) and SOD-KGF2-R (SEQ ID NO. 10) to verify the transformation. The resulting fusion protein expression vector was designated pET28a-SOD-KGF2( Figure 2 The resulting strain was named E. coli BL21(DE3) / pET28a-SOD-KGF2 and stored at -80℃.

[0038] The induction method for E. coli BL21(DE3) / pET28a-SOD-KGF2 expression was the same as in Example 1. The 12% SDS-PAGE results of the SOD-KGF2 fusion protein are shown below. Figure 3 As shown, SDS-PAGE electrophoresis analysis of the SOD-KGF2 fusion protein after IPTG induction revealed a molecular weight of approximately 44 kDa, consistent with the target protein. The SOD-KGF2 fusion protein was mainly present in the supernatant of the sonicated bacterial cells, indicating that the fusion protein was primarily expressed in a soluble form. The primers and the nucleotide sequences of the target protein are detailed in Table 1.

[0039] Table 1. Nucleotide sequences of each primer and target protein.

[0040]

[0041]

[0042] Example 3: Cleavage of SOD-KGF2 fusion protein and purification of EC-SOD and KGF2 proteins

[0043] (1) Cleavage of fusion proteins

[0044] E. coli BL21(DE3) / pET28a-SOD-KGF2 cells expressed by IPTG in Example 2 were collected and suspended in 20mM Tris-HCl buffer (pH 7.5) containing 0.3M NaCl, 10mM EDTA-2Na. The cells were sonicated for 15 minutes on ice using an ultrasonic cell disruptor, with 1-second intervals between each sonication. Recombinant TEV (tobacco mosaic virus) protease (purchased from Yeason) was added to the cell supernatant for lysis. For every 1 mg of fusion protein, add one-tenth volume of 10x TEV protease buffer (500 mM Tris-HCl, pH 8.0, 5 mM EDTA, 1% Tween-20 (v / v), 10 mM DTT) and 200 U of TEV protease. Incubate overnight at 4°C until the fusion protein is completely cleaved, yielding cell lysates containing EC-SOD protein with residual Glu-Asn-Leu-Tyr-Phe-Gln amino acid residues at the carboxyl terminus and KGF2 protein with a residual Gly residue at the amino terminus. Centrifuge the resulting cell lysates at 8000 rpm for 10 min at 4°C, collect the supernatant, and store on ice for later use.

[0045] (2) Purification of human EC-SOD

[0046] Take the supernatant obtained in step (1) and use an UNOsphereS strong cation exchange column (40×12.6mm, Biorad). Equilibration buffer and elution buffer A consist of 6M urea and 0.25M acetic acid, pH 4.0; elution buffer B consists of 6M urea, 0.25M acetic acid, and 1M sodium chloride, pH 4.05. The elution gradient starts with 100% elution buffer A for 5 minutes, then gradually increases to 70% elution buffer A / 30% elution buffer B for 10 minutes, and then continues with a linear gradient of 30-100% elution buffer B for 50 minutes. Elute the column at a flow rate of 1 mL / min and collect the EC-SOD eluent in a 10 mL tube. After elution, wash the column with 100% elution buffer B for 20 minutes and equilibrate with 100% equilibration buffer for 20 minutes. Monitor the elution at 280 nm to obtain the purified SOD protein. The electrophoresis image of the purified SOD protein is shown below. Figure 4 As shown.

[0047] (3) Purification of KGF2 protein

[0048] Using a CM-Sepharose 6FF ion exchange column, flush the column with 3-5 column volumes at a flow rate of 3.0 mL / min to remove the 20% ethanol solution. Equilibrate the ion exchange column by adding 20 mM phosphate buffer (pH 6.0) containing 0.3 M NaCl and 10 mM EDTA-2Na to pump A at a flow rate of 3.0 mL / min. Stop flushing once the UV line is horizontal. Adjust the flow rate to 1.0 mL / min and load crude KGF2. Elute approximately 3-5 column volumes using pumps A and B respectively, adding degassed ddH2O and 1.0 M NaCl and 10 mM EDTA-2Na to pump B at a flow rate of 3.0 mL / min. Collect the elution peak; this is the purified KGF2 protein solution. Store at -20°C. The electrophoresis image of the purified KGF2 protein is shown below. Figure 4 As shown.

[0049] Example 4 EC-SOD enzyme activity assay

[0050] The enzyme activity of EC-SOD obtained by IPTG-induced expression in Example 1, SOD-KGF2 fusion protein obtained by IPTG-induced expression in Example 2, and EC-SOD purified in Example 3 were measured.

[0051] For enzyme activity assay, add buffer solution as shown in Table 2, incubate at 25℃ for 20 min, add pyrogallol, mix quickly with a shaker, place in a 1 cm cuvette, and use a blank as a control. Measure absorbance A at 325 nm wavelength every 0.5 min, continuously recording for 3 min. Control the auto-oxidation rate A0 at 0.060 (±0.001) / min. For SOD activity assay, add sample as shown in Table 1, and record A1 values ​​in the same way. Adjust the SOD sample concentration to control the pyrogallol oxidation rate at 50% of the auto-oxidation rate, i.e., 0.030 (±0.001) / min.

[0052] Table 2

[0053]

[0054] An enzyme activity unit is defined as the amount of enzyme that inhibits the rate of pyrogallol autoxidation by 50% per minute in 1 mL of reaction solution.

[0055] Formula for calculating enzyme activity units:

[0056] SOD activity (U) = (((A0-A1) / A0) / 50%) * (Va / V1) * (N / VE)

[0057] A0 – Auto-oxidation rate; A1 – Oxidation rate after enzyme addition; N – Dilution factor; V1 – Defined volume; Va – Total reaction volume; VE – Volume of enzyme added.

[0058] 1 mg of enzyme protein was taken to determine the enzyme activity, that is, the specific activity of EC-SOD obtained in Example 1, fusion protein obtained in Example 2, and EC-SOD isolated and purified in Example 3 was measured. The results are shown in Table 3.

[0059] Table 3: Results of EC-SOD enzyme specific activity assay

[0060]

[0061]

[0062] The results further indicated that EC-SOD was expressed as an inactive inclusion body in the engineered strain E. coli BL21(DE3) / pET28a-SOD. Therefore, under 28℃ induction conditions, the specific activity of EC-SOD in the supernatant after cell disruption and centrifugation was extremely low. The fusion protein in the engineered strain E. coli BL21(DE3) / pET28a-SOD-KGF2 was mainly expressed in a soluble form. It was mainly present in the supernatant after ultrasonic disruption. Therefore, under 28℃ induction conditions, the fusion protein still showed high EC-SOD activity in the supernatant after ultrasonic disruption. The purified EC-SOD showed a maximum specific activity of 1803.8 U / mg.

[0063] In the description of this specification, references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0064] Although embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the invention. The scope of the present invention is defined by the appended claims and their equivalents.

[0065] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A recombinant Escherichia coli for preparing soluble EC-SOD, characterized in that: The recombinant Escherichia coli is obtained by inserting a gene encoding KGF2 into the downstream of a gene encoding SOD in an expression vector and then transforming the vector into a host Escherichia coli competent cell.

2. The recombinant Escherichia coli for preparing soluble EC-SOD according to claim 1, characterized in that: The recombinant Escherichia coli is obtained by removing the 3'-terminator codon TAA from the SOD gene of SEQ ID NO.1 in an expression vector, connecting it with the KGF2 gene of SEQ ID NO.2 using a connecting peptide coding sequence containing a protease cleavage site, and then transforming it into a host Escherichia coli competent cell.

3. The recombinant Escherichia coli for preparing soluble EC-SOD according to claim 2, characterized in that: The connecting peptide containing the protease cleavage site has a nucleic acid sequence as shown in SEQ ID NO.11 and a TEV protease cleavage sequence as shown in SEQ ID NO.

12.

4. The recombinant Escherichia coli for preparing soluble EC-SOD according to any one of claims 1 to 3, characterized in that: The host Escherichia coli is Escherichia coli BL21 (DE3).

5. The recombinant Escherichia coli for preparing soluble EC-SOD according to any one of claims 1 to 3, characterized in that: The expression vector is the expression vector pET28a.

6. A method for constructing a recombinant Escherichia coli for preparing soluble EC-SOD according to any one of claims 1 to 3, characterized in that: The following steps are involved: (1) The SOD nucleotide sequence was synthesized from the amino acid sequence of superoxide dismutase according to the codon preference of Escherichia coli and cloned into the expression vector pET28a to construct the pET28a-SOD vector; (2) synthesizing the KGF2 nucleotide sequence based on the amino acid sequence of keratinocyte growth factor 2 according to the codon preference of Escherichia coli, and ligating it into the cloning vector pUC19 to construct the pUC19-KGF2 vector; (3) Using the pET28a-SOD vector as a template, primers P1 and P2 were used to amplify the pET28a-SOD vector backbone, which was recorded as fragment 1; (4) Using the pUC19-KGF2 vector as a template, primers P3 and P4 containing the linker peptide coding sequence were used to amplify the KGF2 fragment with the linker peptide coding sequence, which was recorded as fragment 2; (5) Fragment 1 and fragment 2 were incubated using a one-step seamless cloning kit, and then transformed into Escherichia coli BL21 (DE3) competent cells to obtain recombinant E. coli BL21 (DE3) / pET28a-SOD-KGF2.

7. The method for constructing a recombinant Escherichia coli for preparing soluble EC-SOD according to claim 6, characterized in that: The primer P1 in step (3) has the sequence shown in SEQ ID NO.3, and the primer P2 has the sequence shown in SEQ ID NO.4; the primer P3 in step (4) has the sequence shown in SEQ ID NO.5, and the primer P4 has the sequence shown in SEQ ID NO.

6.

8. The method for constructing a recombinant Escherichia coli for preparing soluble EC-SOD according to claim 6, characterized in that: In step (5), there is a homology arm of 20 bases between fragment 1 and fragment 2.

9. Use of the recombinant Escherichia coli expressing the soluble EC-SOD fusion protein according to any one of claims 1 to 3 in the preparation of EC-SOD.

10. The use according to claim 9, characterized in that: The recombinant Escherichia coli is cultured and an inducer is added to induce expression, and the cell lysate is digested by protease to obtain two proteins, EC-SOD and KGF2.

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