WO2023097475A1

WO2023097475A1 - Method for constructing eukaryotic expression vector of human pak1 protein, expression method, and purification method

Info

Publication number: WO2023097475A1
Application number: PCT/CN2021/134500
Authority: WO
Inventors: 张浩洋; 石坚; 徐爱华; 朱国方
Original assignee: 绍兴守仁医疗健康科技有限公司
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2023-06-08

Abstract

A method for constructing an eukaryotic expression vector of a human PAK1 protein, comprising the steps of: 1) carrying out PCR amplification to obtain a PAK1 gene fragment; 2) linking to an eukaryotic expression vector to obtain a recombinant plasmid; and 3) carrying out transfection, transformation, and replication to obtain an eukaryotic expression vector of a human PAK1 protein. Also disclosed is a method for expressing a recombinant human PAK1 protein, further comprising step 4): transfecting a plasmid of the eukaryotic expression vector of the human PAK1 protein obtained in step 3) into eukaryotic cells for expression. Further disclosed is a method for obtaining a recombinant human PAK1 protein, further comprising step 5): culturing the positive clone obtained in the step 4) to enlarge a system, collecting cultured cells, carrying out centrifugation to collect supernatant, and carrying out purification by means of a chromatographic column to obtain a recombinant human PAK1 protein.

Description

Construction method, expression and purification method of eukaryotic expression vector of human PAK1 protein

technical field

The present application relates to the technical field of molecular biology, to eukaryotic expression technology of recombinant protein, and in particular to a construction method, expression and purification method of a eukaryotic expression vector of human PAK1 protein.

Background technique

protein kinase Kinases (PK for short) are enzymes that catalyze the phosphorylation process of proteins. The phosphorylation process of proteins is the last link in the transmission of nerve information in cells, leading to state changes of ion channel proteins and channel gates. It is an extremely important part of signaling pathways in the body, and there are many types in nerve cells. Therefore, the activity of protein kinases is regulated in many ways, one of which is the activation of autophosphorylation. The PAK (p21-activated kinase) protein family plays an important role in a variety of cellular processes and is also subject to very fine regulation; PAK1 is a representative member of this family, and its activity is controlled by its N-terminal regulatory domain and autophosphorylation The regulation of the response, the phosphorylation of a conserved threonine site on its activation loop is necessary for its activation.

technical problem

At present, there are many methods for the recombination and expression of human PAK1 protein, but all of them are expressed in the E. coli system. Another fact is that most of the PAK1 protein monoclonal antibodies currently available on the market have poor specificity when establishing a PAK1 protein detection method. , The disadvantage of low sensitivity. We believe that during the protein expression process using the prokaryotic system, due to the lack of the same expression environment as the natural PAK1 protein, the structure of the recombinant PAK1 protein is different from the natural PAK1 protein, resulting in poor specificity and poor specificity of the prepared monoclonal antibody in recognizing the natural PAK1 protein. The disadvantage of low sensitivity.

technical solution

In view of the above technical problems, the purpose of this application is to provide a method for constructing a eukaryotic expression vector of human PAK1 protein, comprising the following steps:

1) Using the gene sequence expressing PAK1 protein as a template, PCR amplification is used to obtain the PAK1 gene fragment;

2) Link the PAK1 gene fragment obtained in step 1) to a eukaryotic expression vector to obtain a recombinant plasmid;

3) Transfection, transformation and replication of the obtained recombinant plasmid to obtain a eukaryotic expression vector of human PAK1 protein.

In the above technical scheme, the eukaryotic expression vector described in step 2) is pcDNA3.1, and the PAK1 gene fragment and pcDNA3.1 obtained in step 1) are digested with restriction endonucleases BamHI and EcoRI respectively, and ligated and digested The product is a recombinant plasmid.

Alternatively, the eukaryotic expression vector described in step 2) is pCMV-blank, and the PAK1 gene fragment obtained in step 1) is first cloned with a T vector, and the cloned plasmid and pCMV-blank that are sequenced correctly are identified using the restriction endonuclease ApaⅠ After digestion with PstI, the digested products were ligated to obtain recombinant plasmids.

In the above technical scheme, step 3) transform the obtained recombinant plasmid into Escherichia coli and replicate to obtain a large number of recombinant plasmids.

In the above technical scheme, the primers used in the PCR amplification in the step 1) are PAK1-F/PAK1-R, and the primer sequences are:

PAK1-F: 5'-AGCTCAACTATGATCTTTTT-3',

PAK1-R: 5'-GAATATTTTTCATCTCCTTTT-3'.

Another object of the present application is to provide a method for expressing recombinant human PAK1 protein using a eukaryotic expression system. On the basis of the aforementioned construction method, it also includes the following steps:

4) Using the correct recombinant amplification and purification of the eukaryotic expression vector of human PAK1 protein obtained in step 3), the plasmid obtained is transfected into eukaryotic cells for expression.

Preferably, the eukaryotic cells in step 4) include CHO cells, COS-7 cells, NSO cells, yeast, insect cells, and human cells used for exogenous gene expression, and the transfection uses liposome transfection .

Another object of this application is to provide a method for obtaining recombinant human PAK1 protein, which also includes step 5) on the basis of the aforementioned method steps:

5) The positive clones obtained in step 4) were cultured to expand the system, the cultured cells were collected, the cells were broken, and the supernatant was collected after centrifugation, and the target protein was purified through a chromatographic column to obtain a high-purity recombinant human PAK1 protein.

Preferably, in the step 5), the cultured cells are collected, the cells are disrupted by sonication, the supernatant is collected after centrifugation, and the Ni column is used for affinity chromatography.

The final purpose of this application is to provide a eukaryotic expression system of recombinant human PAK1 protein, which is an expression system obtained by transfecting eukaryotic cells with the eukaryotic expression vector of human PAK1 protein constructed by the aforementioned method. The cells are selected from CHO cells, COS-7 cells, NSO cells, yeast, insect cells and human cells for exogenous gene expression.

Beneficial effect

The inventor believes that in the process of protein expression using prokaryotic systems in the prior art, due to the lack of the same expression environment as the natural PAK1 protein, there are differences in the structure of the recombinant PAK1 protein and the natural PAK1 protein, resulting in the existence of the prepared monoclonal antibody that recognizes natural PAK1 Disadvantages of poor protein specificity and low sensitivity. For this reason, we choose eukaryotic expression system to simulate the expression environment of natural PAK1 protein. Eukaryotic expression system such as CHO can be used to express and purify human PAK1 protein. The CHO expression system has the following advantages:

(1) It has accurate post-transcriptional modification function, and the expressed protein is closest to the natural protein molecule in terms of molecular structure, physical and chemical properties and biological function;

(2) It can not only grow on the wall, but also can be cultured in suspension, and has a high ability to withstand shear force and osmotic pressure;

(3) High-efficiency amplification and expression capabilities of recombinant genes, and stable integration of foreign proteins;

(4) It has the function of extracellular secretion of the product, and rarely secretes its own endogenous protein, which is convenient for the separation and purification of downstream products;

(5) High-density culture can be achieved in suspension culture or in serum-free medium. And the culture volume can reach more than 1000L, which can be produced on a large scale.

The eukaryotic expression vector referred to in this application is a molecular biology carrier tool that can carry gene fragments and can regulate the expression of gene fragments carried in the eukaryotic expression system through regulatory elements, and furthermore, can be secreted or not through signal peptides. A vector with a signal peptide for intracellular expression or a vector that can infect a host cell by packaging a virus and integrate the gene into the host genome to express the gene; this application does not limit the selection of eukaryotic expression vectors. The eukaryotic expression system mentioned in this application refers to all eukaryotic cells suitable for exogenous gene expression, and this application does not limit the choice of host cells to be used.

Implementing the embodiment of the present application will have the following beneficial effects:

The eukaryotic expression system of the present application overcomes the shortcomings of prokaryotic expression, obtains highly active recombinant human PAK1 protein, and the protein purified by the method of the present application has a high purity of more than 95%. The recombinant human PAK1 protein obtained by the method of the present application The PAK1 protein has the same structure as the natural PAK1 protein, and the monoclonal antibody prepared by using the recombinant human PAK1 protein obtained by the method of this application has high specificity and high sensitivity to recognize the natural PAK1 protein, which provides a strong basis for obtaining high-quality antibodies.

Embodiments of the present invention

The following will clearly and completely describe the technical solutions in the embodiments of the application with reference to the drawings in the embodiments of the application. Apparently, the described embodiments are only some of the embodiments of the application, not all of them. Based on the embodiments in this application, all other embodiments obtained by persons of ordinary skill in the art without creative efforts fall within the protection scope of this application.

The experimental methods in the following examples, unless otherwise specified, are conventional methods; the biological and chemical reagents used in each example, unless otherwise specified, are conventional reagents and can be obtained commercially.

Main source of reagents:

pcDNA3.1 (+) carrier: purchased from Invitrogen;

Restriction enzymes BamHI, EcoRI, ApaI, PstI: use products from NEB;

Reagents for PCR amplification PCR reaction MIX: use the products of Beijing Quanshijin Company;

lipofectAMINE reagent: brand Invitrogen;

pGEM-T carrier: purchased from PROMEGA;

Strain containing eukaryotic expression vector pCMV-blank: use the product of Beijing Quanshijin Company.

Example 1 Construction of PAK1 protein eukaryotic expression vector and its expression and purification:

1. Construction of PAK1 protein eukaryotic expression vector;

Design amplification primers according to the sequence of PAK1 protein:

Upstream primer PAK1-F (SEQ ID NO.1): 5'-AGCTCAACTATGATCTTTTT-3',

Downstream primer PAK1-R (SEQ ID NO.2): 5'-GAATATTTTTCATCTCCTTTT-3'.

Among them, a BamHI restriction site was introduced into the upstream primer, and an EcoRI restriction site and a 6×histidine tag were introduced into the downstream primer.

Using HELA cells as a template, PAK1-F/PAK1-R was amplified by PCR with primers. The reaction system was: PCR reaction MIX 25μl, forward and reverse primers with a concentration of 10μM each 1μl, DNA template 2μl, and ddH ₂ O to 50 μl. The PCR amplification program is: 95°C for 180 seconds; 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, 35 cycles; finally 72°C for 300 seconds.

After PCR amplification was completed, agarose gel electrophoresis was carried out. After the gel was recovered with a gel recovery kit, it was digested with restriction enzymes BamHI and EcoRI, and then used with the pcDNA3.1(+) vector digested with BamHI and EcoRI. Ligase was used to connect and transform Escherichia coli DH5a competent cells. After PCR identification, positive recombinants were selected for sequencing identification. The sequence of positive recombinants was shown in SEQ ID NO.3.

2. Expression of PAK1 protein in CHO cells

The correct clones were identified by sequencing, and a large number of plasmids were prepared.

Use lipofectAMINE reagent to transfect CHO cells according to the product instructions, add the screening drug G418 Sulfate (0.8 mg/ml) to screen positive clones, and select CHO cells containing the plasmid.

The culture system was increased to 1 L, and the cells were collected after 72 hours of culture, and the cells were broken by ultrasonication, and the supernatant was collected after centrifugation, and affinity chromatography was performed using a Ni column. After SDS-PAGE analysis, the protein size was about 21 kD, which was the same as the theoretical value, and the yield was 3 mg/L. After estimating the protein concentration according to the band gray value of SDS-PAGE, the protein was concentrated to 3 mg/ml.

The purified protein obtained by eukaryotic expression in this example was determined using the rabbit monoclonal antibody [EPR20045] of ABCOM Company, and the operation steps are as follows:

(1) Dilute ABCOM rabbit monoclonal antibody [EPR20045] into carbonic acid buffer at a concentration of 0.1mg/ml, add 0.1ml to each well of the ELISA reaction well, and react overnight at 2-8°C. Antibody was washed away the next day and blocked with BSA.

(2) Dilute the purified protein to about 1.0 μg/ml, 2.0 μg/ml, and 3.0 μg/ml with protein protection solution; use the above ELISA well for detection, and use ABCOM’s human PAK1 protein (ab67946) to also be diluted to 1.0 μg/ml, 2.0 μg/ml, 3.0 μg/ml were tested.

(3) The detection of each concentration was repeated 5 times.

(4) The average value of the above concentration values was measured, and the correlation coefficient R>0.99.

Prokaryotically expressed PAK1 protein was used as a control, and the purified protein expressed in Escherichia coli was determined using ABCOM company rabbit monoclonal antibody [EPR20045]:

(1) Dilute the rabbit monoclonal antibody [EPR20045] from ABCOM Company into carbonic acid buffer at a concentration of 0.1mg/ml, add 0.1ml to each well of the ELISA reaction well, and react overnight at 2-8°C. Antibody was washed away the next day and blocked with BSA.

(2) Dilute the purified protein to about 1.0 μg/ml, 2.0 μg/ml, and 3.0 μg/ml with protein protection solution; use the above ELISA wells for detection, and use ABCOM’s human PAK1 protein (ab67946) to also be diluted to 1.0 μg/ml, 2.0 μg/ml, 3.0 μg/ml were tested.

(3) The detection of each concentration was repeated 5 times.

(4) Take the average value of the above concentration values, and the correlation coefficient R<0.95.

Example 2

1. Molecular cloning of PAK1 gene

Using HELA cells as a template, PAK1-F/PAK1-R was amplified by PCR using the primers in Example 1. The reaction system was: 25 μl of PCR reaction MIX, 1 μl of forward and reverse primers with a concentration of 10 μM, 2 μl of DNA template, Add ddH ₂ O to 50 μl. The PCR amplification program is: 95°C for 180 seconds; 94°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, 35 cycles; finally 72°C for 300 seconds.

After PCR amplification, perform agarose gel electrophoresis detection, use the gel recovery kit to recover and purify the PCR amplification products from the gel, and establish the following ligation reaction according to the pGEM-TEasy vector system product manual: 10×T4 DNA ligation reaction buffer 1 μl, 1 μl (50ng) of pGEM-T vector, 3 μl of purified PCR amplified fragment, 1 μl (3U) of T4 DNA ligase, add ddH ₂ O to a total volume of 10 μl, and ligate at 16°C for 2 hours to obtain a ligation reaction.

2. Preparation of Escherichia coli Competent Cells

Pick a single colony of Escherichia coli DH5α from the plate with an inoculation loop and inoculate it in 3ml of LB liquid medium without antibiotics, culture it with shaking at 37°C overnight, take 0.5ml of the bacterial solution in the logarithmic growth phase and inoculate it in 50ml without antibiotics Continue to shake at 37°C for 4 hours, then transfer the culture liquid into a 50ml centrifuge tube, put it in an ice bath for 10 minutes, and centrifuge at 4000 rpm for 5 minutes at 4°C, discard the culture liquid, and use a pre- Resuspend the cells in cold CaCl ₂ (0.1mol/L) solution, ice-bath for 20 minutes, centrifuge at 4000 rpm for 20 minutes at 4°C, remove the supernatant, add 1ml ice-bathed CaCl ₂ solution (0.1mol/L) Mix gently, dispense into 1.5ml centrifuge tubes, and store in 4°C after 2 hours on ice.

Take 200μl of DH5α competent cell culture solution into a 1.5ml centrifuge tube, add 2μl of the ligation reaction, mix gently, put it in ice bath for 20 minutes, put it in a 42℃ water bath for 45 seconds, take out the centrifuge tube and put it in the ice bath for 2 minutes , add 800 μl of SOC liquid medium without antibiotics, shake at 37°C (150 rpm) for 1 hour, take 100 μl of the transformed bacterial solution and spread it on an LB plate (containing 100 μg/ml of ampicillin, and 40 μl of 20 mg /ml X-gal and 4μl 200mg/ml IPTG) were cultured overnight at 37°C. After the transformation, place the overnight plate cultured at 37°C at 4°C for 4 hours to fully visualize the blue-white colonies. Randomly pick white colonies from the transformation plate, inoculate them in 3ml LB liquid medium containing ampicillin, shake at 37°C for 14 hours, and extract plasmid DNA according to the operating instructions of the plasmid extraction kit. Sequencing confirmed that the contained target sequence was accurate, and the resulting plasmid was PGEM-TEasy-PAK1.

3. Construction and identification of eukaryotic expression vector pCMV-PAK1

Take out the frozen strain containing the eukaryotic expression vector pCMV-blank from the refrigerator and dissolve at room temperature; take 200ul of the frozen bacteria solution and add it to the LB solution of Kana; cultivate overnight in a shaking incubator at 37°C for 16 hours, extract the plasmid, and measure the concentration of the plasmid Finally, mark the plasmid concentration on the EP tube and store it at -20°C for future use.

The cloned plasmid pGEM-TEasy-PAK1 with correct sequencing was digested with double enzymes. The reaction system was: 12 μl plasmid, 1 μl each of ApaI and PstI, 2 μl of 10× reaction buffer, supplemented with ddH ₂ O to 20 μl, digested at 37°C for 2 hours, and stored in -20°C for standby, take 3 μl of digested product and electrophoresis on 1.5% agarose gel to observe the result. Cut off the agar block containing the target fragment with a clean scalpel under ultraviolet light, put it into a 1.5ml centrifuge tube, and recover the target gene PAK1 gene with a gel recovery kit. Take 3 μl for electrophoresis to observe the extraction effect, and store the remaining samples at -20°C for future use.

Double enzyme digestion of pCMV-blank: Take 10 μl of pCMV-blank, add it to a centrifuge tube, add 2 μl of 10× reaction buffer, 1 μl of ApaⅠ and PstⅠ, add pure water to 20 μl, bathe in 37°C water for 3 hours, take 3 μl of digested enzyme The reaction mixture was electrophoresed in 1.5% agarose gel to detect the enzyme cutting effect.

Recover the large fragment of pCMV-blank digested by the gel, and connect the target gene PAK1 gene recovered from the previous gel with the large fragment of pCMV-blank. The reaction system is as follows: target gene 12 μl, pCMV-blank large fragment 3 μl, Solution Ⅰ (TaKaRa DNA Ligation Kit Ver.2) 15 μl, ligated at 16°C for 2 hours to obtain the ligation reaction. Take 200μl of DH5α competent cell culture solution into a 1.5ml centrifuge tube, add 2μl of the ligation reaction, mix gently, put it in ice bath for 20 minutes, put it in a 42℃ water bath for 45 seconds, take out the centrifuge tube and put it in the ice bath for 2 minutes , add 800 μl of SOC liquid medium without antibiotics, shake at 37°C (150 rpm) for 1 hour, take 100 μl of the transformed bacterial solution and spread it on an LB plate containing Kana, and incubate at 37°C. Pick a single colony from the transformation plate, inoculate it in 3ml of Kana-containing LB liquid medium, shake overnight at 37°C, extract the plasmid DNA according to the operating instructions of the plasmid extraction kit, obtain 50 μl of plasmid DNA, and take 3 μl to gel in 1.5% agarose Plasmid DNA was detected by gel electrophoresis. Add 10 μl of the resulting recombinant plasmid DNA to a 1.5ml centrifuge tube, add 2 μl of 10× reaction buffer, 1 μl of ApaI and PstI each, add pure water to 20 μl, bathe in water at 37°C for 3 hours, take 10 μl of the reaction mixture after digestion at 1.5 The enzyme digestion effect was detected by electrophoresis in % agarose gel. Sequence the recombinant plasmid identified as positive after restriction endonuclease double-enzyme digestion, and the sequence obtained is shown in SEQ ID NO.4. The results showed that the recombined target gene sequence was completely consistent with the PAK1 protein gene sequence in Genbank, and the direction was correct.

The results showed that the eukaryotic expression vector pCMV-PAK1 of PAK1 was successfully constructed.

4. Obtain a cell line stably expressing PAK1

COS-7 cells were cultured in DMEM high-glucose complete medium containing 10% fetal bovine serum in a constant temperature and humidity incubator at 37°C and 5% CO2. The day before transfection, spread the cells with confluence of 85% to 95% in a 12-well plate. After culturing for 24 hours, replace it with serum-free and antibiotic-free DMEM high-glucose medium. The recombinant plasmid pCMV-PAK1 was mixed and allowed to stand for 20 minutes, and then evenly dropped into each well of a 12-well plate. After culturing for 6 hours, the culture solution in each well was discarded and replaced with fresh DMEM high-sugar culture solution without antibiotics to continue the culture.

COS-7 cells transfected with blank plasmid pCMV-blank were used as control. After 24 hours of transfection, the cells were replaced with the cell culture medium containing the screening drug G418 with an initial concentration of 400 μg/ml. After 7 days of pressure screening, the cells began to die in large numbers. After washing with PBS twice, the concentration of G418 was adjusted to 200 μg/ml, and the culture was continued for three times. One day later, the remaining cells were planted in a 96-well plate according to 1 cell/well, and maintained at a G418 concentration of 200 μg/ml for expansion and culture for 8 weeks to obtain monoclonal cell lines. Screen the positive monoclonal cell lines of PAK1 by PCR, and then continue to culture the positive monoclonal cell lines in an environment with a G418 concentration of 200 μg/ml. After ten consecutive passages, use PCR again to identify positive cells that still express the target gene PAK1. The monoclonal cell line was used as the cell line stably expressing PAK1 for subsequent experiments. Collect 2ml of the cell culture supernatant of the cell line stably expressing PAK1 protein, lyophilize and concentrate, and use it as a protein sample for Western blotting experiments. The protein detection method was the same as in Example 1, and the detected protein size was about 21 kD, which was the same as the theoretical value.

Claims

A method for constructing a eukaryotic expression vector of human PAK1 protein, comprising the steps of:

1) Using the gene sequence expressing PAK1 protein as a template, PCR amplification is used to obtain the PAK1 gene fragment;

2) Link the PAK1 gene fragment obtained in step 1) to a eukaryotic expression vector to obtain a recombinant plasmid;

3) Transfection, transformation and replication of the obtained recombinant plasmid to obtain a eukaryotic expression vector of human PAK1 protein.
The method for constructing a eukaryotic expression vector of human PAK1 protein according to claim 1, characterized in that: the eukaryotic expression vector in step 2) is pcDNA3.1, and the PAK1 gene fragment obtained in step 1) and pcDNA3 .1 Digest with restriction endonucleases BamHI and EcoRI respectively, and connect the digestion products to obtain recombinant plasmids.
The method for constructing a eukaryotic expression vector of human PAK1 protein according to claim 1, characterized in that: the eukaryotic expression vector in step 2) is pCMV-blank, and the PAK1 gene fragment obtained in step 1) is firstly used in T Vector cloning, identification and sequencing of the correct cloned plasmid and pCMV-blank were digested with restriction endonucleases ApaI and PstI respectively, and the digested products were connected to obtain a recombinant plasmid.
The method for constructing a eukaryotic expression vector of human PAK1 protein according to claim 1, characterized in that: step 3) transforming the obtained recombinant plasmid into Escherichia coli and replicating to obtain a large number of recombinant plasmids.
The method for constructing a eukaryotic expression vector of human PAK1 protein according to any one of claims 1-4, characterized in that: the primers used for PCR amplification in the step 1) are PAK1-F/PAK1-R, and the primers The sequence is:

PAK1-F: 5'-AGCTCAACTATGATCTTTTT-3',

PAK1-R: 5'-GAATATTTTTCATCTCCTTTT-3'.
A method for expressing recombinant human PAK1 protein using a eukaryotic expression system, characterized in that: on the basis of the construction method described in any one of claims 1-5, further comprising the following steps:

4) Using the correct recombinant amplification and purification of the eukaryotic expression vector of human PAK1 protein obtained in step 3), the plasmid obtained is transfected into eukaryotic cells for expression.
The method according to claim 6, characterized in that: step 4) said eukaryotic cells include CHO cells, COS-7 cells, NSO cells, yeast, insect cells and human cells used for exogenous gene expression, so The above transfection was performed by liposome method.
A method for obtaining recombinant human PAK1 protein, characterized in that: on the basis of the method steps described in claim 6 or 7, further comprising step 5):

5) The positive clones obtained in step 4) were cultured to expand the system, the cultured cells were collected, the cells were broken, and the supernatant was collected after centrifugation, and the target protein was purified through a chromatographic column to obtain a high-purity recombinant human PAK1 protein.
The method according to claim 8, characterized in that: in the step 5), the cultured cells are collected, the cells are broken by ultrasonic disruption, the supernatant is collected after centrifugation, and the Ni column is used for affinity chromatography.
A eukaryotic expression system of recombinant human PAK1 protein, characterized in that: it is an expression system obtained by transfecting eukaryotic cells with the eukaryotic expression vector of human PAK1 protein constructed by the method described in any one of claims 1 to 5 , the eukaryotic cells are selected from CHO cells, COS-7 cells, NSO cells, yeast, insect cells and human cells for exogenous gene expression.