CN112279899A - Method for purifying prokaryotic expression protein - Google Patents

Abstract

The invention discloses a method for purifying prokaryotic expression protein, which comprises the following steps: 1) taking prokaryotic expression engineering bacteria, carrying out heavy suspension by using a buffer solution, and homogenizing and crushing the bacteria; 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 for more than 6 hours at the temperature of 2-8 ℃, 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 present invention belongs to the field of recombinant protein purification.

Background

Prokaryotic expression, namely protein expression through prokaryotic expression engineering bacteria, is a common protein production mode at present and is mainly applied to the production of products such as enzyme, subunit vaccines and the like. The cells used for prokaryotic expression include escherichia coli, bacillus, streptomycete, cyanobacteria and the like, wherein the escherichia coli is the most widely used prokaryotic expression cell in the industry at present.

Escherichia coli is gram-negative bacteria, a large amount of endotoxin can be generated when recombinant protein is expressed, the chemical nature of the endotoxin is Lipopolysaccharide (LPS), the LPS has extremely strong heat source, and trace LPS can cause stress reactions such as high heat, diarrhea, shock and the like when entering an animal body, so if prokaryotic expression protein needs to enter a human body or the animal body to play a role, the removal of the endotoxin is one of the keys for purifying the protein.

The current method for removing the bacterial biotin in the solution mainly comprises the following steps: (1) detoxification function: the detoxification mechanism mainly comprises the steps of degrading bacterial endotoxin into non-toxic fragments by enzyme, forming reversible complexes between the bacterial endotoxin and protein, and generating detoxification by acid-base hydrolysis, wherein the method can influence the activity of the protein to different degrees, and the detoxified endotoxin has the possibility of toxicity reversion; (2) sucrose density gradient centrifugation technique: the different existence forms, molecular sizes, densities, sedimentation coefficients and molecular weights of the endotoxin cause certain difficulties in the application of the method; (3) affinity chromatography techniques: the method has the advantages of mild and simple conditions, and has the functions of purifying and concentrating the antigen, but also has the defects of expensive reagent, complex operation, poor ligand and glue bonding stability and the like; (4) ion exchange chromatography: there are problems of expensive equipment, large material loss, and the like.

Porcine circovirus type 2 (PCV2) is an icosahedral, non-enveloped, single-stranded circular DNA virus with a diameter size of about 17-22 nm. PCV2 is able to infect many tissues and organs of pigs, of which lymphoid organs are the most common organ damaged, causing damage to the immune system of pigs and thereby causing secondary infections including porcine reproductive and respiratory syndrome, pseudorabies and other bacterial infectious diseases. Open reading frame 2(ORF2) of PCV2 encodes the only structural protein of the virus, the Cap protein, which is also the major immunoantigenic protein of PCV2, 5 Cap proteins make up a 5-mer, and 12 5-mer Cap proteins can 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 the genetic material of the virus, but have a structure similar to that of the virus particles.

The escherichia coli expression system is an ideal expression system for producing Cap protein due to the advantages of low production cost, simple production condition requirement, short period and the like, but the removal of endotoxin still has various problems of high cost, complex operation and the like involved in the endotoxin removal method, 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, which adopts cation exchange chromatography to purify Cap protein and remove endotoxin, wherein the endotoxin content is below 2.5 EU/mL.

Disclosure of Invention

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

The technical scheme of the invention is as follows:

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

1) taking prokaryotic expression engineering bacteria, carrying out heavy suspension by using a buffer solution, and homogenizing and crushing the bacteria;

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 for more than 6 hours at the temperature of 2-8 ℃, and precipitating protein;

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

As in the previous method, the final concentration of polyethyleneimine in step 2) was 0.2% (m/v).

The molecular weight of the polyethyleneimine in step 2) is 70000, as in the previous method.

The method as described above, wherein the final concentration of polyethylene glycol in step 3) is 4% (m/v).

The method as described above, wherein the polyethylene glycol in step 3) is polyethylene glycol 6000.

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

adding NaCl with a final concentration of 500 +/-50 mM, Triton X-100 with a final concentration of 1% +/-0.2% (v/v) and glycerol with a final concentration of 5% +/-1% (v/v) into 20 +/-5 mM Tris-HCl buffer solution with a pH value of 7.0 +/-0.2;

preferably, the buffer solution of step 1) is prepared by the following method:

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

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

adding NaCl with a final concentration of 500 +/-50 mM, cysteine with a final concentration of 10 +/-2 mM and glycerol with a final concentration of 5% +/-1% (v/v) into 20 +/-5 mM Tris-HCl buffer solution with a pH of 7.0 +/-0.2;

preferably, the buffer solution of step 4) is prepared by the following method:

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

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

Further, the gram-negative bacterium is escherichia coli.

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

The invention has the beneficial effects that:

the purification method can effectively remove the endotoxin of the prokaryotic expression protein product without using expensive reagents and complex operation steps required by the existing endotoxin removal method, the endotoxin removal rate is up to 99.9993 percent, the purified endotoxin concentration is only 22.35EU/mL, and the endotoxin removal effect is obviously better than that of the patent application CN 111233983A.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Detailed Description

Example 1 purification of recombinant Cap protein

Firstly, bacterial strain

The strain is escherichia coli (Chaperone component cells pGro7/BL21(DE3)), 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) position 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

Secondly, fermentation

1. Seed liquid preparation

Preparing LB culture medium of seed liquid, sterilizing at 121 deg.C for 30min, and cooling.

First-stage seed: single colonies were selected, inoculated into 30ml of LB medium, and antibiotics (final concentration: kana (kanamycin sulfate) 50ug/ml, CHL (chloramphenicol) 20ug/ml) were added thereto, and then cultured by shaking at 37 ℃ and 220rpm for 10 hours.

Secondary seeds: according to the inoculation amount of 1:100, the first-level seed liquid is respectively inoculated into a triangular flask with the volume of 1L or 2L, and is placed on a constant temperature shaking table with the temperature of 32 ℃ and the rpm of 220, and is cultured by shaking bacteria until the OD range is between 1.5 and 2.5.

2. Fermentation of

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

The formula of the feed supplement is as follows:

when OD reaches 5 +/-0.5, the flow rate is 1 ml/min; when OD reaches 7.5 +/-2.5, the feeding speed is 3 ml/min; when the OD reached 14. + -.4, the flow rate was 6 ml/min. Feeding was stopped when OD reached 20. + -.1. When there was a sign of pH rebound (pH rise after stopping feeding and base addition), the temperature was initially lowered to 32 ℃ and induction was carried out by adding Isopropylthiogalactoside (IPTG) at a final concentration of 1mM for 5 h. During the induction process, the DO is controlled to be about 30 percent, and the flow rate is 3 ml/min. After the culture, the cells were harvested by centrifugation at 10000g for 7 min.

Note: the OD values were measured at a wavelength of 600 nm.

Thirdly, the purification method of the invention

1) The escherichia coli thallus containing PCV2 Cap protein which is induced to express is resuspended by solution A, the fresh weight of the thallus is 100g, the volume of the solution A is 2L, the thallus is fully crushed by a homogenizing homogenizer under high pressure, polyethyleneimine (PEI, preferably with the molecular weight of 70000) with the final concentration of 0.2 percent (m/v) is added, the mixture is gently mixed, the mixture is placed for 15 minutes at room temperature, 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, 500mM NaCl, 1% (v/v) Triton X-100 and 5% (v/v) glycerol were added to the final concentration.

2) The obtained supernatant solution was added with PEG6000 to a final concentration of 4% (m/v), and left at 4 ℃ for 6 hours or more to precipitate the first-class protein.

3)10000g, 4 ℃ for 5 minutes, removing the supernatant, adding 100mL of solution B to dissolve the precipitate, and obtaining 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 at a final concentration of 500mM, cysteine at 10mM, and glycerol at 5% (v/v).

4) Endotoxin content in protein samples was determined using limulus reagent.

5) The recovery rate and purity of the protein are detected by an Elisa method and SDS-PAGE.

In solution a: triton X-100 plays a role in helping to break Escherichia coli; NaCl and glycerol play a role in stabilizing proteins; Tris-HCl serves to maintain pH.

In solution B: NaCl and glycerol play a role in stabilizing proteins, and cysteine serves as a reducing agent to prevent protein oxidation.

Fourth, the following control experiment was set up

# 1: directly homogenizing the thallus in the solution A without treatment, standing at room temperature for 15 minutes, centrifuging to remove precipitate, taking the supernatant, and measuring the endotoxin content by using a limulus reagent;

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

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

# 4: thirdly, on the basis of the purification method of the invention, the step of PEI treatment in the step 1) is omitted; the remaining steps were unchanged.

Five results

As shown in Table 1, sample #4 corresponds to the purification method of the present invention. Therefore, the PEI treatment can greatly reduce the content of endotoxin, and the PEI and PEG precipitate are used in a matching way, so that most of the endotoxin can be removed, the product purity can be improved, and a certain recovery rate can be ensured.

TABLE 1 endotoxin content for different treatment regimes

In the absence of PEI treatment, the #4 sample had more PEG precipitation steps than the #1 sample, and the endotoxin content of the #4 sample was about 1.9% of the endotoxin content of the #1 sample; the #5 sample also had more PEG precipitation steps than the #3 sample with all 0.2% PEI treatments, but the endotoxin content of the #5 sample was only about 0.3% of the endotoxin content of the #3 sample (table 2). Therefore, the PEI and the PEG precipitate are used in a matching way, and a synergistic effect is generated in the aspect of endotoxin removal.

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

In summary, the purification method of the present invention can effectively remove the endotoxin in the prokaryotic expression protein product and improve the purity of the target protein without involving expensive reagents and complicated operation steps required by the existing endotoxin removal method.

SEQUENCE LISTING

<110> Chengdu Tianbang biological products GmbH

Shiji Biotechnology (Nanjing) Co.,Ltd.

<120> a method for purifying 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 (10)

1. A method for purifying a prokaryotic expression protein, comprising: the method comprises the following steps:

1) taking prokaryotic expression engineering bacteria, carrying out heavy suspension by using a buffer solution, and homogenizing and crushing the bacteria;

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 for more than 6 hours at the temperature of 2-8 ℃, and precipitating protein;

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

2. The method of claim 1, wherein:

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

3. The method of claim 1, wherein:

the molecular weight of the polyethyleneimine in the 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:

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

adding NaCl with a final concentration of 500 +/-50 mM, Triton X-100 with a final concentration of 1% +/-0.2% (v/v) and glycerol with a final concentration of 5% +/-1% (v/v) into 20 +/-5 mM Tris-HCl buffer solution with a pH value of 7.0 +/-0.2;

preferably, the buffer solution of step 1) is prepared by the following method:

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

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

adding NaCl with a final concentration of 500 +/-50 mM, cysteine with a final concentration of 10 +/-2 mM and glycerol with a final concentration of 5% +/-1% (v/v) into 20 +/-5 mM Tris-HCl buffer solution with a pH of 7.0 +/-0.2;

preferably, the buffer solution of step 4) is prepared by the following method:

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

8. The method of claim 1, wherein: the prokaryotic expression engineering bacteria are gram-negative bacteria.

9. The method of claim 8, wherein: the gram-negative bacterium is escherichia coli.

10. The method of any of claims 1 to 9, wherein: the protein is porcine circovirus type 2 recombinant Cap protein.