CN115322978A - High-density fermentation expression and separation and purification method of totipotent nuclease - Google Patents
High-density fermentation expression and separation and purification method of totipotent nuclease Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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- C12N15/09—Recombinant DNA-technology
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- C07K2319/35—Fusion polypeptide containing a fusion for enhanced stability/folding during expression, e.g. fusions with chaperones or thioredoxin
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Abstract
The invention relates to the technical field of nuclease processing, and discloses a high-density fermentation expression and separation and purification method of totipotent nuclease, which comprises the following steps: s1, constructing thalli, and constructing a plasmid containing a totipotent nuclease target gene; s2, fermenting and expressing; s3, pretreating fermentation thalli; s4, chromatographic purification, and purifying the supernatant containing the target protein by affinity chromatography. According to the method for high-density fermentation expression and separation and purification of the totipotent nuclease, the high-expression-amount secreted nuclease is obtained through escherichia coli fermentation for expressing the totipotent nuclease, the high-purity totipotent nuclease is obtained by adopting a chromatography purification method, and the 6 XHis-tag and SUMO-tag double tags are added on a gene sequence for expressing the nuclease, so that the production efficiency and the protein purity of the totipotent nuclease can be obviously improved, the production time is shortened, the cost is reduced, and the industrial production and application of the totipotent nuclease are facilitated.
Description
Technical Field
The invention relates to the technical field of nuclease processing, in particular to a high-density fermentation expression and separation and purification method of totipotent nuclease.
Background
The totipotent nuclease is also called broad-spectrum nuclease, is a genetically engineered endonuclease derived from Serratia marcocens, can degrade all forms of DNA and RNA (including single-stranded, double-stranded, linear, circular, natural and denatured nucleic acids) to generate 5-monophosphate oligonucleotide with the length of 3-5 bases, and does not have base recognition specificity. Therefore, the method is mainly applied to efficiently removing nucleic acid residues in samples or products in the field of biological medicine, improves the purity of the samples and the biological efficacy of the products, and is widely applied to various life science researches and vaccine, protein and polysaccharide pharmaceutical industries.
At present, part of commercial totipotent nuclease comes from secretion expression of recombinant pichia pastoris, and part of commercial totipotent nuclease adopts an escherichia coli inclusion body for expression. The culture conditions and the fermentation process of the pichia pastoris are complex, the culture period is long, and the cost is high; the adoption of the expression of the escherichia coli inclusion body requires the adoption of an inclusion body renaturation purification process, the process is complex, the purification period is long, the degradation of the obtained enzyme protein is fast, the production cost is high, and the enzyme activity yield is low, so that a method for high-density fermentation expression and separation purification of the totipotent nuclease is provided.
Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a method for high-density fermentation expression and separation and purification of totipotent nuclease, which has the advantages of remarkably improving the production efficiency and protein purity of totipotent nuclease, shortening the production time, reducing the cost, being beneficial to industrial production and application of totipotent nuclease and solving the problems of improving the secretory expression and high-purity separation of totipotent nuclease.
(II) technical scheme
The invention provides the following technical scheme: a method for high-density fermentation expression and separation and purification of totipotent nuclease, wherein the purification method comprises the following steps:
s1, constructing thalli, and constructing a plasmid containing a totipotent nuclease target gene, wherein the target gene contains a totipotent nuclease sequence, and the front end of the sequence is provided with a sequence for coding a 6XHis tag and a SUMO tag.
S2, fermentation expression, namely inoculating the selected monoclonal strains on the resistant plate to a shake flask culture medium for growth, inoculating the strains to a fermentation tank (the inoculation amount is 1-10%) when the strain concentration meets the requirement of transferring the strains, performing fermentation culture for growth, adding an inducer isopropyl-P-D-thiogalactose for induction expression when the strain amount reaches a proper concentration, wherein the expressed protein is soluble expression, harvesting bacteria liquid after the fermentation expression is finished, centrifuging, and collecting bacteria.
S3, pretreating the fermentation thalli, resuspending the thalli obtained in the step two in a proper resuspension solution, crushing the thalli to ensure that the treated thalli are all crushed, centrifuging the crushed solution, removing the precipitate, and collecting the supernatant containing the target protein.
And S4, performing chromatographic purification, namely purifying the supernatant containing the target protein through affinity chromatography, performing enzyme digestion by SUMO protease, then performing affinity chromatography again, and collecting flow-through liquid to obtain the high-purity totipotent nuclease.
Further, in the step S1, the expression strain which can be used includes but is not limited to BL21 (DE 3), rosetta-gami (DE 3), origimiB (DE 3) or Rosetta (DE 3) E.coli modified expression strain, the expression plasmid is pET28a-6XHis-SUMO-NucA which is self-constructed, and the plasmid resistance is kanamycin resistance.
Further, the method described in step S2 is characterized in that the seed shake flask culture medium and the fermentation culture medium contain five substances including carbon sources, nitrogen sources, water, inorganic salts, and trace elements, and the fermentation process requires a feed supplement, and the specific formula is as follows: glycerol: 1-10g/L, peptone: 1-20g/L, yeast powder: 1-30g/L, potassium dihydrogen phosphate: 1-10g/L, magnesium sulfate: 1-5g/L and zinc chloride: 1-3g/L, calcium sulfate: 1-5g/L.
Further, in step S2, the induction start time: fermenting and culturing until OD600 reaches 15-20, and adding inducer;
and (3) inducing conditions: after the inducer is added, the final concentration of isopropyl-P-D-thiogalactose (TPTG) after the reactor is 0.1-1mM/L, and the induction temperature is 30-18 ℃.
Furthermore, in step S2, the time from inoculation to induction culture starting is 0-8h, the induction time after the induction culture starting is 5-20h, and the OD600 is 50-120 when the tank is placed.
Further, in step S1 or S2, the culture temperature is 35-37 ℃ before the plate monoclonal picking, the seed shake flask culture and the fermenter induction.
Further, in step S3, PBS or Tris-HCl buffer solution is selected as the re-suspension solution of the thalli, and the pH is adjusted to 7.0-8.5.
Further, in step S3, the cell bodies may be disrupted as follows: 1) Crushing by a homogenizer; 2) Carrying out ultrasonic crushing; 3) Lysozyme cracking;
the broken liquid after the thalli are broken can be centrifuged by adopting the following centrifugal machines: 1) A tubular centrifuge; 2) A disk centrifuge; 3) A cup centrifuge, which ensures that the centrifugal force is more than or equal to 7000g.
Furthermore, in step S4, the affinity chromatography fillers selected are chelating metal affinity chromatography fillers, the metal ions may be any one of Cu2+, ni2+, zn2+, and Co2+, and the matrix may be a cross-linked product of dextran or agarose.
And further, sequentially carrying out affinity chromatography on the supernatant obtained in the step S3, collecting eluent, adding SUMO enzyme into the collected eluent for carrying out label enzyme digestion, carrying out affinity chromatography on the enzyme digestion solution through the same chromatographic column as the first step after the completion of the electrophoretic detection enzyme digestion, and collecting flow-through solution to obtain the high-purity all-round nuclease.
Further, according to the method described in step S4, the affinity chromatography step is divided into: balancing the chromatographic column, loading a sample, washing impurities and eluting. The eluent comprises the following components: 20mM Tri-HCl (pH 6.5-8.5), 0.5M sodium chloride, 10-300mM imidazole.
Compared with the prior art, the invention provides a method for high-density fermentation expression and separation and purification of totipotent nuclease, which has the following beneficial effects:
1. according to the method for high-density fermentation expression and separation and purification of the totipotent nuclease, the high-expression-amount secreted nuclease is obtained through escherichia coli fermentation for expressing the totipotent nuclease, the high-purity totipotent nuclease is obtained by adopting a chromatography purification method, and the 6 XHis-tag and SUMO-tag double tags are added on a gene sequence for expressing the nuclease, so that the production efficiency and the protein purity of the totipotent nuclease can be obviously improved, the production time is shortened, the cost is reduced, and the industrial production and application of the totipotent nuclease are facilitated.
2. The process realizes the soluble expression of the totipotent nuclease in the escherichia coli thallus, and the supernatant containing the target protein can be obtained by centrifugation after the thallus is crushed.
Drawings
FIG. 1 is a schematic chromatogram of example 3 of the present invention;
FIG. 2 is a schematic chromatogram of example 4 of the present invention;
FIG. 3 is a schematic diagram showing the completion of enzyme digestion in the electrophoresis detection after dialysis according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to FIGS. 1-3, a method for high-density fermentation expression and separation and purification of a totipotent nuclease, the purification method comprising the following steps:
s1, constructing thalli, and constructing a plasmid containing a totipotent nuclease target gene, wherein the target gene contains a totipotent nuclease sequence, and the front end of the sequence is provided with a sequence for coding a 6XHis tag and a SUMO tag.
S2, fermentation expression, namely inoculating the selected monoclonal strain on the resistant plate to a shake flask culture medium for growth, inoculating the strain to a fermentation tank (the inoculation amount is 1% -10%) when the strain concentration meets the requirement of transferring the strain, performing fermentation culture for growth, adding an inducer isopropyl-P-D-thiogalactose for induction expression when the strain amount reaches the proper concentration, wherein the expressed protein is soluble expression, harvesting a bacterial liquid after the fermentation expression is finished, centrifuging, and collecting thalli.
S3, pretreating the fermentation thalli, resuspending the thalli obtained in the step two in a proper resuspension solution, crushing the thalli to ensure that the treated thalli are all crushed, centrifuging the crushed solution, removing the precipitate, and collecting the supernatant containing the target protein.
And S4, performing chromatographic purification, namely purifying the supernatant containing the target protein by affinity chromatography, performing enzyme digestion by SUMO protease, and then performing affinity chromatography again to collect flow-through liquid so as to obtain the high-purity totipotent nuclease.
EXAMPLE 1 selection of target Strain
1. And (4) screening qualified target cloning bacteria.
The plasmid pET28a-6xHis-SUMO-NucA identified by restriction enzyme is transformed into competent cells of Escherichia coli host bacteria BL21 (DE 3), coated on a kanamycin resistant plate, cultured overnight at 37 ℃ and screened to obtain positive clones.
2. Media preparation
1) 100ml seed medium: 10g/L of Tryptone; yeast extract,5g/L; naCl,10g/L.
2) 200ml fermenter feed medium: yeast extract,150g/L; 300g/L of glycerol;
3) Preparing a fermentation liquid culture medium: trypone, 28.32g; yeast extract,56.64g; glycerol, 12.00g; 5.20g of monopotassium phosphate; dipotassium hydrogen phosphate trihydrate, 32.86; magnesium sulfate, 0.96g; antifoam, 1mL (added directly to the fermentor); dissolving and metering to 2.4L, and pouring into a cleaned 5L glass fermentation tank.
| Name of material | Standard ratio g/L | Instruction amount (g) |
| Glycerol | 5.00 | 12.00 |
| Peptone | 11.8 | 28.32 |
| Yeast powder | 23.6 | 56.64 |
| KH 2 PO 4 | 2.20 | 5.20 |
| K 2 HPO 4 ·3H 2 0 | 12.6 | 32.86 |
| Magnesium sulfate | 0.4 | 0.96 |
| Defoaming agent | 1ml | 1ml |
Sterilizing the above culture medium at 121 deg.C for 30min, respectively placing the seed shake flask and the fermentation tank under a clean bench at room temperature after sterilization, and adding kanamycin to final concentration of 50mg/L. The 5L glass fermentor was connected and the electrode calibration was adjusted to the inoculation state for fermentation use.
EXAMPLE 2 culturing and expression of the target bacterium
1. Inoculating the screened positive clone single colony to a seed shake flask, and carrying out shake culture at 37 ℃ and 250rpm overnight to obtain a shake flask seed solution with OD of 0.5-1.5.
2. Inoculating the qualified seed solution to a 5L glass fermentation tank, culturing at 37 ℃, controlling DO to be more than or equal to 30%, adding an inducer IPTG to a final concentration of 0.5mmol/L when culturing is carried out until OD600=15-20, cooling to 30 ℃, continuing culturing for 6h (feeding in a middle stream) until OD600=30-35 after fermentation is finished, placing the tank into the tank, and centrifuging to collect thalli (10000 rpm multiplied by 10 min).
3. Crushing of thallus
Adding 50g of thalli into a resuspension (20 mM Tris-HCl,500mM NaCl, pH8.4), wherein the mass ratio of the thalli to the resuspension is 1:5, mixing uniformly, taking 1ml of reserved sample for electrophoresis detection, homogenizing and crushing for 3 times by using a homogenizer (800 bar pressure), collecting a bacteria breaking liquid, adjusting the pH to 8.0, centrifuging at low temperature and high speed (10000rpm, 30min and 4 ℃), collecting a supernatant after centrifugation, and carrying out chromatography purification (4 ℃), and taking 1ml of reserved sample for electrophoresis detection.
EXAMPLE 3 isolation and purification of target protease
1. One-step chromatographic purification of target protease
1) Balancing: the TED Ni column was washed with purified water for 15CV and then equilibrated with an equilibration solution (20 mM Tris-HCl,500mM NaCl, pH 8.4) for 10CV until the UV absorption was zeroed (A280)
2) Sampling: and (4) loading the bacteria-breaking supernatant at the speed of 10ml/min, and collecting the effluent for electrophoresis.
3) Balancing: the equilibrium solution (20 mM Tris-HCl,500mM NaCl,20mM imidazole, pH 8.4) was added in an amount of 3ml
And (4) balancing 10CV at the flow rate/min until the ultraviolet absorption reaches a base line, and collecting the balance liquid for electrophoresis.
4) Elution 1: the eluate (20 mM Tris-HCl,500mM NaCl,50mM imidazole, pH 8.4) was eluted at 2ml/min, and the eluted peak was collected for electrophoresis.
5) And (3) elution 2: the eluate (20 mM Tris-HCl,500mM NaCl,300mM imidazole, pH 8.4) was eluted at 2ml/min, and the eluted peak was collected for electrophoresis.
6) Regeneration and regeneration, washing the column with 100% eluent (20 mM Tris-HCl,500mM NaCl,500mM imidazole, pH 8.4) for 5CV, washing with ultrapure water for 10CV, and washing with 20% ethanol for 5CV to preserve the column and packing.
2; and (3) determining target protease by electrophoresis: and (3) reserving a sample for the heavy suspension, loading the sample for reserving the sample after homogenizing, loading the sample for flowing through the solution, and carrying out SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) on the eluate, wherein the electrophoresis result is shown in the third picture. The electrophoresis result shows that the target protease is concentrated in the eluent 2, and the protein concentration and the protein amount in the collected eluent are measured by adopting an ultraviolet absorption method (see table 1).
Enzyme digestion: purification was carried out by adding Sumo enzyme (1 unit of SUMO enzyme can cleave 100ug of protein) to the eluate according to the weight of protein in the eluate, overnight in 20mM Tris-HCl,300mM NaCl, pH 8.1 dialysis solution, and then replacing the dialysis solution 20mM Tris-HCl,500mM NaCl, pH 8.1 the next day, and then detecting the completion of cleavage by electrophoresis (see FIG. 3).
The lane information in FIG. 3 is as follows:
1: bacterial suspension
2: sample loading liquid after bacteria breaking and centrifugation
3: first step nickel column chromatography flow-through
4: eluent 1 (50 mM imidazole elution)
5: eluent 2 (300 mM imidazole elution)
6: eluent 2 concentrated small sample
7: eluent 2 after overnight digestion
8: second step nickel column equilibrium liquid
9: second step nickel column sample loading flow-through liquid
10: second step nickel column sample loading balance liquid
11: second step nickel column cleaning solution
12: protein Marker
13: universal nuclease reference substance
Example 4 Secondary chromatography of target protease
1) And (3) secondary chromatography: and (4) performing nickel column chromatography as same as the first time, and collecting flow-through liquid to obtain the high-purity totipotent nuclease protein liquid (with the purity of 99 percent, see electrophoresis chart III). After completion, the column and the packing were directly washed with 100% washing solution (20 mM Tris-HCl,500mM NaCl,500mM imidazole, pH 8.4) for 5CV, washed with ultrapure water for 10CV, and washed with 20% ethanol for 5CV preservation.
2) The concentration and amount of the collected fluid protein were measured by UV absorption (see Table I).
Table one: chromatography yield (50 g of cells)
| Chromatography step | Concentration (mg/ml) | Volume (ml) | Total protein amount (mg) |
| First step of nickel column eluent | 6 | 60 | 360 |
| Second step nickel column flow-through liquid | 3 | 58 | 174 |
Remarking: finally, the target protein 174mg is obtained from 50g of the thalli, and the yield is 3.48mg/g
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (9)
1. A method for high-density fermentation expression and separation and purification of totipotent nuclease is characterized in that: the purification method comprises the following steps:
s1, constructing thalli, namely constructing a plasmid containing a totipotent nuclease target gene, wherein the target gene contains a totipotent nuclease sequence, and the front end of the sequence is provided with a sequence for coding a 6xHis tag and a SUMO tag;
s2, performing fermentation expression, namely inoculating the selected monoclonal strain on the resistant plate to a shake flask culture medium for growth, inoculating the strain to a fermentation tank (the inoculation amount is 1% -10%) when the strain concentration meets the requirement of transferring the strain, performing fermentation culture for growth, adding an inducer isopropyl-P-D-thiogalactose for induction expression when the strain amount reaches the proper concentration, wherein the expressed protein is soluble expression, harvesting a bacterial liquid after the fermentation expression is finished, centrifuging, and collecting thalli;
s3, pretreating the fermented thalli, namely resuspending the thalli obtained in the step two in a proper resuspension solution, crushing the thalli to ensure that the treated thalli are all crushed, then centrifuging the crushed solution, removing precipitates, and collecting a supernatant containing the target protein;
and S4, performing chromatographic purification, namely purifying the supernatant containing the target protein by affinity chromatography, performing enzyme digestion by SUMO protease, and then performing affinity chromatography again to collect flow-through liquid so as to obtain the high-purity totipotent nuclease.
2. The method for high-density fermentation expression, separation and purification of totipotent nuclease according to claim 1, wherein the method comprises the following steps: in the step S1, the expression strain which can be used includes but is not limited to BL21 (DE 3), rosetta-gami (DE 3), originib (DE 3) or Rosetta (DE 3) E.coli modified expression strain, the expression plasmid is pET28a-6XHis-SUMO-NucA which is self-constructed, and the plasmid resistance is kanamycin resistance.
3. The method for high-density fermentation expression and separation and purification of a pluripotent nuclease according to claim 1, wherein the method comprises the following steps: the method in the step S2 is characterized in that the seed shake flask culture medium and the fermentation culture medium contain five substances including a carbon source, a nitrogen source, water, inorganic salt and trace elements, and feeding materials are required in the fermentation process, and the specific formula is as follows: glycerin: 1-10g/L, peptone: 1-20g/L, yeast powder: 1-30g/L, monopotassium phosphate: 1-10g/L, magnesium sulfate: 1-5g/L and zinc chloride: 1-3g/L, calcium sulfate: 1-5g/L.
4. The method for high-density fermentation expression, separation and purification of totipotent nuclease according to claim 1, wherein the method comprises the following steps: in step S2, the induction start time: fermenting and culturing until OD600 reaches 15-20, and adding inducer;
induction conditions are as follows: after the inducer is added, the final concentration of isopropyl-P-D-thiogalactose (TPTG) in the reactor is 0.1-1mM/L, and the induction temperature is 30-18 ℃.
5. The method for high-density fermentation expression, separation and purification of totipotent nuclease according to claim 1, wherein the method comprises the following steps: in the step S2, the time from inoculation to induction culture starting is 0-8h, the induction time after the induction culture starting is 5-20h, and the OD600 is 50-120 when the tank is placed.
6. The method for high-density fermentation expression, separation and purification of totipotent nuclease according to claim 1, wherein the method comprises the following steps: in step S1 or S2, the culture temperature is 35-37 ℃ before the monoclonal is picked from the flat plate, and the seeds are cultured in a shake flask and are induced in a fermentation tank.
7. The method for high-density fermentation expression and separation and purification of a pluripotent nuclease according to claim 1, wherein the method comprises the following steps: in step S3, PBS or Tris-HCl buffer solution is selected as the re-suspension solution of the thalli, and the pH is adjusted to 7.0-8.5.
8. The method for high-density fermentation expression and separation and purification of a pluripotent nuclease according to claim 1, wherein the method comprises the following steps: in step S3, the following method can be adopted for disrupting the cells: 1) Crushing by a homogenizer; 2) Ultrasonic crushing; 3) Lysozyme cracking;
the broken liquid after the thalli are broken can be centrifuged by adopting the following centrifuges: 1) A tubular centrifuge; 2) A disk centrifuge; 3) A cup centrifuge, which ensures that the centrifugal force is more than or equal to 7000g.
9. The method for high-density fermentation expression and separation and purification of a pluripotent nuclease according to claim 1, wherein the method comprises the following steps: in the step S4, the selected affinity chromatography fillers are chelating metal affinity chromatography fillers, the metal ions can be any one of Cu < 2+ >, ni < 2+ >, zn < 2+ > and Co < 2+ >, and the matrix can be a cross-linked product of glucan or agarose.
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