A method for purifying protein

Abstract

The present invention discloses a method for purifying a protein, which comprises operably connecting a nucleic acid encoding a signal peptide, a tag, and a protease cleavage site to a nucleic acid sequence encoding a target protein, thereby generating an expression vector encoding a fusion protein; and introducing the expression vector into a host cell. The present invention also provides a protein constructed by the above method, a nucleic acid encoding a protein, an expression vector, and a host cell. Experiments have shown that the protein purified by the method of the present invention has a high expression amount and purity.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine, and in particular relates to a method for purifying protein. Background Art

[0002] At present, protein purification is a key link in genetic engineering and protein engineering, both in laboratory scale and in industrial production. According to statistics, the separation and purification cost of genetic engineering products accounts for about 60%-80% of their total cost. Efficient prokaryotic, fungal and eukaryotic cell protein expression systems have been developed. However, no matter which system is used for expression, the target protein may interact with other proteins in tissues and cells to form a complex mixture, making separation and purification very difficult.

[0003] Therefore, providing a method for purifying protein with high purity and simple operation is an urgent problem to be solved in the art. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides a method for purifying protein.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] A first aspect of the present invention provides a method for purifying a protein, the method comprising:

[0007] (1) operably linking a nucleic acid encoding a signal peptide, a tag, and a protease cleavage site to a nucleic acid sequence encoding a target protein, thereby generating an expression vector encoding a fusion protein;

[0008] (2) Introducing the expression vector into host cells.

[0009] Furthermore, the signal peptide, tag, and protease cleavage site described in step (1) are sequentially connected to the target protein.

[0010] Furthermore, the signal peptide includes CD33 signal peptide, human growth hormone signal peptide, hIG1 Kappa light chain signal peptide, β2 microglobulin signal peptide or IL2 signal peptide.

[0011] Furthermore, the signal peptide is selected from CD33 signal peptide.

[0012] Furthermore, the CD33 signal peptide sequence is shown as SEQ ID NO.3.

[0013] Furthermore, the tag includes an HA tag, a His tag, a Flag tag, a c-Myc tag, a V5 tag or a C9 tag.

[0014] Furthermore, the tag is selected from a His tag.

[0015] Furthermore, the His tag includes 1-20 histidine residues.

[0016] Furthermore, the His tag includes 6-10 histidine residues.

[0017] Furthermore, the His tag is selected from 10 histidine residues.

[0018] Furthermore, the His-tag also includes (His x -R y ) z , where R is a non-His residue, and x, y, and z are positive integers.

[0019] Furthermore, the R is an alanine residue.

[0020] Furthermore, the His tag sequence is shown as SEQ ID NO.4.

[0021] Furthermore, the protease cleavage site is shown in SEQ ID NO.5.

[0022] Furthermore, the target protein is selected from KLKB1 protein.

[0023] Furthermore, the KLKB1 protein includes full-length, partially truncated or truncated KLKB1 protein.

[0024] Furthermore, the KLKB1 protein is selected from truncated KLKB1 proteins.

[0025] Furthermore, the truncated KLKB1 protein sequence is shown in SEQ ID NO.6.

[0026] Furthermore, the host cell in step (2) includes a prokaryotic host cell and a eukaryotic host cell.

[0027] Furthermore, the host cell is selected from eukaryotic host cells.

[0028] Furthermore, the eukaryotic host cells include mammalian cells, insects, and yeast.

[0029] Furthermore, the eukaryotic host cell is selected from mammalian cells.

[0030] Furthermore, the mammalian cells include rodent cells and human cells.

[0031] Furthermore, the mammalian cells are selected from human cells.

[0032] Furthermore, the human cells include Hela, HEK293, H9, Per.C6, and Jurkat cells.

[0033] Furthermore, the human cells are selected from HEK293 cells.

[0034] The second aspect of the present invention provides a protein, which is prepared by the method described in the first aspect of the present invention.

[0035] The third aspect of the present invention provides a nucleic acid comprising a nucleotide sequence encoding the protein described in the second aspect of the present invention.

[0036] The fourth aspect of the present invention provides an expression vector, which comprises the nucleic acid described in the third aspect of the present invention.

[0037] The fifth aspect of the present invention provides a host cell, wherein the host cell comprises the expression vector described in the fourth aspect of the present invention.

[0038] Advantages and beneficial effects of the present invention:

[0039] The present invention sequentially connects the CD33 signal peptide, the His tag, the protease cleavage site and the KLKB1 protein, and purifies the fused protein. The results show that the protein purified by the protein purification method provided by the present invention has a high expression amount and purity, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a graph showing the results of electrophoresis detection after protein expression and purification of sequence 1;

[0041] Figure 2 This is a graph showing the results of electrophoresis detection after protein expression and purification of sequence 2;

[0042] Figure 3 This is a graph showing the results of electrophoresis detection of sequence 3 protein after expression and purification;

[0043] Figure 4 This is a graph showing the results of electrophoresis detection of sequence 4 protein after expression and purification;

[0044] Figure 5 This is the result of electrophoresis detection of sequence 5 protein after expression and purification. DETAILED DESCRIPTION

[0045] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0046] The present invention provides a method for purifying a protein, the method comprising:

[0047] (1) operably linking a nucleic acid encoding a signal peptide, a tag, and a protease cleavage site to a nucleic acid sequence encoding a target protein, thereby generating an expression vector encoding a fusion protein;

[0048] (2) Introducing the expression vector into host cells.

[0049] Furthermore, the signal peptide, tag, and protease cleavage site described in step (1) are sequentially connected to the target protein.

[0050] In the present invention, the signal peptide (also referred to as signal sequence, leader sequence or leader peptide) is structurally characterized by a hydrophobic amino acid segment that has a tendency to form a single alpha-helix. This hydrophobic segment is usually immediately followed by a shorter segment rich in positively charged amino acids (especially lysine). The signal peptide cut from the mature polypeptide usually ends at a stretch of amino acids recognized and cut by a signal peptidase. The signal peptide that directs the insertion of the polypeptide gene product into the membrane, sometimes referred to as a signal anchor sequence, may lack an amino acid sequence cut by a signal peptidase, and in this case remains in the polypeptide gene product. The signal peptide can usually be functionally characterized by directing the polypeptide to be transported outside the cytoplasm during or after translation, and for example, through the plasma membrane of a prokaryotic organism (or the inner membrane of a gram-negative bacterium such as Escherichia coli), or the ability to be transported to the endoplasmic reticulum of a eukaryotic cell.

[0051] The signal peptide of the present invention includes but is not limited to human growth hormone signal peptide, hIG1Kappa light chain signal peptide, β2 microglobulin signal peptide, CD33 signal peptide or IL2 signal peptide, and the length of the signal peptide can be between 3-33, 3-10, 10-30, 10-20, 15-30 or 20-30 amino acids.

[0052] In a specific embodiment of the present invention, the signal peptide is selected from CD33 signal peptide. In the present invention, CD33 signal peptide can be used interchangeably with SP1.

[0053] In the present invention, a tag refers to any chemical structure that can be used as a marker, and the tag of the present invention includes but is not limited to a histidine (His) tag (especially a polyhistidine tag), an arginine tag (especially a polyarginine tag), a peptide substrate of an antibody, a chitin binding domain, an RNAse S peptide, protein A, β-galactosidase, a Flag tag, a Strep II tag, a streptavidin binding peptide (SBP) tag, a calmodulin binding peptide (CBP), a glutathione S-transferase (GST), a maltose binding protein (MBP), an S-tag, an HA tag or a c-Myc tag, a V5 tag, a C9 tag or any other known tag that can be used for efficient purification of a protein fused thereto.

[0054] In embodiments of the present invention, the tag typically has a low molecular weight of about 1-3 kDa. Exemplary non-limiting low molecular weight tags include HA tags, His tags, Flag tags, c-Myc tags, V5 tags or C9 tags.

[0055] In an embodiment of the present invention, the tag is selected from a His tag.

[0056] The His tag in the present invention preferably includes 1-20 histidine residues, and is even more preferably a 1-His, 2-His, 3-His, 4-His, 5-His, 6-His, 7-His, 8-His, 9-His, 10-His, 11-His, 12-His, 13-His, 14-His, 15-His, 16-His, 17-His, 18-His, 19-His or 20-His tag sequence.

[0057] In one embodiment of the present invention, the His tag comprises 6-10 histidine residues, preferably 6-His, 7-His, 8-His, 9-His, 10-His.

[0058] In a preferred embodiment of the present invention, the His tag is selected from 10 histidine residues, ie, 10-His.

[0059] In some embodiments, the tag includes other residues besides His. See, for example, Knecht (2008) J. Mol. Recognit. 22: 270-279; Charlton (2008) Methods in Molecular Biology 421: 25-36. In some embodiments, the His-tag may include (His x -R y ) z , where R is a non-His residue, and x, y, and z are positive integers.

[0060] In a particular embodiment of the invention, R is an alanine residue (A).

[0061] In some embodiments, the His tag can be used in combination with one or more different affinity tags. In some embodiments, the His tag can be located at or near the N-terminus of the protein of interest, represented as HHHHHHHHHHA. In other embodiments, the His tag can be located at or near the C-terminus of the protein of interest, represented as AHHHHHHHHHH.

[0062] In a specific embodiment of the invention, the His tag is located at or near the N-terminus of the protein of interest.

[0063] In the present invention, protease cleavage sites include, but are not limited to, IEGR, DDDDK, LVPRGS, ENLYFQG, and LEVLFQGP.

[0064] In a specific embodiment of the present invention, the protease cleavage site is selected from LEVLFQGP (SEQ ID NO. 5).

[0065] Furthermore, the target protein of the present invention is selected from KLKB1 protein.

[0066] The KLKB1 protein may include a full-length, partially truncated or truncated KLKB1 protein, or any fragment thereof that includes one or more epitopes capable of eliciting an immune response to KLKB1.

[0067] In an embodiment of the present invention, the KLKB1 protein is selected from truncated KLKB1 proteins.

[0068] In a preferred embodiment of the present invention, the target protein comprises SEQ ID NO.6 or an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.6. In another preferred embodiment of the present invention, the target protein comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.6, which is modified so that the amino acid sequence undergoes amino acid substitutions and deletions of residues in non-conserved regions, wherein the positions correspond to equivalent positions within the amino acid sequence of SEQ ID NO.6 that is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO.6.

[0069] In the present invention, deletion is the removal of one or more amino acid residues from a protein sequence. Typically, no more than about 1 to 6 residues (e.g., 1 to 4 residues) are deleted at any one site within a protein molecule.

[0070] In a specific embodiment of the present invention, the truncated KLKB1 protein sequence is shown as SEQ ID NO.6.

[0071] In a specific embodiment of the present invention, the protein is connected in sequence with SP1 (SEQ ID NO.3)-His tag (SEQ ID NO.4)-protease cleavage site (SEQ ID NO.5)-KLKB1 protein sequence (SEQ ID NO.6) to form sequence 1 (SEQ ID NO.7).

[0072] In the present invention, host cells are cells that parasitize other microorganisms or genes to provide nutrition, and refer to cells in which vectors are transformed into host cells to exert various gene or molecular effects in host cells, which is the same as the protein expression system in the embodiments of the present invention.

[0073] In the present invention, transformation refers to the phenomenon of introducing deoxyribonucleic acid (DNA) into a host to replicate the deoxyribonucleic acid as a chromosomal factor or by chromosome integration, and introducing external deoxyribonucleic acid into cells to artificially cause genetic changes. The above-mentioned transformation methods include CaCl2 precipitation method, Hanahan method using a reducing substance called dimethyl sulfoxide (DMSO) in the CaCl2 method to improve efficiency, electroporation, calcium phosphate precipitation method, protoplast fusion method, stirring method using silicon carbide fiber, Agrobacterium-mediated transformation method, transformation method using PEG, dextran sulfate, liposome and drying / inhibition-mediated transformation method, but are not limited to these.

[0074] The growth medium, growth temperature and other growth conditions can be selected according to the type of host cell used. Various methods for harvesting host cells from cell cultures expressing proteins are well known to those skilled in the art, and include, for example, centrifugation of a culture medium containing host cells, and filtration of a culture medium containing host cells. Typically, the cells are then lysed, either by chemical or mechanical methods, or by a combination thereof.

[0075] Certain changes may be made to the gene function of host cells containing inducible expression constructs to facilitate efficient and homogenous induction of the host cell population by the inducing agent. Preferably, the combination of expression construct, host cell genotype and inducing conditions results in at least 75% (more preferably at least 85%, most preferably at least 95%) of the cells in the culture expressing the gene product from each induced promoter.

[0076] In the present invention, host cells include prokaryotic host cells and eukaryotic host cells.

[0077] Among them, prokaryotic host cells include, but are not limited to, archaea (such as Haloferax volcanii, Sulfolobus solfataricus), Gram-positive bacteria (such as Bacillus subtilis, Bacillus licheniformis, Brevibacillus choshinensis, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis or Streptomyces lividans), or Gram-negative bacteria, including α-Proteobacteria

[0078] Alphaproteobacteria (Agrobacterium tumefaciens, Caulobacter crescentus, Rhodobacter sphaeroides, and Sinorhizobium meliloti), Betaproteobacteria (Alcaligenes eutrophus), and Gammaproteobacteria (Acinetobacter calcoaceticusus, Azotobacter vinelandii, Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida). putida), gammaproteobacteria of the family Enterobacteriaceae, such as Enterobacter, Erwinia, Escherichia (including Escherichia coli), Klebsiella, Proteus, Salmonella (including Salmonella typhimurium), Serratia (including Serratia marcescans), and Shigella.

[0079] In an embodiment of the present invention, the host cell is selected from eukaryotic host cells.

[0080] The eukaryotic host cells include mammalian cells, insects, and yeast.

[0081] The yeast includes but is not limited to Pichia pastoris, Kluyveromyces lactis, Saccharomyces cerevisiae, and Schizosaccharomyces pombe.

[0082] Insects include but are not limited to Sf9, Mimic Sf9, Sf21, HighFive.

[0083] In a preferred embodiment of the present invention, the eukaryotic host cell is selected from mammalian cells.

[0084] Mammalian cells include rodent cells and human cells.

[0085] Among them, rodent cells include but are not limited to mouse NIH3T3, NS0 and C127 cells, COS 1, COS 7 and CV1, L cells, sarcoma cells, Bowes melanoma cells and Chinese hamster ovary (CHO) cells.

[0086] Human cells include, but are not limited to, Hela, HEK293, H9, Per.C6 or Jurkat cells.

[0087] In the most preferred embodiment of the present invention, the mammalian cells are selected from human cells.

[0088] In a specific embodiment of the present invention, the human cells are selected from HEK293 cells.

[0089] In the present invention, purification refers to separating the target protein from one or more contaminants. Contaminants are any substances different from the protein or target protein or its conjugates (e.g., bioconjugates). Contaminants can be, for example, cell debris, nucleic acids, lipids, proteins other than fusion proteins or target proteins, polysaccharides, and other cellular components.

[0090] The methods for recovering and purifying the proteins linked by the methods described in the present application include, but are not limited to, affinity chromatography using microproteins (e.g., protein A or protein G affinity chromatography), affinity chromatography using recombinant proteins as ligands (e.g., single-chain Fv as ligands, e.g., Kappa selection), ion exchange chromatography (e.g., cation exchange (carboxymethyl resin)), anion exchange (aminoethyl resin) and mixed mode exchange), thiophilic adsorption (e.g., with β-mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., with phenyl-agarose, aza-arenophilic resins or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., with Ni(II)- and Cu(II)-affinity materials), size exclusion chromatography and electrophoretic methods (e.g., gel electrophoresis, capillary electrophoresis). These methods can be independently combined in different embodiments reported in the present invention.

[0091] In an embodiment of the invention, the method is selected from gel electrophoresis.

[0092] The present invention provides a nucleic acid comprising a nucleotide sequence encoding the above protein.

[0093] In the present invention, nucleic acid and nucleic acid molecule can be used interchangeably. The nucleic acid of the present invention also includes complementary sequences of the above nucleic acids, such as antisense nucleotides or probes.

[0094] Nucleic acid of the present invention can be in the form of RNA (e.g., mRNA) or DNA, including, for example, cDNA, synthetic DNA, or genomic DNA. These nucleic acids can be obtained by cloning, chemical synthesis techniques, or a combination thereof. For example, solid phase phosphoramidite chemical synthesis techniques can be used to chemically synthesize, obtain from genomes or cDNA libraries, or separate from organisms to prepare these nucleic acids. RNA molecules can usually be produced by transcribing DNA sequences in vitro or in vivo.

[0095] The nucleic acid may be double-stranded or single-stranded. Single-stranded DNA may be a coding strand (also called a sense strand) or a non-coding strand (also called an antisense strand).

[0096] The nucleic acid encoding the protein of the present invention may be identical to the coding sequence of one or more nucleic acids of the present invention.

[0097] The present invention provides an expression vector, which comprises the above nucleic acid.

[0098] In the present invention, expression vector and vector are used interchangeably, and are expression vectors that can express target proteins in appropriate host cells, and refer to nucleic acid constructs that are operably linked to necessary regulatory elements to express nucleic acid inserts. The above-mentioned vectors include plasmids, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses or other vectors commonly used in the art.

[0099] Among them, plasmids include but are not limited to pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series and pUC19.

[0100] Bacteriophages include, but are not limited to, λgt4λB, λ-Charon, λΔz1, and M1.

[0101] Viruses include but are not limited to CMV and SV40.

[0102] Typically, the vector may contain one or more origins of replication (ori) and a genetic system for cloning or expression, one or more markers for selection in a host, such as antibiotic resistance and one or more expression cassettes. Examples of suitable origins of replication include, for example, full-length ColE1, truncated forms thereof, such as those present on pUC plasmids, SV40 virus and M13 phage origins of replication. Non-limiting examples of selectable markers include ampicillin, chloramphenicol, tetracycline, kanamycin, dhfr, gpt, neomycin, hygromycin, blasticidin or geneticin.

[0103] In addition, the vector comprises a regulatory sequence operably linked to the nucleic acid of the present invention. The vector of the present invention is capable of directing the replication and expression of the nucleic acid provided by the present invention, such as a nucleic acid comprising a nucleotide sequence encoding a protein provided by the present invention.

[0104] The nucleic acids and / or vectors described above can be designed for introduction into cells by, for example, non-chemical methods (electroporation, sonoporation, optical transfection, gene electrotransfer, hydrodynamic delivery, or naturally occurring transformation when cells are contacted with the nucleic acids of the invention), chemical-based methods (calcium phosphate, DMSO, PEG, liposomes, DEAE-dextrane, polyethyleneimine, nucleofection, etc.), particle-based methods (gene gun, magnetofection, impalefection), phage or phagemid vector-based methods, and viral methods. For example, expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, Semliki Forest Virus, or bovine papilloma virus can be used to deliver nucleic acids to a target cell population.

[0105] If not explicitly stated otherwise, the term "comprising" is used in the present invention to indicate that in addition to the components listed under "comprising", other components may optionally be present. However, it is considered that a specific embodiment includes the possibility that no other components are present, that is, within the scope of this specific embodiment, the term "comprising" is understood as the term "consisting of..."

[0106] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0107] Example 1 Protein purification method

[0108] 1.1 Experimental Materials

[0109] HEK293 cells were purchased from ATCC

[0110] 1.1 Experimental methods

[0111] 1.1.1 Expression vector construction

[0112] 1. PCR amplification

[0113] The primer sequences used to amplify the target gene are shown in Table 1 ;

[0114] Table 1 Primer sequences

[0115]

[0116] The primers are in dry powder form. Centrifuge at 13,000 rpm for 5 min, dilute the primers to 10 pmol / L, prepare a working solution, and mix it on a vortex mixer.

[0117] The RCR reaction system is shown in Table 2;

[0118] Table 2 PCR reaction system

[0119]

[0120] PCR reaction procedure:

[0121]

[0122] 2. Rubber recovery and purification

[0123] 1) Turn on the water bath and adjust the temperature to 55℃~65℃;

[0124] 2) After electrophoresis is completed, carefully cut out the gel containing the DNA to be recovered under visible light or ultraviolet light (ultraviolet light is used if the band concentration is weak), place it in a 1.5 mL EP tube, and weigh it;

[0125] 3) Add 10 μL of membrane binding solution to every 10 mg of gel recovery tape, place in a 65°C water bath, take out the EP tube after 2-3 minutes, turn it upside down to mix, if the gel is not completely dissolved, repeat the above steps;

[0126] 4) Place the column on a 2 mL collection tube, add the dissolved gel solution to the column, place at room temperature for 1 minute, and centrifuge at 16,000 g for 1 minute;

[0127] 5) Add the liquid in the collection tube back to the column, let it stand for 1 minute, centrifuge at 16000g for 1 minute, discard the liquid in the collection tube, add 700 μL membrane elution buffer, centrifuge at 16000g for 1 minute, and discard the liquid in the collection tube;

[0128] 6) Add 500 μL of membrane elution buffer, centrifuge at 16000 g for 5 minutes, discard the liquid in the collection tube, and centrifuge the empty column at 16000 g for 1.5 minutes;

[0129] 7) Place the column in a new 1.5 mL centrifuge tube, add 40 μL ddH2O to the center of the column membrane, leave at room temperature for 1 min, and centrifuge at 16,000 g for 2 minutes.

[0130] 3. Connect the expression vector

[0131] 1) Take out the ligase and vector from -20℃ and place them on an ice box to thaw;

[0132] 2) Adjust the PCR instrument to 50°C. Add the reaction system according to Table 3;

[0133] Table 3 Ligation reaction system

[0134]

[0135] 3) Place the PCR tube into a PCR instrument and react at 50°C for 20 minutes.

[0136] 4. Heat shock method to transform competent cells

[0137] 1) Take out the prepared plate culture medium from 4°C and place it on the clean bench 1 hour before transformation;

[0138] 2) Take out the prepared competent cells from -80℃ and thaw them on an ice box at 4℃ for about 30 minutes;

[0139] 3) Adjust the water bath to 45°C;

[0140] 4) Add approximately 10 μL of ligation product per 100 μL of competent cells into a 1.5 mL EP tube;

[0141] 5) Place on ice at 4°C for 30 minutes;

[0142] 6) Heat shock in a 42°C water bath for 90 seconds;

[0143] 7) Quickly transfer the centrifuge tube to an ice box and place it at 4°C for 3 minutes;

[0144] 8) Add 500 μL SOC to the centrifuge tube and resuscitate at 37°C, 200 rpm for 45 min;

[0145] 9) Centrifuge at 6000 rpm for 1 min, take out 500 μL and discard it on the clean bench, mix the rest, spread it on the plate culture medium with a coating rod, and put it upside down in the incubator for culture. Culture conditions: 37°C, 16-18 hours.

[0146] 5. Sequencing

[0147] A single clone was picked from the overnight culture plate for testing, and the sequencing results were analyzed to obtain the plasmid containing the correct sequence.

[0148] 6. Scale up and cultivate

[0149] 1) Transform the plasmid with correct sequencing;

[0150] 2) Pick a single clone from the overnight culture plate and culture it in the corresponding resistance culture medium for amplification. The culture conditions are: culture temperature: 37°C, rotation speed: 250 rpm; culture time: 16-18 hours.

[0151] 7. Plasmid extraction

[0152] 1) Centrifuge the overnight cultured bacteria at 6000 rpm for 10 min and discard the supernatant;

[0153] 2) Add 1 / 10 volume of suspension to resuspend the bacterial solution;

[0154] 3) Add the same volume of alkaline lysis solution, gently invert upside down until the liquid becomes clear, and let it stand for 3 minutes;

[0155] 4) Add 1.4 times the volume of acid neutralization solution and mix by inversion;

[0156] 5) Centrifuge at 6000 rpm for 30 min, purify the supernatant through a column, and elute with buffer;

[0157] 6) Submit the obtained plasmid for downstream expression.

[0158] 8. Cell culture and protein expression

[0159] HEK293 cells were subcultured using 293 serum-free CD medium (Cat. No. SMM 293-TI). The target protein expression plasmid was mixed with transfection reagent TF1 and added to the cells. 293 serum-free feed solution (Cat. No. M293-SUPI-100) was added on days 1, 3, and 5 after transfection. Protein purification was performed after 7 days of cell culture.

[0160] 9. Protein purification

[0161] 1) After the culture medium is centrifuged, the remaining insoluble matter is removed by filtration and then the target protein is purified using an affinity chromatography column;

[0162] 2) Metal chelate affinity chromatography purification: After the Ni-ion affinity chromatography column is equilibrated with the loading buffer, the culture supernatant is loaded onto the column. After the impurity protein flows through, the target protein is eluted using an imidazole gradient. After the purified target protein is replaced with a buffer, the protein concentration is calibrated and the purity is tested.

[0163] 1.3 Experimental Results

[0164] 1.3.1 Protein name code: 22ZJY17-2, His-KLKB1-HEK293

[0165] Sequence features: SP1 (SEQ ID NO.3) - His tag (SEQ ID NO.4) - protease cleavage site (SEQ ID NO.5) - KLKB1 protein sequence (SEQ ID NO.6) are connected in the order to form sequence 1 (SEQ ID NO.7). The specific sequence is shown in Table 4.

[0166] The protein expression system is the 293 cell expression system. The electrophoresis results after protein expression and purification are as follows: Figure 1 As shown, the protein purity was 98.4%.

[0167] Table 4 Sequence 1

[0168]

[0169]

[0170] Comparative Example 1 Protein name code: 22ZJY17-2, His-KLKB1-HEK293

[0171] Sequence features: SP1 (SEQ ID NO.3) - KLKB1 protein sequence (SEQ ID NO.6) - protease cleavage site (SEQ ID NO.5) - His tag (SEQ ID NO.8) are connected in the order to form sequence 2 (SEQ ID NO.9). The specific sequence is shown in Table 5.

[0172] The protein expression system is the 293 cell expression system. The electrophoresis results after protein expression and purification are as follows: Figure 2 As shown, the protein yield after purification was 0.1 mg and the purity was 81.8%.

[0173] Table 5 Sequence 2

[0174]

[0175] Comparative Example 2 Protein name code: SP-KLKB1(376-638aa)-3C-10his-22ZJY17

[0176] Sequence features: SP2 (SEQ ID NO.10) - KLKB1 protein sequence (SEQ ID NO.6) - protease cleavage site (SEQ ID NO.5) - His tag (SEQ ID NO.8) are connected in the order to form sequence 3 (SEQ ID NO.11). The specific sequence is shown in Table 6.

[0177] The protein expression system is the 293 cell expression system. The results of electrophoresis detection after protein expression and purification are as follows Figure 3 As shown, the protein yield after purification was 0.25 mg and the purity was 82%.

[0178] Table 6 Sequence 3

[0179]

[0180] Comparative Example 3 Protein name code: BSP-10His-3C-KLKB1(376-638aa)-22ZJY18-2

[0181] Sequence features: SP3 (SEQ ID NO.12) - His tag (SEQ ID NO.4) - protease cleavage site (SEQ ID NO.5) - KLKB1 protein sequence (SEQ ID NO.6) are connected in the order to form sequence 4 (SEQ ID NO.13). The specific sequence is shown in Table 7.

[0182] The protein expression system is an insect cell expression system. The electrophoresis test results after protein expression and purification are as follows Figure 4 As shown, the protein purity was 91.2% and the yield was 0.6 mg.

[0183] Table 7 Sequence 4

[0184]

[0185]

[0186] Comparative Example 4 Protein name code: BSP-10His-3C-KLKB1(376-638aa)-22ZJY18-2

[0187] Sequence features: SP3 (SEQ ID NO.12) - KLKB1 protein sequence (SEQ ID NO.6) - protease cleavage site (SEQ ID NO.5) - His tag (SEQ ID NO.8) are connected in the order to form sequence 5 (SEQ ID NO.14). The specific sequence is shown in Table 8.

[0188] The protein expression system is an insect cell expression system. The electrophoresis test results after protein expression and purification are as follows Figure 5 As shown, the protein purity was 79.9% and the yield was 0.08 mg.

[0189] Table 8 Sequence 5

[0190]

[0191]

[0192] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for purifying a protein, characterized in that: The method comprises: (1) Operably linking a nucleic acid encoding a signal peptide, a tag, or a protease cleavage site to a nucleic acid sequence encoding a target protein, thereby generating an expression vector encoding a fusion protein; (2) introducing the expression vector into a host cell; The signal peptide, tag, protease cleavage site and target protein described in step (1) are sequentially connected; The signal peptide is selected from CD33 signal peptide; The tag is selected from a His tag; The target protein is selected from KLKB1 protein; The His tag sequence is shown in SEQ ID NO.4; The protease cleavage site is shown in SEQ ID NO.5; The KLKB1 protein is selected from a truncated KLKB1 protein; The truncated KLKB1 protein sequence is shown in SEQ ID NO.6; The CD33 signal peptide sequence is shown in SEQ ID NO.3; The host cell is selected from HEK293 cells.

2. An expression vector as claimed in claim 1.

3. A host cell, characterized in that The host cell comprises the expression vector according to claim 2, and the host cell is a HEK293 cell.

Images