Preparation method and application of recombinant staphylococcus aureus leukocidin E

Abstract

The invention provides a preparation method and application of recombinant staphylococcus aureus leukocidin E. The preparation method comprises fine purification, wherein the fine purification comprises a first step of purification, a second step of desalination replacement, a third step of purification and a fourth step of purification. The recombinant staphylococcus aureus leukocidin E prepared by the invention not only meets the quality standard required by pharmacopeia, but also has the advantages of simple process, high yield, easiness in amplification and low cost, and has wide application prospects in preparation of human vaccines, veterinary vaccines and antibody drugs and development of in-vitro diagnostic reagents.

Description

Technical Field

[0001] This invention relates to the production of recombinant protein drugs using genetic engineering technology, specifically to a method for preparing recombinant Staphylococcus aureus leukocidin E and its application, belonging to the fields of genetic engineering technology and pharmaceutical technology. Background Technology

[0002] Staphylococcus aureus (S. aureus) is a Gram-positive bacterium widely distributed in nature and is an important pathogen causing infections in humans and animals. It can cause pneumonia, endocarditis, and other diseases through respiratory tract infection; it can also cause sepsis, septicemia, and other diseases through bloodstream infection; and it can cause vomiting, diarrhea, toxic shock syndrome, and other diseases through food contamination.

[0003] S. aureus secretes numerous virulence factors, among which perforating toxins (PFTs) are a class of virulence factors. These are expressed as water-soluble monomeric proteins and accumulate on the target cell membrane to form pores. Based on the secondary structure of the transmembrane region, PFTs can be divided into two families: α-helical PFTs (α-PFTs) and β-barrel PFTs (β-PFTs). Seven β-PFTs have been the most popular subjects of research: α-toxin (Hla), γ-hemolysin AB (HlgAB), γ-hemolysin CB (HlgCB), Panton-Valentine leukocidin (LukSF-PV), leukocidin ED (LukED), leukocidin AB (LukAB, also known as LukGH), and leukocidin MF' (LukMF'). Woodin classified LukSF by ion-exchange column chromatography, naming the slower-eluting subunit "S" (LukS-PV) and the faster-eluting subunit "F" (LukF-PV). This naming convention has also been applied to other two-component toxins. The "S" subunit is responsible for binding to cell surface receptors, inducing a conformational change, which then assembles with the "F" subunit to form aggregates, ultimately achieving membrane perforation.

[0004] PVL has long been a research hotspot for Staphylococcus aureus virulence factors. However, in recent years, the important role of LukED in the pathogenic mechanism of Staphylococcus aureus has been increasingly confirmed by experiments. Studies have shown that LukED can bind to the chemokine receptor CCR5 on the surface of macrophages, T cells, and dendritic cells, the CXCR1 / 2 receptor on the surface of neutrophils, monocytes, and natural killer cells, and the DARC receptor on the surface of erythrocytes, causing corresponding target cell lysis and promoting infection. Epidemiological data show that LukED is closely related to Staphylococcus aureus-induced impetigo and antibiotic-associated diarrhea, and exhibits a high prevalence in clinical Staphylococcus aureus cases.

[0005] Based on its mechanism of action—binding to cell surface molecules via the "S" subunit—LukE is widely used in research on candidate antigens for S. aureus vaccines. Currently, there are few studies on the preparation of recombinant LukE (rLukE) protein detoxified through key amino acid mutations; most studies involve shake-flask-scale sample preparation. For example, non-patent literature 1 constructed various LukE mutant strains, created recombinant expression strains by fusing a His tag to the N-terminus, and obtained rLukE protein through one-step affinity purification for activity studies.

[0006] rLukE's poor stability and low yield limit its development as a candidate antigen for S. aureus vaccines; while fusion tags such as GST generally have residual Gly or Ser at the N-terminus of the target protein, which may lead to safety issues due to incomplete tag removal.

[0007] Non-patent literature 1: Peng Z, Takeshita M, Shibata N, Tada H, Tanaka Y, KanekoJ. Rim domain loops of staphylococcal β-pore forming bi-component toxin S-components recognize target human erythrocytes in a coordinated manner. JBiochem. 2018 Aug 1;164(2):93-102. Summary of the Invention

[0008] The purpose of this invention is to provide a method for preparing recombinant Staphylococcus aureus leukocidin E and its application, so as to solve the technical problem of difficulty in preparing pure rLukE protein in the prior art.

[0009] Specifically, the present invention provides a method for preparing recombinant Staphylococcus aureus leukocidin E, the method comprising purification, the purification comprising the following steps:

[0010] (1) First step purification: collect the first step purification eluent sample, wherein the chromatography column used for the first step purification is Ni Sepharose HP or TA-Ni FF;

[0011] (2) Perform a second-step desalting replacement on the eluent sample obtained in step (1) and collect the second-step replacement sample. The second-step desalting replacement method is to use a Bestdex G 25-F or Sephadex G-25F desalting column, ultrafiltration membrane pack or dialysis.

[0012] (3) The replacement sample obtained in step (2) is purified in the third step, and the purified flow-through sample is collected. The chromatography column used in the third step purification is Ni Sepharose HP or TA-Ni FF.

[0013] (4) The flow-through sample obtained in step (3) is purified in the fourth step, and the eluent sample of the fourth step purification is collected. The chromatography column used in the fourth step purification is Phenyl Bestarose HP or Phenyl Sepharose HP.

[0014] In some implementations, the chromatography column used for the first step of purification is Ni Sepharose HP or TA-Ni FF.

[0015] In some specific implementations, the chromatography column used for the first step of purification is TA-NiFF.

[0016] In some specific implementations, the chromatographic conditions for the first step of purification are selected from:

[0017] Equilibration buffer: 20-50 mmol / L PB at pH 7.4 containing 1 mol / L NaCl and 157 mmol / L imidazole;

[0018] Elution buffer: 20-50 mmol / L PB at pH 7.4 containing 1 mol / L NaCl and 353 mmol / L imidazole;

[0019] Alkaline washing buffer: 0.5 mol / L NaOH solution;

[0020] Column chromatography procedure: Equilibrate 3CV, load sample, wash 3~10CV, elute 3CV, wash with alkali 3CV, reequilibrate 3CV;

[0021] Sample loading volume: 5~30mg target protein / ml packing material.

[0022] In some implementations, the second step of desalting is performed using a Bestdex G 25-F or Sephadex G-25F desalting column, an ultrafiltration membrane pack, or dialysis.

[0023] In some specific implementations, the desalting column for the second step of desalting replacement is Bestdex G 25-F.

[0024] In some specific implementation schemes, the conditions for the second step of desalination and replacement are selected from:

[0025] Replacement buffer: 20–50 mmol / L PB at pH 7.4, containing 0.05–0.2 mol / L argon;

[0026] Alkaline wash buffer: 0.2M NaOH solution;

[0027] Column chromatography procedure: Equilibrate to 1.5 CV, load sample, elute to 3 CV, wash with alkali to 3 CV, reequilibrate to 3 CV;

[0028] Sample loading: 25% of column volume;

[0029] In some implementations, the chromatography column used for the third purification step is Ni Sepharose HP or TA-Ni FF.

[0030] In some specific implementations, the chromatography column used for the third purification step is TA-NiFF.

[0031] In some specific implementations, the chromatographic conditions for the third purification step are selected from:

[0032] Equilibration buffer: 20-50 mmol / L PB at pH 7.4, containing 1 mol / L NaCl and 40-80 mmol / L imidazole;

[0033] Sample conditioning solutions: 20-50 mmol / L PB at pH 7.4, containing 4 mol / L NaCl; 20-50 mmol / L PB at pH 7.4, containing 1 mol / L NaCl and 1 mol / L imidazole.

[0034] Alkaline wash buffer: 0.5M NaOH solution;

[0035] Column chromatography procedure: Equilibrate to 3 CV, load sample, elute 3-8 CV, wash with alkali for 3 CV, then equilibrate to 3 CV;

[0036] Loading volume: 5~30 mg target protein / ml filler;

[0037] In some implementations, the chromatography column used for the fourth step of purification is Phenyl Bestarose HP or Phenyl Sepharose HP.

[0038] In some specific implementations, the chromatography column used for the fourth step of purification is Phenyl Bestarose HP.

[0039] In some specific implementations, the chromatographic conditions for the fourth purification step are selected from:

[0040] Equilibration buffer: 20-50 mmol / L PB at pH 7.4, containing 1.5 mol / L NaCl;

[0041] Sample conditioning solution: 20-50 mmol / L PB at pH 7.4, containing 4 mol / L NaCl;

[0042] Elution buffer: 20–50 mmol / L PB at pH 7.4, containing 0.05–0.2 mol / L Arg;

[0043] Wash buffer: Water for injection;

[0044] Alkaline wash buffer: 0.5M NaOH solution;

[0045] Column chromatography procedure: Equilibration 3CV, sample loading, elution 3CV, elution 8CV, water washing 3CV, alkali washing 3CV, reequilibration 3CV;

[0046] Sample loading volume: 5~10 mg target protein / ml packing material.

[0047] In some implementations, the method further includes the following steps:

[0048] The replacement sample obtained in step (2) is digested with enzymes, and the enzyme digestion product sample is collected. The enzyme digestion product sample is then purified in the third step described in step (3).

[0049] In some specific implementations, the enzyme digestion reaction conditions are: protein:SUMO enzyme mass ratio = 500~1000:1, final DTT concentration of 2~5 mmol / L, and digestion at room temperature in the dark for 6~30 h.

[0050] In some embodiments, the method further includes one or more of the steps of constructing the expression strain, fermentation, crude purification, and preserving the purified protein, wherein the crude purification is to separate recombinant Staphylococcus aureus leukocidin E from the bacterial cells.

[0051] In some implementations, the construction of the expression strain includes one or more of the following steps:

[0052] (1) Obtain a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 with codon optimization; preferably, the codon optimization is performed according to the preferences of Escherichia coli;

[0053] (2) Synthesize nucleic acids encoding the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively;

[0054] (3) Nucleic acids encoding the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 are inserted into the expression vector through enzyme restriction sites to obtain recombinant plasmids; preferably, the expression vector is pET28a or pET30a; more preferably, the expression vector is pET28a;

[0055] (4) The recombinant plasmid containing the nucleic acid fragment encoding the amino acid sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 is transformed into the host strain, and the transformants are screened to obtain the transformants; preferably, the host strain is BL21(DE3) or BL21(DE3) mutant strain; more preferably, the host strain is BL21(DE3);

[0056] (5) Transformers containing nucleic acid fragments encoding amino acid sequences as shown in SEQ ID NO: 1 and SEQ ID NO: 2 were inoculated into culture medium for expansion culture to obtain glycerol bacteria strains.

[0057] In some implementations, the fermentation includes one or more of the following steps:

[0058] (1) Inoculate the glycerol bacteria into a shake flask culture medium and culture overnight to obtain a primary seed culture; preferably, the shake flask culture conditions are 20~32℃, 120~220rpm overnight.

[0059] (2) Inoculate the primary seed culture into the basal culture medium and start the fermentation and feeding program; preferably, the fermentation parameters are 35~37℃, 100~600rpm, aeration 15~25L / h, and tank pressure 0.05MPa; when OD 600nm When the value reaches 1 or above, start replenishing materials in batches, once per hour, in stages;

[0060] (3) Cooling and inducing the growth of bacteria in the logarithmic growth phase; preferably, the OD corresponding to the logarithmic growth phase is... 600nm The value is 8~12, the temperature after cooling is 25~32℃, the inducing agent is IPTG stock solution, and the final IPTG concentration is 0.01~0.1mmol / L;

[0061] (4) Stop fermentation and feeding of the cells during the plateau phase; preferably, the OD corresponding to the plateau phase is... 600nm The value is 15~35.

[0062] In some implementations, the crude purification includes the following steps:

[0063] (1) The obtained fermentation liquid is crushed to obtain a crushed sample; preferably, the crushing method is homogenization crushing, ultrasonic crushing or osmotic shock crushing; more preferably, the crushing is homogenization crushing, and the crushing conditions are crushing 3 times at 800~1000 bar under low temperature conditions.

[0064] (2) Clarify the obtained broken sample to obtain a first-clarified flow-through sample and a first-clarified retentate sample; preferably, the clarification is carried out by ultrafiltration membrane clarification, and the membrane pore size is 0.45μm, 0.2μm or 500KDa; more preferably, the membrane pore size is 500KDa.

[0065] In some specific implementations, the crude purification further includes:

[0066] (a) The obtained primary clarified retentate sample is subjected to secondary crushing to obtain a secondary crushed sample;

[0067] (b) The obtained secondary crushed sample is further clarified, and the secondary clarified flow-through sample is collected;

[0068] (c) The obtained primary clarified flow-through sample and secondary clarified flow-through sample are combined and then concentrated and replaced to obtain a concentrated replacement collection sample; the concentration and replacement is preferably carried out using an ultrafiltration membrane with a pore size of 5 kDa or 3 kDa; more preferably, the pore size of the membrane is 3 kDa.

[0069] In some implementations, the preservation of the purified protein includes the following steps:

[0070] (1) The eluent sample purified in the fourth step is concentrated and replaced to obtain the replaced sample; preferably, the concentration and replacement is carried out by using an ultrafiltration membrane or an ultrafiltration centrifuge tube; more preferably, the pore size of the membrane or centrifuge tube is 5 kDa or 3 kDa; even more preferably, the pore size of the centrifuge tube is 3 kDa.

[0071] (2) The obtained replacement sample was sterilized, filtered and dispensed, and the obtained pure protein sample was stored at -80℃;

[0072] In some specific implementations, the sterilization filtration is performed using a 0.2 μm syringe filter.

[0073] In some embodiments, each elution step in the method includes starting to collect the eluent when the A280 absorption peak rises to 50 mAU after the elution program is started, and stopping the collection when the A280 absorption peak falls to 50 mAU.

[0074] The present invention also provides the application of a purification device in purifying recombinant Staphylococcus aureus leukocidin E, the purification device comprising:

[0075] (Ⅰ) Ni Sepharose HP or TA-Ni FF;

[0076] (II) Bestdex G 25-F or Sephadex G-25F or 3 kDa membrane pack;

[0077] (Ⅲ) Ni Sepharose HP or TA-Ni FF;

[0078] (IV) Phenyl Bestarose HP or Phenyl Sepharose HP.

[0079] In some specific implementations, the purification apparatus includes:

[0080] (I) TA-Ni FF;

[0081] (II) Bestdex G 25-F;

[0082] (III) TA-Ni FF;

[0083] (IV) Phenyl Bestarose HP.

[0084] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0085] The reagents and raw materials used in this invention are all commercially available.

[0086] The significant advancements of this invention are as follows: This invention provides a method for preparing recombinant Staphylococcus aureus leukocyte-killing protein E and its applications. Through codon optimization, amino acid sequence modification, and fermentation condition optimization, this invention achieves a soluble expression yield of 1.84 g target protein / L fermentation broth in a 30L fermenter. After crude purification, one-step replacement, and three-step column purification, the yield of purified rLukE protein reaches approximately 560 mg / L fermentation broth. The purified protein, as detected by SDS-PAGE and capillary electrophoresis, exhibits a purity exceeding 95%, an endotoxin content below 0.1 EU / μg, and an N-terminal sequence completely consistent with the theoretical sequence, indicating that this invention has broad application prospects in the scale-up production of rLukE. Attached Figure Description

[0087] Figure 1 This is an SDS-PAGE electrophoresis pattern of the rLukE expression strain after shake-flask induction to verify its expression. The arrows indicate the target protein. M stands for Marker. Figure 1 Part A and Figure 1 Part B represents the expression of the BL21(DE3) pET28a-His-SUMO-rLukE expression strain after overnight induction at 20℃ and 4h induction at 32℃, respectively: Figure 1 In section A, lane 1 is the crushed supernatant sample; lane 2 is the crushed precipitate sample. Figure 1 In section B, lane 1 is the crushed supernatant sample; lane 2 is the crushed precipitate sample. Figure 1 Part C represents the expression of the BL21(DE3) pET30a-rLukE expression strain after induction at 32℃ for 4 hours: lane 1 is the sample before induction; lane 2 is the sample after 0 hours of induction; lane 3 is the sample after 1 hour of induction; lane 4 is the sample after 2 hours of induction; lane 5 is the sample after 3 hours of induction; lane 6 is the supernatant of the sample after 4 hours of induction; lane 7 is the precipitate of the sample after 4 hours of induction; lane 8 is the precipitate after 4 hours of induction; and lane 9 is the supernatant after 4 hours of induction.

[0088] Figure 2 This is the SDS-PAGE electrophoresis pattern of the sample from the crude purification process of rLukE fermentation broth. M is the marker; lane 1 is the broken supernatant; lane 2 is the broken precipitate; lane 3 is the primary clarification retentate; lanes 4-6 are the primary clarification permeate; lane 7 is the secondary clarification retentate; lane 8 is the secondary clarification permeate; lane 9 is the concentrated permeate; and lanes 10-12 are the concentrated displacement collection solution.

[0089] Figure 3 This is a chromatography chromatogram of the rLukE protein during column chromatography purification; the arrow indicates the target peak. Figure 3 Part a is the first step purification chromatography chromatogram; Figure 3 Part b is the displacement tomography spectrum of the third step; Figure 3 Part c is the chromatogram of the second purification step; Figure 3 Part d is the chromatogram of the fourth purification step; Figure 3 Part E is the SDA-PAGE spectrum of the first step purified chromatographic sample, where M is the marker, lane 1 is the flow-through, lane 2 is the elution, and lane 3 is the salt wash. Figure 3 Part F is the SDS-PAGE pattern of the second step of enzyme replacement digestion sample, where M is the marker, lane1 is before replacement, lane2 is after replacement, and lane3 is after enzyme digestion. Figure 3Part G is the SDS-PAGE pattern of the third purification step, where M is the marker, lane 1 is flow-through, lane 2 is elution, lane 3 is salt washing, and lane 4 is the standard protein. Figure 3 The H section represents the SDS-PAGE chromatogram of the purified sample from the fourth step of chromatography, where M is the marker, lane 1 is the flow-through, lanes 2-3 are the elution, and lane 4 is the water wash.

[0090] Figure 4 This is a chromatography chromatogram of the rLukE protein during column chromatography purification; the arrow indicates the target peak. Figure 4 Part A is the first step purification chromatography chromatogram; Figure 4 Part B is the chromatogram of the second purification step; Figure 4 Part C is the chromatogram of the third purification step; Figure 4 Part D is the chromatogram of the fourth purification step. Figure 4 Part E is the SDA-PAGE chromatogram of the first step purified chromatography sample, where M is the marker, lane 1 is the rLukE standard, and lanes 2-8 are the samples in the purification process. Figure 4 Part F is the SDS-PAGE spectrum of the purified chromatographic sample from the second step, where M is the marker, lane 1 is the loading solution, and lanes 2-10 are the samples from the purification process. Figure 4 Part G is the SDS-PAGE spectrum of the third-step purified chromatography sample, where M is the marker, lane 1 is the loading solution, and lanes 2-12 are the samples in the purification process. Figure 4 The H section represents the SDS-PAGE chromatogram of the purified chromatographic sample from the fourth step, where M is the marker, lane 1 is the loading solution, and lanes 2-13 are the samples from the purification process.

[0091] Figure 5 This is the SDS-PAGE electrophoresis purity analysis chromatogram of rLukE purified protein. M is the marker; lanes 1-3 are for 10 μg loading reduction electrophoresis; lanes 4-6 are for 10 μg loading non-reduction electrophoresis.

[0092] Figure 6 This is the CE-SDS electrophoresis pattern of the purified rLukE protein.

[0093] Figure 7 This is a graph showing the N-terminal sequencing results of the purified rLukE protein. Detailed Implementation

[0094] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0095] The following embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the following embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0096] Example 1: Construction of the rLukE expression line

[0097] Based on the amino acid sequence of the LukE protein published in GenBank BAB57982.1, and referring to the mutation method in Non-Patent Literature 2, amino acids FPRTG at positions 245-249 of the mature LukE protein peptide were replaced with YGNSY, resulting in the rLukE amino acid sequence. Furthermore, a His-SUMO tag was fused to the N-terminus of the rLukE protein. Codon optimization was performed according to the codon preference of *E. coli*, followed by full-fragment chemical synthesis. The synthesized nucleotide sequence was seamlessly cloned into the pET28a vector to obtain the pET28a-His-SUMO-rLukE recombinant plasmid, which was then transformed into DH5α competent cells. Plasmids with correct sequencing were extracted from the bacterial culture using a kit, and the plasmid was then transformed into BL21(DE3) competent cells. Plasmid extraction was performed again, and after correct sequencing, the culture was preserved and named BL21(DE3) pET28a-His-SUMO-rLukE, stored at -80℃.

[0098] Comparative Example: Based on the amino acid sequence of LukE protein published in GenBank BAB57982.1, and referring to the mutation method in Non-Patent Literature 2, amino acids FPRTG at positions 245-249 of the mature LukE protein peptide were replaced with YGNSY, resulting in the rLukE amino acid sequence. Codon optimization was performed according to the codon preference of *E. coli*, and the entire fragment was chemically synthesized after optimization. The synthesized nucleotide sequence was seamlessly cloned into the pET30a vector to obtain the pET30a-rLukE recombinant plasmid, which was then transformed into DH5α competent cells. The plasmid with correct sequencing was extracted using a kit, and the plasmid was then transformed into BL21(DE3) competent cells. The plasmid was extracted again, and after correct sequencing, the strain was preserved and named BL21(DE3) pET30a-rLukE, and stored at -80℃.

[0099] Example 2: Verification of rLukE expression line induction

[0100] The BL21(DE3) pET28a-His-SUMO-rLukE and BL21(DE3) pET30a-rLukE glycerol bacteria from Example 1 were inoculated into solid LB (containing Kana) medium using the three-line inoculation loop method and cultured overnight at 37°C with the medium inverted.

[0101] Select a single colony with good growth from the plate and transfer it to LB (containing Kana) liquid medium (3 mL), and incubate overnight at 32°C and 140 rpm.

[0102] The overnight culture was inoculated into 100 mL of fresh liquid LB (containing Kana) medium at a ratio of 1:500 and incubated at 35°C and 220 rpm until OD500 was reached. 600nm =0.6-0.8, approximately 3-4 h; add IPTG to achieve a final IPTG concentration of 0.1 mmol / L, cool to 20℃ and 32℃ for 4 h, then sample and measure OD. 600nm Absorbance; the sample after induction was ultrasonically disrupted, and the supernatant and precipitate were collected by centrifugation. The sample was processed according to the corresponding OD value. 600nm The solution was reconstituted with 0.85% NaCl to a concentration of 10A. 600nm / tube, and perform reduction SDS-PAGE electrophoresis analysis.

[0103] like Figure 1 As shown in the gel imaging analysis, the soluble expression levels of the target protein in the supernatant samples of the BL21(DE3) pET28a-His-SUMO-rLukE expression strain after overnight induction at 20℃ for 19 h and after induction at 32℃ for 4 h were approximately 71.24% and 41.16%, respectively, indicating that rLukE showed good soluble expression under overnight induction at 20℃. In contrast, the soluble expression level of the target protein in the supernatant sample of the BL21(DE3) pET30a-rLukE expression strain after induction at 32℃ for 4 h was approximately 11.34%, significantly lower than that of the BL21(DE3) pET28a-His-SUMO-rLukE expression strain. These results indicate that the N-terminal fusion of a His-SUMO tag with rLukE can significantly improve the soluble expression level of the target protein.

[0104] Example 3: Fermentation of the rLukE expression strain in a 30L fermenter

[0105] Take 1.0 mL of BL21(DE3) pET28a-His-SUMO-rLukE glycerol bacteria from Example 1 and inoculate it evenly into four 1 L sterile LB broths for culture; add 1 mL of 30 mg / mL kanamycin sulfate to each bottle to a final concentration of 30 μg / mL. Set the shaker temperature to 32°C and the rotation speed to 140 rpm, and incubate overnight. OD 600nm1-3. Add 10L of basal culture medium to the fermenter for sterilization. Before inoculation, add 20 mL of sterile 30 mg / mL kanamycin sulfate (final concentration 30 μg / mL) and sterile inorganic salt solution (K2HPO4, MgSO4). Inoculate under flame-protected aseptic conditions after the temperature reaches the set temperature of 35±0.5℃. The seed culture inoculation ratio is 1:5 (final volume 20 L). Fermentation process parameters are: pH 7.0; rotation speed 200-600 rpm; aeration 15-25 L / H; tank pressure 0.05 MPa; dissolved oxygen ≥30%. Add an antifoaming agent to eliminate foam during the process. Begin batch feeding after 1 hour of fermentation, feeding once per hour, in stages, completing feeding during the logarithmic phase; measure A every hour. 600nm Staining and microscopic examination. When OD 600nm When the pH value reaches around 10, the temperature is lowered to 32℃, and sterile IPTG is added to a final concentration of 0.1 mmol / L to begin induction. Fermentation is completed after 4 hours of induction, fermentation is stopped, and the fermentation broth is discharged and harvested.

[0106] The expression of the samples during the fermentation process is shown in Table 1. The target protein content in the sample after 4 hours of induction was approximately 60.98 g, and the yield reached 3.39 g / L of fermentation broth.

[0107] surface Target protein expression in samples from a 30L fermentation scale fermentation process using the rLukE expression strain.

[0108]

[0109] Note: The target protein content of each sample in Table 1 was estimated based on SDS-PAGE electrophoresis.

[0110] Example 4: Crude purification of rLukE fermentation broth

[0111] Approximately 18 L of fermentation broth was harvested. The bacterial culture was homogenized using a high-pressure homogenizer at 900–1000 bar for three cycles. After homogenization, 600 ml of a 20 mmol / L PB (pH 7.4) buffer solution containing 1 mol / L NaCl and 157 mmol / L imidazole was added to clean the equipment. The homogenized bacterial culture was then collected. A 500 kDa membrane pack (Copatiel, 0.5m) was used. 2 *4) Clarify the lysate and collect the clarified permeate; the clarified retentate is subjected to a second high-pressure homogenization and clarification. The second clarified retentate is washed with an equal volume of 20 mmol / L PB (pH 7.4) buffer containing 1 mol / L NaCl and 157 mmol / L imidazole, and the combined clarified permeate is collected. A 3 kDa ultrafiltration membrane (Cobati, 0.5m) is used. 2*2) The clarified permeate was concentrated. After being concentrated to a certain volume, the concentrated retentate was washed and replaced with an equal volume of 20 mmol / L PB (pH 7.4) buffer containing 1 mol / L NaCl and 157 mmol / L imidazole. Finally, a certain volume of buffer was added to rinse the membrane. This process was repeated 3 times, and the concentrated replacement solution was collected.

[0112] The expression of samples during the crude purification process is as follows: Figure 2 As shown in Table 2, the supernatant sample after 4 hours of induction contained 58.32 g of target protein. After clarification and concentration, the concentrated and replaced liquid contained 33.37 g of target protein, the soluble protein recovery rate was 54.72%, and the target protein yield was 1.85 g / L fermentation broth.

[0113] Table 2. Analysis of target protein content in samples from the crude purification process of rLukE fermentation broth.

[0114]

[0115] Note: The target protein content of each sample in Table 2 was estimated based on SDS-PAGE electrophoresis.

[0116] Example 5: Purification of the concentrated replacement collection solution of rLukE

[0117] 5.1 First step purification

[0118] Equilibrate a TANi FF (NTA) (Chutian, BXK26 / 20) chromatography column for 3 CV using 50 mmol / L PB (pH 7.4) buffer containing 1 mol / L NaCl and 157 mmol / L imidazole. Load the concentrated replacement sample obtained from the crude purification onto the TANi FF (NTA) column, controlling the target protein loading volume to 5–30 mg rLukE / mL gel. Elute with equilibration buffer for 3 CV. Elute with 50 mmol / L PB (pH 7.4) buffer containing 1 mol / L NaCl and 353 mmol / L imidazole for 3 CV, and collect the eluent. Then, wash the column with 0.5 mol / L NaOH solution for 3 CV, and equilibrate with equilibration buffer for 3 CV.

[0119] 5.2 Second step of replacement

[0120] Equilibrate a Bestarose G 25-F (Borglon, BXK50 / 30) column for 1.5 CV until the baseline is flat using 20 mmol / L PB (pH 7.4) containing 0.2 mol / L Arg equilibration buffer. Load approximately 25% CV of the first-step purification flow-through onto the Bestarose G 25-F column, then wash with equilibration buffer for 3 CV. Begin collecting the eluent when the A280 absorption peak is above 50 mAU. After collection, equilibrate the column again for 3 CV. Repeat the above steps until all sample is eluted. Subsequently, wash the column with 0.2 mol / L NaOH solution for 3 CV, and equilibrate with equilibration buffer for 3 CV.

[0121] 5.3 Enzyme digestion

[0122] The replacement samples obtained in the second step were combined and digested with a self-made recombinant UPL1 protease. The digestion conditions were: protein:SUMO enzyme mass ratio = 1000:1, final DTT concentration of 2 mmol / L, and digestion was carried out overnight at room temperature in the dark.

[0123] 5.4 Third step purification

[0124] The enzyme digestion products were adjusted to achieve a final imidazole concentration of 80 mmol / L, a final NaCl concentration of 1 mol / L, and a DTT concentration <2 mmol / L. The TA Ni FF (NTA) (Chutian, BXK26 / 40) column was equilibrated with 50 mmol / L PB (pH 7.4) containing 1 mol / L NaCl and 80 mmol / L imidazole for 3 CV. The adjusted enzyme digestion products were then loaded onto the TA Ni FF (NTA) column, with the target protein loading volume controlled at 5–30 mg rLukE / mL gel. The column was washed with equilibration buffer for 3 CV, and flow-through was collected when the A280 absorption peak was above 50 mAU. After collection, the column was washed with 0.5 mol / L NaOH solution for 3 CV, and then equilibrated with equilibration buffer for 3 CV.

[0125] 5.5 Fourth step purification

[0126] The third-step purification flow-through buffer was adjusted to a conductivity of 120 ms / cm using sample conditioning buffer containing 50 mmol / L PB (pH 7.4) and 4 mol / L NaCl. A PhenylBestarose HP chromatography column (Borglon, XK16 / 40) was equilibrated for 3 CVs using equilibration buffer containing 50 mmol / L PB (pH 7.4) and 1.5 mol / L NaCl. The adjusted third-step purification flow-through buffer was then loaded onto the column, with the target protein loading volume controlled at 5–10 mg rLukE / mL gel. The column was washed with equilibration buffer for 3 CVs, followed by elution with 50 mmol / L PB (pH 7.4) and 0.05 mol / L Arg for 8 CVs. The eluent was collected. The column was then washed with water for 3 CVs, followed by alkaline washing with 0.5 mol / L NaOH for 3 CVs, and then equilibrated for 3 CVs.

[0127] Four-step column chromatography chromatography pattern as follows Figure 3 As shown in Table 3, the purity and recovery rate of the samples at each step are as follows: the purification recovery rate of each step is above 70%, and the sample purity is above 95%.

[0128] Table 3 Purity and recovery rate of target protein during rLukE purification process

[0129]

[0130] Note: The target protein content and purity of each sample in Table 3 were estimated based on SDS-PAGE electrophoresis, and the endotoxin content was determined by the Limulus Amebocyte Lysate (LAL) reagent method.

[0131] The following are comparison examples:

[0132] Enzyme digestion: The concentrated replacement collection solution obtained from crude purification was digested with the self-made recombinant UPL1 protease. The digestion conditions were: protein:SUMO enzyme mass ratio = 1000:1, final DTT concentration of 2 mmol / L, and digestion was carried out overnight at room temperature in the dark.

[0133] Step 1 Purification: Equilibrate the DEAE Bestarose FF column (Borglon, XK10 / 30) with 50 mmol / L PB (pH 7.4) buffer for 3CV. Load the concentrated replacement collection solution after enzyme digestion onto the column. After loading, wash the 3CV with 50 mmol / L PB (pH 7.4) buffer and collect the loading flow-through and the washing flow-through. Wash the column with 1.5 mol / L NaCl solution for 3CV, then wash the 3CV with 0.5 mol / L NaOH solution, and then equilibrate the 3CV.

[0134] Second purification step: Equilibrate the Monemix HC30 SP column (Saifen Technology, XK10 / 30) with 50 mmol / L PB (pH 7.4) buffer for 3 CV. Load the purified flow-through solution from step 1 onto the column. After loading, wash with 50 mmol / L PB (pH 7.4) buffer for 3 CV, then perform linear elution with 50 mmol / L PB (pH 7.4) buffer containing 1 mol / L NaCl for 20 CV. Collect the eluent in separate tubes. Wash the column with 1.5 mol / L NaCl solution for 3 CV, then wash with 0.5 mol / L NaOH for 3 CV, and then equilibrate with 3 CV.

[0135] Step 3 purification: Equilibrate the PhenylBestarose HP chromatography column (Borglon, XK10 / 30) for 3 CV using 50 mmol / L PB (pH 7.4) buffer containing 1 mol / L NaCl. Combine the eluent from Step 2 purification in tubes B10-B6, adjust the conductivity of the combined eluent with 50 mmol / L PB (pH 7.4) buffer containing 4 mol / L NaCl until it is consistent with the loading buffer, and then load it onto the chromatography column. Elute for 3 CV using 50 mmol / L PB (pH 7.4) buffer containing 1 mol / L NaCl, followed by linear elution for 20 CV using 50 mmol / L PB (pH 7.4) buffer, and collect the eluent in separate tubes. Wash the chromatography column with purified water for 3 CV, then wash with 0.5 mol / L NaOH for 3 CV, and then equilibrate for 3 CV.

[0136] Step 4 Purification: Equilibrate the Polar MC30HIC Butyl chromatography column (Saifen Technology, XK10 / 30) for 3 CV using 50 mmol / L PB (pH 7.4) buffer containing 1.5 mol / L NaCl. Combine the eluent from Step 3 (tubes B7-C4), adjust the conductivity of the combined eluent with 50 mmol / L PB (pH 7.4) buffer containing 4 mol / L NaCl until it is consistent with the loading buffer, and then load the sample onto the chromatography column. Elute for 3 CV using 50 mmol / L PB (pH 7.4) buffer containing 1.5 mol / L NaCl, followed by linear elution for 20 CV using 50 mmol / L PB (pH 7.4) buffer. Collect the eluent in separate tubes. Wash the chromatography column with purified water for 3 CV, then wash with 0.5 mol / L NaOH for 3 CV, and then equilibrate for 3 CV.

[0137] Four-step column chromatography chromatography pattern as follows Figure 4 As shown in Table 4, the purity and recovery rate of samples at each step are as follows. After the four-step column chromatography process was explored and developed, there are still obvious impurities.

[0138] Table 4 Purity and recovery rate of target protein during rLukE purification process

[0139]

[0140] Note: The target protein content and purity of each sample in Table 4 were estimated based on SDS-PAGE electrophoresis, and the endotoxin content was determined by the Limulus Amebocyte Lysate (LIL) method.

[0141] Example 6: Preparation of purified rLukE protein

[0142] The 3 kDa ultrafiltration membrane pack (Cobati, 0.1m) was replaced with 0.2 mol / L borate buffer (pH 8.4) containing 0.85% NaCl. 2 *4) Perform a rinse, then concentrate the eluent from Example 5.5. When the concentration reaches a certain volume, begin the washing and filtration process until the pH and conductivity of the concentrated permeate are essentially the same as the replacement solution, at which point the replacement is complete. Continue concentrating to a certain volume, and when the OD... 280nm When the concentration value is greater than 10, concentration is stopped. After concentration, the protein is sterilized and filtered through a 0.2μm syringe filter to obtain purified protein; the purified protein is then aliquoted and stored at -80℃.

[0143] Calculations show that rLukE produces approximately 560 mg / L of purified protein in the fermentation broth.

[0144] Example 7: Quantitative Analysis of Purified Proteins (rLukE)

[0145] 7.1 Determination of Endotoxin Content by Dynamic Turbidimetric Method

[0146] Prepare four sets of bacterial endotoxin working standard solutions with endotoxin concentrations of 10 EU / mL, 2.5 EU / mL, 0.625 EU / mL, and 0.156 EU / mL, respectively. Prepare three replicates for each concentration, and include a negative control. Dilute the samples with water for bacterial endotoxin detection to a concentration between 0.156 and 10 EU / mL, preparing two replicates of the test solution. The dilution factor of the test solution should not exceed the maximum effective dilution factor. Set up a positive spiking solution and use Limulus Amebocyte Lysate (LAL) reagent to detect the samples, plotting a standard curve. The absolute value of the correlation coefficient of the standard curve should be ≥0.980, the spiked recovery rate should be between 50% and 200%, and the endotoxin concentration in the negative control should be below the detection limit for the sample detection data to be valid.

[0147] The results showed that the rLukE endotoxin content was 0.05 EU / μg, indicating that the four-step purification method could effectively remove the endotoxin.

[0148] 7.2 Determination of purity by SDS-PAGE method

[0149] 10 μg of purified rLukE protein was subjected to reduced and non-reduced SDS-PAGE electrophoresis (separating gel concentration 15%) at 150 V for 60 min until the bromophenol blue band reached the bottom of the separating gel. The gel was then peeled off and stained using a gel staining and destaining instrument. SDS-PAGE pattern analysis was performed using a gel imaging system.

[0150] The results are as follows Figure 5 As shown, under reducing electrophoresis conditions, the purity of rLukE protamine is 100%; under non-reducing electrophoresis conditions, the purity of rLukE protamine is 100%.

[0151] 7.3 Purity determination by CE-SDS method

[0152] Desalted the purified rLukE protein with purified water. Then, 5 μL of sample, 90 μL of sample buffer (SCIEX, A10663), and 2 μL of internal standard (SCIEX, A26487) were placed in a 0.2 ml centrifuge tube. After mixing, the mixture was centrifuged at 5200 rpm for 1 min, incubated in a 70°C water bath for 10 min, and cooled for 3 min. 100 μL of the mixture was then transferred to a microsample tube, which was then placed in a general-purpose bottle and capped. The IgG Purity mode was selected on the capillary electrophoresis instrument (Beckman, PA800plus), and the program was started.

[0153] The results are as follows Figure 6 As shown, the purity of rLukE protamine is 98.84%.

[0154] 7.4 N-terminal sequencing of rLukE protein

[0155] The purified rLukE protein was sent to Suzhou Wenli Biotechnology Co., Ltd. for analysis of the first 15 amino acids at the N-terminus using the Edman degradation method.

[0156] like Figure 7 As shown, the results indicate that the first 15 amino acids at the N-terminus of the purified rLukE protein are Asn-Thr-Asn-Ile-Glu-Asn-Ile-Gly-Asp-Gly-Ala-Glu-Val-Ile-Lys (SEQ ID NO: 3), which is completely consistent with the theoretical sequence, indicating that the N-terminal fused His-SUMO tag has been completely removed.

Claims

1. A method for preparing recombinant Staphylococcus aureus leukocidin E, characterized in that, The method includes purification, which includes the following steps: (1) First step purification: collect the first step purification eluent sample, wherein the chromatography column used for the first step purification is NiSepharose HP or TA-Ni FF; (2) Perform a second-step desalting replacement on the eluent sample obtained in step (1) and collect the second-step replacement sample. The second-step desalting replacement method is to use a Bestdex G 25-F or Sephadex G-25F desalting column, ultrafiltration membrane pack or dialysis. (3) The replacement sample obtained in step (2) is purified in the third step, and the purified flow-through sample is collected. The chromatography column used in the third step purification is Ni Sepharose HP or TA-Ni FF. (4) The flow-through sample obtained in step (3) is purified in the fourth step, and the eluent sample of the fourth step purification is collected. The chromatography column used in the fourth step purification is Phenyl Bestarose HP or Phenyl Sepharose HP.

2. The method as described in claim 1, characterized in that, The method includes one or more of the following conditions: （a） The chromatography column used for the first purification step is TA-NiFF; the preferred chromatography conditions for the first purification step are: Equilibration buffer: 20-50 mmol / L PB at pH 7.4 containing 1 mol / L NaCl and 157 mmol / L imidazole; Elution buffer: 20-50 mmol / L PB at pH 7.4 containing 1 mol / L NaCl and 353 mmol / L imidazole; Alkaline washing buffer: 0.5 mol / L NaOH solution; Column chromatography procedure: Equilibrate 3CV, load sample, wash 3~10CV, elute 3CV, wash with alkali 3CV, reequilibrate 3CV; Sample loading volume: 5~30 mg target protein / ml packing material; （b） The desalting column used in the second step of desalting replacement was a Bestdex G 25-F; the conditions for the second step of desalting replacement were preferably selected from: Replacement buffer: 20–50 mmol / L PB at pH 7.4, containing 0.05–0.2 mol / L argon; Alkaline wash buffer: 0.2M NaOH solution; Column chromatography procedure: Equilibrate to 1.5 CV, load sample, elute to 3 CV, wash with alkali to 3 CV, reequilibrate to 3 CV; Sample loading: 25% of column volume; （c） The chromatography column used for the third purification step is TA-NiFF; the preferred chromatography conditions for the third purification step are: Equilibration buffer: 20-50 mmol / L PB at pH 7.4, containing 1 mol / L NaCl and 40-80 mmol / L imidazole; Sample conditioning solution: 20-50 mmol / L PB at pH 7.4, containing 4 mol / L NaCl; 20-50 mmol / L PB at pH 7.4, containing 1 mol / L NaCl and 1 mol / L imidazole. Alkaline wash buffer: 0.5M NaOH solution; Column chromatography procedure: Equilibrate to 3 CV, load sample, elute 3-8 CV, wash with alkali for 3 CV, then equilibrate to 3 CV; Loading volume: 5~30 mg target protein / ml filler; （d） The chromatography column used for the fourth purification step was Phenyl Bestarose HP; the preferred chromatography conditions for the fourth purification step were: Equilibration buffer: 20-50 mmol / L PB at pH 7.4, containing 1.5 mol / L NaCl; Sample conditioning solution: 20-50 mmol / L PB at pH 7.4, containing 4 mol / L NaCl; Elution buffer: 20–50 mmol / L PB at pH 7.4, containing 0.05–0.2 mol / L Arg; Wash buffer: Water for injection; Alkaline wash buffer: 0.5M NaOH solution; Column chromatography procedure: Equilibration 3CV, sample loading, elution 3CV, elution 8CV, water washing 3CV, alkali washing 3CV, reequilibration 3CV; Sample loading volume: 5~10 mg target protein / ml packing material; Preferably, The method further includes the following steps: The eluent sample obtained in step (2) is subjected to enzyme digestion, and the enzyme digestion product sample is collected. The enzyme digestion product sample is then purified in the third step described in step (3). The preferred enzyme digestion conditions are a protein:SUMO enzyme mass ratio of 500-1000:1, a final DTT concentration of 2-5 mmol / L, and digestion at room temperature in the dark for 6-30 hours.

3. The method as described in claim 1 or 2, characterized in that, The method further includes one or more of the following steps: construction of the expression strain, fermentation, crude purification, and preservation of purified protein, wherein the crude purification is to separate recombinant Staphylococcus aureus leukocidin E from the bacterial cells.

4. The method as described in claim 3, characterized in that, The construction of the expression strain includes one or more of the following steps: (1) Obtain a nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 1 with codon optimization; preferably, the codon optimization is performed according to the preferences of Escherichia coli; (2) Synthesize a nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 1; (3) Insert the nucleic acid encoding the amino acid sequence shown in SEQ ID NO: 1 into the expression vector through the restriction enzyme site to obtain a recombinant plasmid; preferably, the expression vector is pET28a or pET30a; more preferably, the expression vector is pET28a; (4) Transform a recombinant plasmid containing a nucleic acid fragment encoding the amino acid sequence shown in SEQ ID NO: 1 into a host strain and screen to obtain transformants; preferably, the host strain is BL21(DE3) or a BL21(DE3) mutant strain; more preferably, the host strain is BL21(DE3); (5) Inoculate the bacterial culture containing the nucleic acid fragment encoding the amino acid sequence shown in SEQ ID NO: 1 into the culture medium for expansion culture to obtain the glycerol bacteria strain.

5. The method as described in claim 3, characterized in that, The fermentation includes one or more of the following steps: (1) Inoculate the glycerol bacteria into a shake flask culture medium and culture overnight to obtain a primary seed culture; preferably, the shake flask culture conditions are 20~32℃, 120~220rpm overnight. (2) Inoculate the primary seed culture into the basal culture medium and start the fermentation and feeding program; preferably, the fermentation parameters are 35~37℃, 100~600rpm, aeration 15~25L / h, and tank pressure 0.05MPa; when OD 600nm When the value reaches 1 or above, start replenishing materials in batches, once per hour, in stages; (3) Cooling and inducing the growth of bacteria in the logarithmic growth phase; preferably, the OD corresponding to the logarithmic growth phase is... 600nm The value is 8~12, the temperature after cooling is 25~32℃, the inducing agent is IPTG stock solution, and the final IPTG concentration is 0.01~0.1mmol / L; (4) Stop fermentation and feeding of the cells during the plateau phase; preferably, the OD corresponding to the plateau phase is... 600nm The value is 15~35.

6. The method as described in claim 3, characterized in that, The crude purification includes the following steps: (1) The obtained fermentation liquid is crushed to obtain a crushed sample; preferably, the crushing method is homogenization crushing, ultrasonic crushing or osmotic shock crushing; more preferably, the crushing is homogenization crushing, and the crushing conditions are crushing 3 times at 800~1000 bar under low temperature conditions. (2) Clarify the obtained broken sample to obtain a first-clarified flow-through sample and a first-clarified retentate sample; preferably, the clarification is carried out by ultrafiltration membrane clarification, and the membrane pore size is 0.45μm, 0.2μm or 500KDa; more preferably, the membrane pore size is 500KDa.

7. The method as described in claim 6, characterized in that, The crude purification also includes: (a) The obtained primary clarified retentate sample is subjected to secondary crushing to obtain a secondary crushed sample; (b) The obtained secondary crushed sample is further clarified, and the secondary clarified flow-through sample is collected; (c) The obtained primary clarified flow-through sample and secondary clarified flow-through sample are combined and then concentrated and replaced to obtain a concentrated replacement collection sample; the concentration and replacement is preferably carried out using an ultrafiltration membrane with a pore size of 5 kDa or 3 kDa; more preferably, the pore size of the membrane is 3 kDa.

8. The method as described in claim 3, characterized in that, The preservation of the purified protein includes the following steps: (1) The eluent sample purified in the fourth step is concentrated and replaced to obtain the replaced sample; preferably, the concentration and replacement is carried out by using an ultrafiltration membrane or an ultrafiltration centrifuge tube; more preferably, the pore size of the membrane or centrifuge tube is 5 kDa or 3 kDa; even more preferably, the pore size of the centrifuge tube is 3 kDa. (2) The obtained replacement sample was sterilized, filtered and dispensed, and the obtained pure protein sample was stored at -80℃; Preferably, the sterilization filtration is performed using a 0.2 μm needle filter.

9. The method as described in claim 1 or 2, characterized in that, Each elution step in the method includes starting to collect the eluent when the A280 absorption peak rises to 50 mAU after the elution program is started, and stopping the collection when the A280 absorption peak drops to 50 mAU.

10. The application of a purification device in purifying recombinant Staphylococcus aureus leukocidin E, characterized in that, The purification device includes: (I) Ni Sepharose HP or TA-Ni FF, preferably TA-Ni FF; (II) Bestdex G 25-F, Sephadex G-25F or 3 kDa membrane pack, preferably Bestdex G 25-F; (III) Ni Sepharose HP or TA-Ni FF, preferably TA-Ni FF; (Ⅳ) Phenyl Bestarose HP or Phenyl Sepharose HP, preferably Phenyl Bestarose HP.

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