A method for synthesizing, purifying, and applying a calprotectin fusion protein.

Abstract

This invention provides a method for synthesizing, purifying, and applying a calprotectin fusion protein, belonging to the field of molecular biology. The calprotectin fusion protein of this invention is a heterodimer or heterotetramer formed by the polymerization of the S100A8 and S100A9 subunits, with a six-histidine affinity tag introduced at the amino terminus of the S100A8 subunit. This invention enables the simultaneous synthesis of both the S100A8 and S100A9 subunits of calprotectin using a single recombinant *E. coli* strain. The two subunits can self-assemble calprotectin within the bacterial cell, eliminating the step of artificially mixing subunit solutions in existing microbial fermentation synthesis methods. Furthermore, by introducing a six-histidine affinity tag at the amino terminus of the S100A8 subunit, this invention facilitates the purification of the calprotectin fusion protein, improving production efficiency and product purity. The calprotectin fusion protein of this invention retains the transcriptional regulatory activity of natural calprotectin in *E. coli*.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to a method for synthesizing, purifying, and applying a calprotectin fusion protein. Background Technology

[0002] Calprotectin, a member of the S100 protein family, is a multifunctional heterozygous calcium-binding protein. It is formed by the self-assembly of the S100A8 and S100A9 subunits and mainly exists in neutrophils as a dimer or tetramer.

[0003] Calprotectin synthesis methods include in vivo isolation, cellular synthesis, and microbial fermentation synthesis. Microbial fermentation synthesis requires the separate synthesis of two calprotectin subunits using two *E. coli* strains, followed by mixing the two subunits in a solution system to allow them to self-assemble into a complete calprotectin. This method yields biologically active calprotectin with relatively low production costs, making it a promising strategy for calprotectin synthesis. However, this approach requires constructing two recombinant bacteria to synthesize the S100A8 and S100A9 subunits separately, and then mixing and assembling the synthesized S100A8 and S100A9 subunits, making the process rather cumbersome. Summary of the Invention

[0004] The purpose of this invention is to provide a method for synthesizing, purifying, and applying calprotectin fusion protein, which achieves the synthesis and assembly of two subunits of calprotectin through a single strain, while optimizing the downstream purification process.

[0005] The present invention provides a calprotectin fusion protein, wherein the calprotectin fusion protein is a heterodimer or heterotetramer formed by the polymerization of S100A8 subunit and S100A9 subunit, wherein the amino terminus of the S100A8 subunit is introduced with an affinity tag of 6 histidine residues.

[0006] The present invention also provides a recombinant vector for expressing the calprotectin fusion protein described in the above scheme, using pRSFDuet-1 as the backbone plasmid, and inserting the coding genes of the S100A8 subunit and the S100A9 subunit; the coding genes of the S100A8 subunit and the S100A9 subunit are optimized according to the codon preference of Escherichia coli.

[0007] Preferably, the nucleotide sequence of the gene encoding the S100A8 subunit is shown in SEQ ID NO.1; the gene encoding the S100A8 subunit is constructed between the BamHI and SalI restriction sites of pRSFDuet-1.

[0008] Preferably, the nucleotide sequence of the coding gene of the S100A9 subunit is shown in SEQ ID NO.2; the coding gene of the S100A9 subunit is constructed between the Nde I and Mfe I restriction sites of the pRSFDuet-1 vector.

[0009] Preferably, the recombinant vector nucleotide sequence is as described in SEQ ID NO.4.

[0010] The present invention also provides a recombinant Escherichia coli comprising the recombinant vector described in the above-described scheme.

[0011] The present invention also provides a method for synthesizing the calprotectin fusion protein described in the above-described scheme, comprising the following steps:

[0012] After fermentation culture of the recombinant Escherichia coli described in the above scheme, bacterial cells containing calprotectin fusion protein were obtained by induction with isopropyl-β-D-thiogalactopyranoside.

[0013] The present invention also provides a purification method for the calprotectin fusion protein described in the above-described scheme, comprising the following steps: purifying the bacterial cells or crude enzyme solution containing the calprotectin fusion protein obtained by the synthesis method described in the above-described scheme using a nickel affinity column to obtain the calprotectin fusion protein.

[0014] Preferably, the purification method includes the following steps: the bacterial cells containing calprotectin fusion protein obtained by the synthesis method described above are ultrasonically disrupted and centrifuged to obtain a cell lysate supernatant; the cell lysate supernatant is sequentially subjected to nickel affinity column affinity adsorption and imidazole gradient elution to obtain a calprotectin fusion protein solution; the calprotectin fusion protein solution is sequentially subjected to ultrafiltration washing to remove imidazole to obtain a purified calprotectin fusion protein solution; the purified calprotectin fusion protein solution is lyophilized to obtain purified calprotectin fusion protein; the imidazole gradient elution includes sequential elution with phosphate buffer containing 20, 50, 100, 200, and 500 mM imidazole.

[0015] This invention also provides the calprotectin fusion protein described in the above-described scheme, the recombinant vector, the recombinant *E. coli*, bacterial cells containing the calprotectin fusion protein obtained by the synthesis method, or the purified calprotectin fusion protein obtained by the purification method, inducing *E. coli* P... ykgMO Applications of the P in downstream gene expression; ykgMO The promoter nucleotide sequence is shown in SEQ ID NO.3.

[0016] This invention provides a calprotectin fusion protein, which is a heterodimer or heterotetramer formed by the polymerization of S100A8 and S100A9 subunits. The S100A8 subunit has a six-histidine affinity tag introduced at its amino terminus. This invention enables the simultaneous synthesis of both the S100A8 and S100A9 subunits of calprotectin using a single recombinant *E. coli* strain. The two subunits can self-assemble calprotectin within the bacterial cell, eliminating the need for the artificial mixing of subunit solutions in existing microbial fermentation synthesis methods, thus simplifying the preparation process. Furthermore, by introducing a six-histidine affinity tag at the amino terminus of the S100A8 subunit, this invention facilitates the purification of the calprotectin fusion protein, further improving production efficiency and product purity. The calprotectin fusion protein of this invention retains the transcriptional regulatory activity of natural calprotectin in *E. coli*, which is of great significance for the functional research and application of calprotectin. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments 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.

[0018] Figure 1 Flowchart for the fermentation synthesis and purification of calprotectin fusion protein;

[0019] Figure 2 The figure shows the concentration-dependent induction intensity gradient of calprotectin fusion protein inducing downstream fluorescent protein expression of the PykgMO promoter at different concentrations. Detailed Implementation

[0020] The present invention provides a calprotectin fusion protein, wherein the calprotectin fusion protein is a heterodimer or heterotetramer formed by the polymerization of S100A8 subunit and S100A9 subunit, wherein the amino terminus of the S100A8 subunit is introduced with an affinity tag of 6 histidine residues.

[0021] The present invention also provides a recombinant vector pRSFDuet-His-S100A8 / A9 for expressing the calprotectin fusion protein described above, using pRSFDuet-1 as the backbone plasmid and inserting the coding genes for the S100A8 subunit and the S100A9 subunit; the coding genes for the S100A8 subunit and the S100A9 subunit are optimized according to the codon preference of Escherichia coli (E. coli).

[0022] In this invention, pRSFDuet-1 is a dual-gene expression vector.

[0023] In the specific implementation of this invention, the nucleotide sequence of the gene encoding the S100A8 subunit is shown in SEQ ID NO.1, specifically as follows:

[0024]

[0025] In the specific implementation of this invention, the coding gene of the S100A8 subunit is constructed between the BamHI and SalI restriction sites of pRSFDuet-1, and the complete expression frame gene sequence is shown in SEQ ID NO.1.

[0026] In a specific implementation of this invention, the nucleotide sequence of the gene encoding the S100A9 subunit is shown in SEQ ID NO.2, specifically as follows:

[0027]

[0028]

[0029] In the specific implementation of this invention, the encoding gene of the S100A9 subunit is constructed between the Nde I and Mfe I restriction sites of the pRSFDuet-1 vector, and the complete expression frame gene sequence is shown in SEQ ID NO.2.

[0030] In one embodiment of the present invention, the recombinant vector nucleotide sequence is as described in SEQ ID NO.4, specifically as follows:

[0031]

[0032]

[0033]

[0034]

[0035] The present invention also provides a recombinant Escherichia coli comprising the recombinant vector described in the above-described scheme.

[0036] In this invention, the original strain of the recombinant Escherichia coli is preferably Escherichia coli BL21(DE3).

[0037] In the specific implementation of this invention, the recombinant vector pRSFDuet-His-S1100A8 / A9 is transformed into BL21(DE3) to obtain recombinant Escherichia coli. This invention does not impose any special restrictions on the transformation conditions; conventional transformation conditions of 42°C heat shock for 90 seconds are sufficient.

[0038] The present invention also provides a method for synthesizing the calprotectin fusion protein described in the above-described scheme, comprising the following steps:

[0039] After fermentation culture of the recombinant Escherichia coli described in the above scheme, bacterial cells containing calprotectin fusion protein were obtained by induction with isopropyl-β-D-thiogalactopyranoside (IPTG).

[0040] This invention uses Escherichia coli as the fermentation substrate strain to synthesize calprotectin fusion protein.

[0041] In a specific implementation of this invention, the recombinant *E. coli* is cultured and activated to obtain a seed culture; the seed culture is inoculated into a fermentation medium, and after fermentation, IPTG-induced expression yields bacterial cells containing the calprotectin fusion protein. In this invention, the fermentation medium is preferably TB medium; the fermentation is carried out until the bacterial cell OD... 600 The concentration of IPTG reaches 0.4–0.6; the fermentation culture temperature is 37°C; the final concentration of IPTG is 500 μM; the induction time is 22 h; the induction temperature is 20°C; and the induction process is carried out with shaking.

[0042] The method for synthesizing calprotectin fusion protein provided by this invention enables the fermentation of Escherichia coli BL21(DE3) on TB medium to obtain high-purity calprotectin fusion protein. The method for synthesizing calprotectin fusion protein provided by this invention is simple, has low E. coli culture costs, rapid cell growth, and high fermentation production intensity.

[0043] This invention also provides a method for purifying the calprotectin fusion protein described above, comprising the following steps:

[0044] The bacterial cells or crude enzyme solution containing calprotectin fusion protein obtained by the synthesis method described above are purified by nickel affinity column to obtain calprotectin fusion protein.

[0045] In this invention, the purification method includes the following steps:

[0046] The bacterial cells containing calprotectin fusion protein obtained by the synthesis method described above were ultrasonically disrupted and then centrifuged to obtain the cell lysate supernatant.

[0047] The cell lysate supernatant was sequentially subjected to nickel affinity column affinity adsorption and imidazole solution gradient elution to obtain a calcium protectin fusion protein solution;

[0048] The calcium protectant fusion protein solution was sequentially subjected to ultrafiltration washing to remove imidazole, resulting in a purified calcium protectant fusion protein solution.

[0049] The purified calprotectin fusion protein solution was lyophilized to obtain the purified calprotectin fusion protein.

[0050] First, the bacterial cells containing calprotectin fusion protein obtained by the synthesis method described above are ultrasonically disrupted and then centrifuged to obtain the cell lysate supernatant.

[0051] In the specific implementation of this invention, the ultrasonic crushing power is 150W; the ultrasonic crushing time is 25-35min; the centrifugation temperature is 4℃; the centrifugation speed is 8000rpm; and the centrifugation time is 10min.

[0052] After obtaining the cell lysate supernatant, the cell lysate supernatant was sequentially subjected to nickel affinity adsorption and imidazole gradient elution to obtain the calprotectin fusion protein solution.

[0053] In a specific implementation of this invention, the imidazole gradient elution involves sequentially eluting with phosphate buffer solutions containing 20, 50, 100, 200, and 500 mM imidazole. In this invention, the concentration of phosphate ions in the phosphate buffer solution is 50 mM; the pH of the phosphate buffer solution is 7.4.

[0054] After obtaining the calprotectin fusion protein solution, the calprotectin fusion protein solution was washed with ultrafiltration to remove imidazole, resulting in a purified calprotectin fusion protein solution.

[0055] In the specific implementation of this invention, the ultrafiltration tube used in the ultrafiltration process has a molecular weight of 10 kDa.

[0056] The calprotectin fusion protein of this invention, after purification using a nickel affinity column and ultrafiltration to remove imidazole, has fewer impurities, and the product has high purity and yield. 38 mg of high-purity calprotectin fusion protein can be obtained from every 100 mL of fermentation broth after purification using a nickel affinity column and ultrafiltration.

[0057] After obtaining the purified calprotectin fusion protein solution, the purified calprotectin fusion protein solution was lyophilized to obtain the purified calprotectin fusion protein.

[0058] The purification method provided by this invention can be achieved using only a nickel affinity column, and the gradient elution process is simple and fast. At the same time, the obtained calprotectin fusion protein retains the biological activity of the natural calprotectin.

[0059] This invention also provides the calprotectin fusion protein described in the above-described scheme, the recombinant vector, the recombinant *E. coli*, bacterial cells containing the calprotectin fusion protein obtained by the synthesis method, or the purified calprotectin fusion protein obtained by the purification method, inducing *E. coli* P... ykgMO Applications of the P in downstream gene expression;ykgMO The promoter nucleotide sequence is shown in SEQ ID NO.3, specifically as follows:

[0060]

[0061]

[0062] In the specific implementation of this invention, P ykgMO The downstream genes of the promoter were analyzed using mNeongreen fluorescent protein (GenBank EU482389) as a reporter gene to demonstrate the induced expression intensity. The nucleotide sequence of the gene encoding mNeonGreen fluorescent protein is shown in SEQ ID NO.5, specifically:

[0063]

[0064] In this invention, the final concentration of the calprotectin fusion protein is preferably ≥100 μg / mL, more preferably 100-800 μg / mL, and most preferably 100, 200, 400 or 800 μg / mL.

[0065] In the specific implementation of this invention, P is induced with a final concentration of 0, 100, 200, 400 or 800 μg / mL of calprotectin fusion protein. ykgMO Downstream expression of mNeongreen fluorescent protein was induced by a concentration-dependent gradient of increasing intensities.

[0066] The calprotectin fusion protein of the present invention retains the activity of the original natural calprotectin in regulating the transcription of Escherichia coli.

[0067] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes the synthesis, purification, and application of a calprotectin fusion protein provided by the present invention, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0068] Example 1

[0069] like Figure 1 As shown, this embodiment provides a method for synthesizing and purifying calprotectin fusion protein using Escherichia coli, including the following steps:

[0070] (1) Construction of pRSFDuet-His-S100A8 / A9 vector:

[0071] The nucleotide sequences of the S100A8 and S100A9 subunits were optimized according to the codon preference of *E. coli*. The optimized coding sequence of S100A8 is shown in SEQ ID NO.1, and the optimized coding sequence of S100A9 is shown in SEQ ID NO.2. The coding sequence of the S100A8 subunit was synthesized between the BamHI and SalI restriction sites of the *E. coli* dual-gene expression vector pRSFDuet-1, enabling it to form a fusion protein with a 6-histidine tag at the amino terminus during protein translation. The coding sequence of the S100A8 subunit was synthesized between the Nde I and Mfe I restriction sites of the pRSFDuet-1 vector. The recombinant vector pRSFDuet-His-S100A8 / A9 was obtained, and its sequence is shown in SEQ ID NO.4.

[0072] (2) Construction of recombinant Escherichia coli E21(DE3)-pRSFDuet-His-S100A8 / A9:

[0073] The recombinant vector pRSFDuet-His-S100A8 / A9 solution described in step (1) was mixed in competent E. coli BL21(DE3) cells, incubated on ice for 30 min, followed by heat shock at 42°C for 60 s, and then incubated on ice for 2 min. 1 mL of resuscitation medium was added to revive the competent cells for 1–2 h. The competent cells were then plated on kanamycin-resistant plates and cultured overnight for screening. Single colonies of recombinant E. coli E. coli BL21(DE3)-pRSFDuet-His-S100A8 / A9 were obtained.

[0074] (3) Preparation of fermentation seed liquid:

[0075] Pick a single colony obtained in step (2) and inoculate it into LB medium containing 50 μg / mL kanamycin. Incubate at 37°C with shaking for 10–12 h to obtain recombinant Escherichia coli seed culture.

[0076] (4) Fermentation culture and induced expression:

[0077] The recombinant Escherichia coli seed culture obtained in step (3) was transferred to TB medium at an inoculation rate of 1% (v / v) and cultured at 37°C with shaking until the bacterial cell OD reached 1%. 600 When the concentration reaches 0.4–0.6, add 500 μM isopropyl-β-D-thiopyranoside (IPTG) to induce the expression of calprotectin fusion protein, and culture at 20°C with shaking for 22 h.

[0078] (5) Preparation of cell lysate supernatant:

[0079] Centrifuge the recombinant E. coli culture medium obtained as described in step (4), collect the bacterial cells containing the calprotectin fusion protein, and resuspend them in PBS buffer. Use an ultrasonic disruptor at 150W for 25–35 min to disrupt the cells. Centrifuge at 8000 rpm for 10 min at 4°C to separate the cell lysis supernatant.

[0080] (6) Nickel gradient elution of calprotectin fusion protein:

[0081] After removing impurities by passing the cell lysate supernatant obtained by the method described in step (5) through a membrane, the supernatant is loaded into a nickel purification column and eluted with gradients of 20, 50, 100, 200, and 500 mM imidazole phosphate buffer to obtain a gradient elution buffer of calprotectin fusion protein.

[0082] (7) SDS-PAGE electrophoresis:

[0083] The gradient elution buffer described in step (6) was mixed with the protein loading buffer, heated at 100°C for 10 min, and loaded onto a 12% protein gel for SDS-PAGE electrophoresis. Based on the electrophoresis results, protein samples eluted with 200 mM and 500 mM imidazole were collected to obtain a high-purity calprotectin fusion protein solution.

[0084] (8) Removal of impurities from the calprotectin fusion protein solution:

[0085] The high-purity calprotectin fusion protein solution described in step (7) was centrifuged through a 10 kDa ultrafiltration tube, followed by washing and centrifugation three times with PBS buffer to obtain a purified calprotectin fusion protein solution free of imidazole.

[0086] (9) Lyophilization and preservation of purified calprotectin fusion protein:

[0087] The calcium protector fusion protein solution described in step (8) was refrigerated overnight at -80°C and then freeze-dried under vacuum at -80°C for 12 hours to obtain freeze-dried powder of calcium protector fusion protein. The protein sample was then sealed and stored at 4°C.

[0088] Example 2

[0089] The induction and application of the calprotectin fusion protein in Example 1 includes the following steps:

[0090] (1) pET28a-P ykgMO Construction of the -mNeongreen plasmid:

[0091] P ykgMOThe promoter (as shown in SEQ ID NO.3) and the gene encoding mNeonGreen fluorescent protein (as shown in SEQ ID NO.5) were synthesized between the BglII and Xho I restriction sites of the pET28a vector to obtain the pET28a-PykgMO-mNeongreen plasmid.

[0092] (2) Recombinant Escherichia coli EcN-pET28a-P ykgMO Build of -mNeongreen:

[0093] The recombinant vector pET28a-PykgMO-mNeongreen plasmid solution described in step (1) was mixed in competent E. coli Nissle 1917 (EcN) cells, electroporated at 1.8 kV for 6 ms, and screened for recombinant single colonies on kanamycin resistance plates.

[0094] (3) Preparation of fermentation seed liquid:

[0095] Pick a single colony obtained in step (2) and inoculate it into LB medium containing 50 μg / mL kanamycin. Incubate at 37°C with shaking for 10-12 h to obtain recombinant Escherichia coli seed culture.

[0096] (4) Fermentation culture and induced expression:

[0097] The recombinant Escherichia coli seed culture obtained in step (3) was transferred to LB medium at an inoculation rate of 1% (v / v). At the time of inoculation, calprotectin fusion protein with a final concentration of 0 / 100 / 200 / 400 / 800 μg / mL was added to each group, with 3 replicates per group.

[0098] (5) Detection of induction intensity:

[0099] From the start of cell culture, samples were taken hourly, and the fluorescence signal and cell OD of each fermentation sample were measured using an enzyme-linked immunosorbent assay (ELISA) reader at 485 / 530 nm excitation / absorption light. 600 Data, based on fluorescent signal bacterial OD 600 The ratio of the fluorescence intensity was used to plot the fluorescence intensity change curve, and the data are as follows: Figure 2 As shown, the fluorescence intensity exhibits a concentration-dependent difference in the calprotectin fusion protein.

[0100] 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. Calprotectin fusion protein, a recombinant vector expressing the calprotectin fusion protein, or recombinant Escherichia coli containing the recombinant vector, inducing Escherichia coli P ykgMO Applications of the P in downstream gene expression; ykgMO The promoter nucleotide sequence is shown in SEQ ID NO.3; The calprotectin fusion protein is a heterodimer or heterotetramer formed by the polymerization of the S100A8 subunit and the S100A9 subunit; the amino terminus of the S100A8 subunit is introduced with an affinity tag of 6 histidine residues. The nucleotide sequence of the gene encoding the S100A8 subunit is shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the S100A9 subunit is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The recombinant vector uses pRSFDuet-1 as the backbone plasmid and inserts the coding genes for the S100A8 subunit and the S100A9 subunit.

3. The application according to claim 2, characterized in that, The gene encoding the S100A8 subunit was constructed from pRSFDuet-1. BamH I and Sal Between I restriction enzyme sites.

4. The application according to claim 2, characterized in that, The gene encoding the S100A9 subunit was constructed in the pRSFDuet-1 vector. Nde I and Mfe Between I restriction enzyme sites.

5. The application according to any one of claims 2 to 4, characterized in that, The nucleotide sequence of the recombinant vector is as described in SEQ ID NO.

4.

6. The application according to claim 1, characterized in that, The method for synthesizing the calprotectin fusion protein includes the following steps: After fermentation culture of recombinant Escherichia coli, bacterial cells containing calprotectin fusion protein were obtained by induction with isopropyl-β-D-thiogalactopyranoside. The recombinant Escherichia coli contains a recombinant vector expressing the calprotectin fusion protein.

7. The application according to claim 6, characterized in that, The purification method for the calprotectin fusion protein includes the following steps: The bacterial cells containing the calprotectin fusion protein or the crude enzyme solution of the bacterial cells were purified by nickel affinity column to obtain the calprotectin fusion protein.

8. The application according to claim 7, characterized in that, The purification method includes the following steps: The bacterial cells containing the calprotectin fusion protein were ultrasonically disrupted and then centrifuged to obtain the cell lysate supernatant. The cell lysate supernatant was sequentially subjected to nickel affinity column affinity adsorption and imidazole solution gradient elution to obtain a calcium protectin fusion protein solution; The calcium protectant fusion protein solution was sequentially subjected to ultrafiltration washing to remove imidazole, resulting in a purified calcium protectant fusion protein solution. The purified calprotectin fusion protein solution was lyophilized to obtain the purified calprotectin fusion protein. The imidazole gradient elution involves sequential elution with phosphate buffer containing 20, 50, 100, 200, and 500 mM imidazole.

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