CN112279899B

CN112279899B - Method for purifying prokaryotic expression protein

Info

Publication number: CN112279899B
Application number: CN202011114481.0A
Authority: CN
Inventors: 刘汉平; 张冬冬; 岳丰雄; 欧仁芳; 潘倩
Original assignee: Chengdu Shiji Biopharmaceutical Co ltd
Current assignee: Chengdu Shiji Biopharmaceutical Co ltd
Priority date: 2020-10-16
Filing date: 2020-10-16
Publication date: 2023-08-29
Anticipated expiration: 2040-10-16
Also published as: CN112279899A

Abstract

The invention discloses a method for purifying prokaryotic expression protein, which comprises the following steps: 1) Taking prokaryotic expression engineering bacteria, and homogenizing and crushing the bacteria after re-suspending the prokaryotic expression engineering bacteria by using a buffer solution; 2) Adding polyethyleneimine with the final concentration of 0.1-0.3% (m/v), uniformly mixing, standing for 10-30 minutes, and centrifuging to obtain a supernatant; 3) Adding polyethylene glycol with the final concentration of 3% -5% (m/v) into the obtained supernatant, standing at 2-8 ℃ for more than 6 hours, and precipitating protein; 4) Centrifuging, removing supernatant, and dissolving precipitate with buffer solution. The method can simplify the purification operation, achieve the effect of effectively removing endotoxin, and has good application prospect.

Description

Method for purifying prokaryotic expression protein

Technical Field

The invention belongs to the field of recombinant protein purification.

Background

Prokaryotic expression, namely protein expression by prokaryotic expression engineering bacteria, is a common protein production mode at present and is mainly applied to the production of products such as enzymes, subunit vaccines and the like. Examples of the cells used for prokaryotic expression include E.coli, bacillus, streptomyces and cyanobacteria, and E.coli is the most widely used prokaryotic expression cell in the industry at present.

Coli is gram negative bacteria, and when recombinant proteins are expressed, a large amount of endotoxin is produced, the chemical nature of the endotoxin is Lipopolysaccharide (LPS), the lipopolysaccharide has extremely strong heat source, and trace LPS can cause stress reactions such as hyperthermia, diarrhea, shock and the like when entering an animal body, so that if prokaryotic expressed proteins need to enter a human body or the animal body to play a role, the removal of the endotoxin is one of the keys of purifying the proteins.

The current method for removing the bacterial endotoxins in the solution mainly comprises the following steps: (1) detoxification: the detoxification mechanism mainly comprises that bacterial endotoxin is degraded by enzyme into a nontoxic fragment, the bacterial endotoxin and protein form a reversible complex, and acid-base hydrolysis generates detoxification, the method can influence the activity of the protein to different degrees, and simultaneously the detoxified endotoxin has possibility of toxicity reversion; (2) sucrose density gradient centrifugation technique: the endotoxin has different existence forms, different molecular sizes, different densities, different sedimentation coefficients and different molecular weights, so that certain difficulty is caused to the application of the method; (3) affinity chromatography technique: the method has mild conditions, is simpler, has the functions of purifying and concentrating antigen, but also has the defects of expensive reagent, complex operation, poor ligand and cement stability and the like; (4) ion exchange chromatography technique: there are problems of expensive equipment, large material loss, etc.

Porcine circovirus type 2 (PCV 2) is an icosahedral, non-enveloped, single-stranded circular DNA virus with a diameter of about 17-22nm. PCV2 infects many tissues and organs of pigs, with lymphoid organs being the most common organ to be compromised, resulting in impaired immune system in pigs, leading to secondary infections including porcine reproductive and respiratory syndrome, pseudorabies and other bacterial infectious diseases. The open reading frame 2 (ORF 2) of PCV2 encodes the unique structural protein of the virus-Cap protein, which is also the major immunogenic protein of PCV2, 5 Cap proteins constitute 5 mers, and 12 5 mer Cap proteins spontaneously assemble into icosahedral virus-like particles (VLPs). Virus-like particles are very effective antigens for the preparation of viral vaccines because they do not contain genetic material of the virus, but have a structure similar to that of a virus particle.

The escherichia coli expression system is an ideal expression system for producing Cap proteins due to the advantages of low production cost, simple production condition requirements, short period and the like, but the endotoxin removal method still has various problems of high cost, complex operation and the like, and the removal effect is not ideal. For example, chinese patent application CN111233983a discloses a one-step large-scale purification and endotoxin removal process for Cap protein, wherein cation exchange chromatography is used to purify Cap protein and remove endotoxin, and the endotoxin content is below 2.5 EU/mL.

Disclosure of Invention

The invention aims to solve the problems that: provides a simple and convenient purification method of prokaryotic expression protein with good endotoxin removal effect.

The technical scheme of the invention is as follows:

the invention first provides a method for purifying prokaryotic expression protein, which comprises the following steps:

1) Taking prokaryotic expression engineering bacteria, and homogenizing and crushing the bacteria after re-suspending the prokaryotic expression engineering bacteria by using a buffer solution;

2) Adding polyethyleneimine with the final concentration of 0.1-0.3% (m/v), uniformly mixing, standing for 10-30 minutes, and centrifuging to obtain a supernatant;

3) Adding polyethylene glycol with the final concentration of 3% -5% (m/v) into the obtained supernatant, standing at 2-8 ℃ for more than 6 hours, and precipitating protein;

4) Centrifuging, removing supernatant, and dissolving precipitate with buffer solution.

The final concentration of polyethylenimine in step 2) was 0.2% (m/v) as described above.

The molecular weight of the polyethyleneimine in step 2) is 70000, as described above.

The final concentration of polyethylene glycol as described in step 3) was 4% (m/v) according to the method described above.

The polyethylene glycol in step 3) is polyethylene glycol 6000 according to the above method.

The preparation method of the buffer solution in the step 1) comprises the following steps:

to 20.+ -. 5mM Tris-HCl buffer pH 7.0.+ -. 0.2, 500.+ -. 50mM NaCl, 1%.+ -. 0.2% (v/v) Triton X-100 and 5%.+ -. 1% (v/v) glycerol were added at a final concentration;

preferably, the buffer solution in step 1) is prepared by the following steps:

to 20mM Tris-HCl buffer pH7.0 was added NaCl at a final concentration of 500mM, 1% (v/v) Triton X-100 and 5% (v/v) glycerol.

The preparation method of the buffer solution in the step 4) comprises the following steps:

adding 500+ -50 mM NaCl, 10+ -2 mM cysteine and 5% + -1% (v/v) glycerol to 20+ -5 mM Tris-HCl buffer at pH 7.0+ -0.2;

preferably, the buffer solution in step 4) is prepared by the following steps:

to 20mM Tris-HCl buffer pH7.0 was added NaCl, 10mM cysteine and 5% (v/v) glycerol at a final concentration of 500 mM.

Further, the prokaryotic expression engineering bacteria are gram-negative bacteria.

Further, the gram-negative bacterium is E.coli.

Further, the protein is porcine circovirus type 2 recombinant Cap protein.

The beneficial effects of the invention are as follows:

the purification method can effectively remove the endotoxin of the prokaryotic expression protein product without using expensive reagents and complicated operation steps required by the existing endotoxin removal method, the endotoxin removal rate is up to 99.9993 percent, the endotoxin concentration after purification is only 22.35EU/mL, and the endotoxin removal effect is obviously better than that of the patent application CN111233983A.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Detailed Description

EXAMPLE 1 recombinant Cap protein purification method

1. Bacterial strain

The strain is escherichia coli (Chaperone competent cells pGro/BL 21 (DE 3)), and the strain comprises an expression vector pET28a carrying a target gene (a gene for expressing Cap protein), wherein the sequence of the target gene is shown as SEQ ID NO.1 and is positioned at the XhoI (enzyme cutting site) of the vector.

SEQ ID NO.1:

atgacctacccgcgtcgtcgtttccgtcgtcgtcgtcaccgtccgcgttctcacctgggt 60

cagatcctgcgtcgtcgtccgtggctggttcacccgcgtcaccgttaccgttggcgtcgt 120

aaaaacggtatcttcaacacccgtctgtctcgtaccatcggttacaccgttaaaaaaacc 180

accgttcgtaccccgtcttggaacgttgacatgatgcgtttcaacatcaacgacttcctg 240

ccgccgggtggtggttctaacccgctgaccgttccgttcgaatactaccgtatccgtaaa 300

gttaaagttgaattctggccgtgctctccgatcacccagggtgaccgtggtgttggttct 360

accgctgttatcctggacgacaacttcgttaccaaagctaacgctctgacctacgacccg 420

tacgttaactactcttctcgtcacaccatcacccagccgttctcttaccactctcgttac 480

ttcaccccgaaaccggttctggaccgtaccatcgactacttccagccgaacaacaaacgt 540

aaccagctgtggctgcgtctgcagaccaccggtaacgttgaccacgttggtctgggtacc 600

gctttcgaaaactctatctacgaccaggactacaacatccgtatcaccatgtacgttcag 660

ttccgtgaattcaacctgaaagacccgccgctgaacccgaaataa 705

2. Fermentation

1. Seed liquid preparation

Preparing a seed liquid LB culture medium, sterilizing at 121 ℃ for 30min, and cooling for later use.

First-stage seed: single colonies were selected, inoculated with 30ml of LB medium, and simultaneously added with an antibiotic (final concentration: kana (kanamycin sulfate) 50ug/ml, CHL (chloramphenicol) 20 ug/ml), placed on a thermostatic shaker at 37℃and 220rpm, and cultured by shaking for 10 hours.

Secondary seed: according to the inoculation amount of 1:100, the first-stage seed liquid is respectively inoculated into a 1L or 2L triangular flask, and is placed in a constant-temperature shaking table at 32 ℃ and 220rpm, and the shaking culture is carried out until the OD range is between 1.5 and 2.5.

2. Fermentation

According to the volume ratio of 6:100, inoculating the secondary seeds into an escherichia coli culture medium, fermenting in a 75L fermentation tank (the liquid loading amount is 50L), simultaneously adding antibiotics (the final concentration is kana 50ug/ml, CHL 20ug/ml and arabinose 0.7 g/L), controlling pH to 6.90+/-0.1 by ammonia water, culturing at 37 ℃ by basic rotating speed and basic ventilation (200 rpm, 35L/min), and controlling DO to 40% +/-5% by adjusting DO (dissolved oxygen) when DO (dissolved oxygen) is lower than 40% for the first time. And simultaneously feeding the feed supplement liquid.

The formula of the feed is as follows:

when OD reaches 5+ -0.5, the flow acceleration is 1ml/min; when the OD reaches 7.5+/-2.5, the flow acceleration is 3ml/min; when the OD reached 14.+ -. 4, the flow acceleration was 6ml/min. When the OD reached 20.+ -. 1, the feed was stopped. When the pH showed signs of rebound (when the feed and alkali addition were stopped, the pH rose), the temperature was initially lowered to 32℃and the induction was carried out by adding isopropyl thiogalactoside (IPTG) at a final concentration of 1mM for 5 hours. During the induction, DO was controlled to be about 30% and the flow acceleration was 3ml/min. After the completion of the culture, 10000g of the cells were collected by centrifugation for 7 min.

Note that: the OD above was measured at a wavelength of 600 nm.

3. The purification method of the invention

1) E.coli cells containing PCV2 Cap protein which are induced to express are resuspended in solution A, the fresh weight of the cells is 100g, the volume of the solution A is 2L, the cells are fully crushed under high pressure by a homogenizing homogenizer, polyethylenimine (PEI, preferably molecular weight 70000) with the final concentration of 0.2% (m/v) is added, gently mixed, left at room temperature for 15 minutes, and the precipitate is removed by centrifugation.

The preparation method of the solution A comprises the following steps: to 20mM Tris-HCl buffer pH7.0 was added NaCl at a final concentration of 500mM, 1% (v/v) Triton X-100 and 5% (v/v) glycerol.

2) Adding PEG6000 with the final concentration of 4% (m/v) into the obtained supernatant solution, and standing at 4 ℃ for more than 6 hours to precipitate out the isoprotein.

3) 10000g, centrifuging at 4 ℃ for 5 minutes, removing the supernatant, and adding 100mL of solution B to dissolve the precipitate to obtain the purified PCV2 VLP sample.

The preparation method of the solution B comprises the following steps: to 20mM Tris-HCl buffer pH7.0 was added NaCl, 10mM cysteine and 5% (v/v) glycerol at a final concentration of 500 mM.

4) The endotoxin content of the protein sample was measured by using a limulus reagent.

5) Protein recovery and purity were checked by Elisa method and SDS-PAGE.

In solution a: triton X-100 functions to aid in the disruption of E.coli; naCl and glycerol play a role in stabilizing proteins; tris-HCl plays a role in maintaining pH.

In solution B: naCl and glycerol play a role in stabilizing proteins, and cysteine is used as a reducing agent to prevent the proteins from being oxidized.

4. The following control experiments were set up

#1: homogenizing thallus in solution A directly without treatment, standing at room temperature for 15 min, centrifuging to remove precipitate, collecting supernatant, and measuring endotoxin content with limulus reagent;

#2: on the basis of the purification method of the present invention, the concentration of PEI was adjusted to 0.02% (m/v), and the supernatant was collected by centrifugation and the endotoxin content was measured using a limulus reagent;

#3: on the basis of "third, purification method of the present invention", steps 2) and 3) are omitted, and the endotoxin content of the supernatant is measured with a limulus reagent;

#4: thirdly, omitting the PEI treatment step in the step 1) on the basis of the purification method of the invention; the rest steps are unchanged.

5. Results

As shown in Table 1, sample #4 corresponds to the purification mode of the method of the present invention. The PEI treatment can greatly reduce the endotoxin content, and PEI and PEG precipitation are matched for use, so that most of endotoxin can be removed, the purity of the product can be improved, and a certain recovery rate is ensured.

TABLE 1 endotoxin content for different treatments

In the absence of PEI treatment, sample #4 had more PEG precipitation steps than sample #1, with endotoxin content of sample #4 accounting for about 1.9% of the endotoxin content of sample # 1; in the case of both 0.2% PEI treatments, sample #5 was also a PEG precipitation step more than sample #3, but the endotoxin content of sample #5 was only about 0.3% of the endotoxin content of sample #3 (Table 2). It can be seen that PEI and PEG precipitation work together to produce a synergistic effect in endotoxin removal.

TABLE 2 endotoxin differentiation of samples before and after PEG precipitation without PEI treatment or with PEI treatment

In summary, the purification method of the invention does not involve expensive reagents and complicated operation steps required by the existing endotoxin removal method, and can effectively remove endotoxin of prokaryotic expression protein products and improve the purity of target proteins.

SEQUENCE LISTING

<110> Chengdu Tianbang biological products Co., ltd

Shiji Biotechnology (Nanjing) Co.,Ltd.

<120> a method for purifying a prokaryotic expression protein

<130> GY768-2020P0111760CC

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 705

<212> DNA

<213> porcine circovirus type 2 (porcine circovirus type 2)

<400> 1

atgacctacc cgcgtcgtcg tttccgtcgt cgtcgtcacc gtccgcgttc tcacctgggt 60

cagatcctgc gtcgtcgtcc gtggctggtt cacccgcgtc accgttaccg ttggcgtcgt 120

aaaaacggta tcttcaacac ccgtctgtct cgtaccatcg gttacaccgt taaaaaaacc 180

accgttcgta ccccgtcttg gaacgttgac atgatgcgtt tcaacatcaa cgacttcctg 240

ccgccgggtg gtggttctaa cccgctgacc gttccgttcg aatactaccg tatccgtaaa 300

gttaaagttg aattctggcc gtgctctccg atcacccagg gtgaccgtgg tgttggttct 360

accgctgtta tcctggacga caacttcgtt accaaagcta acgctctgac ctacgacccg 420

tacgttaact actcttctcg tcacaccatc acccagccgt tctcttacca ctctcgttac 480

ttcaccccga aaccggttct ggaccgtacc atcgactact tccagccgaa caacaaacgt 540

aaccagctgt ggctgcgtct gcagaccacc ggtaacgttg accacgttgg tctgggtacc 600

gctttcgaaa actctatcta cgaccaggac tacaacatcc gtatcaccat gtacgttcag 660

ttccgtgaat tcaacctgaa agacccgccg ctgaacccga aataa 705

Claims

1. A method for removing endotoxin from a prokaryotic expressed protein product, comprising: the protein is porcine circovirus type 2 recombinant Cap protein, and the method comprises the following steps:

1) Taking prokaryotic expression engineering bacteria, and homogenizing and crushing the bacteria after re-suspending the prokaryotic expression engineering bacteria by using a buffer solution; the prokaryotic expression engineering bacteria are escherichia coli; the preparation method of the buffer solution comprises the following steps: to 20.+ -. 5mM Tris-HCl buffer pH 7.0.+ -. 0.2, naCl, 1%.+ -. 0.2% (v/v) Triton X-100 and 5%.+ -. 1% (v/v) glycerol were added at a final concentration of 500.+ -. 50 mM;

2) Adding polyethyleneimine with the final concentration of 0.1% -0.3% (m/v), uniformly mixing, standing for 10-30 minutes, and centrifuging to obtain a supernatant;

3) Adding polyethylene glycol with a final concentration of 3% -5% (m/v) into the obtained supernatant, standing at 2-8 ℃ for more than 6 hours, and precipitating proteins;

4) Centrifuging, removing supernatant, and adding buffer solution to dissolve and precipitate; the preparation method of the buffer solution comprises the following steps: naCl, 10+ -2 mM cysteine and 5% + -1% (v/v) glycerol were added to a final concentration of 500+ -50 mM in 20+ -5 mM Tris-HCl buffer at pH 7.0+ -0.2.

2. The method of claim 1, wherein:

the final concentration of polyethylenimine in step 2) was 0.2% (m/v).

3. The method of claim 1, wherein:

the molecular weight of the polyethyleneimine in step 2) is 70000.

4. The method of claim 1, wherein:

the final concentration of the polyethylene glycol in the step 3) is 4% (m/v).

5. The method of claim 1, wherein:

the polyethylene glycol in the step 3) is polyethylene glycol 6000.

6. The method of claim 1, wherein:

to 20mM Tris-HCl buffer pH7.0, naCl, 1% (v/v) Triton X-100 and 5% (v/v) glycerol were added at a final concentration of 500. 500 mM.

7. The method of claim 1, wherein: the preparation method of the buffer solution in the step 4) comprises the following steps:

NaCl, cysteine 10mM and 5% (v/v) glycerol were added to 20mM Tris-HCl buffer at pH7.0 at a final concentration of 500. 500 mM.