CN106636024A - Purified renaturation method of L-aspartic acid oxidase inclusion bodies - Google Patents
Purified renaturation method of L-aspartic acid oxidase inclusion bodies Download PDFInfo
- Publication number
- CN106636024A CN106636024A CN201610864870.2A CN201610864870A CN106636024A CN 106636024 A CN106636024 A CN 106636024A CN 201610864870 A CN201610864870 A CN 201610864870A CN 106636024 A CN106636024 A CN 106636024A
- Authority
- CN
- China
- Prior art keywords
- aspartic acid
- inclusion body
- buffer solution
- purified
- renaturation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
- C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
- C12N9/0022—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
- C12Y104/03—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with oxygen as acceptor (1.4.3)
- C12Y104/03016—L-Aspartate oxidase (1.4.3.16)
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a purified renaturation method of L-aspartic acid oxidase inclusion bodies. The purified renaturation method comprises the following steps that precipitates of the inclusion bodies are collected, and fully washed, denatured proteins are purified by a nickel column, the purified denatured proteins are diluted with a buffer solution D until the protein concentration is lower than 1mg/ml, and placed in a dialysis buffer solution, the dialysis temperature is kept at 4 DEG C, dialysis lasts for more than 12 hours, and supernatants are centrifugally collected. Finally, the supernatants are concentrated by an ultrafiltration tube, and then monomeric proteins and agrin are purified and separated by a molecular sieve to obtain homogeneous natural active proteins. The purified renaturation method of the L-aspartic acid oxidase inclusion bodies disclosed by the invention solves the problem of L-aspartic acid oxidase renaturation, purification and renaturation on the L-aspartic acid oxidase inclusion bodies are carried out with a convenient and fast method, the inclusion bodies are further rinsed and purified by the nickel column to obtain the denatured proteins with higher purity, denaturants are removed gradually through a one-step dialysis renaturation process, the proteins are folded to be protein molecules with activity, the renaturation efficiency of the proteins is not less than10%, and then the protein molecules are further dialyzed and purified by the molecular sieve to obtain high-purity enzymes with activity.
Description
Technical field
The present invention relates to a kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method, belongs to the egg of bioengineering field
White matter expression and purification technique.
Background technology
In protein engineering field, the albumen for how obtaining BA is a critical problem, and this is for grinding
Study carefully the function of gene, develop protein therapeutic and prepare biomaterial and all have great importance.Most popular table
Up to host e. coli, albumen often occurs with insoluble inclusion bodies, how to obtain from renaturation in inactive inclusion body
There must be the soluble protein of biologically active, become a great problem of protein engineering field.
When albumen great expression in prokaryotic hosts, due to the restriction of host cell interior folding factor, such as albumen table
Improper up to too fast or protein folding physicochemical environment, causing Partial Protein false folding or fail that folding completes to be formed can not
Molten state and form the inclusion body of precipitation.Inclusion body can be assembled in the cytoplasm of bacterium and periplasmic space, generally spherical,
Due to host and the difference of condition of culture, its size is also differed, and general diameter range is 0.1-0.8 μm, and compact structure can
Opposing intracellular protease enzymolysis.Spongiform inclusion body is simultaneously containing active component and inactive component, it may have part is raw
Thing activity.
L-Aspartic acid oxidizing ferment is present in bacterium, ancient bacterium, and in plant and zooblast, the enzyme is nicotinoyl in bacterium
First enzyme of amine dinucleotides de novo synthesis.At present, various L-Aspartic acid oxidizing ferment without source and function exist
Heterogenous expression has been carried out in Escherichia coli, but yield is relatively low, and most of albumen exists with inactive inclusion bodies.But
There is no effective method to carry out the renaturation of inclusion body, seriously restrict fermentoid application industrially.
The content of the invention
It is an object of the invention to solve above-mentioned technical problem, there is provided a kind of effective L-Aspartic acid oxidizing ferment is forgiven
Body purification renaturation method.
The purpose of the present invention is achieved through the following technical solutions：
A kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method, comprises the steps：
The collection of S1, inclusion body precipitation, L-Aspartic acid oxidase gene is carried out to optimize for e. coli codon, is passed through
Full genome synthesis is implemented on coli expression carrier, by abduction delivering, obtains expression thalline；Expression thalline is used
6000rpm is centrifuged 15 minutes collects thallines, is washed using buffer A and has been hanged, and thalline high pressure is crushed, and inclusion body is collected by centrifugation and sinks
Form sediment and obtain inclusion body precipitation；
The abundant washing of S2, inclusion body precipitation, uses the inclusion body precipitation in S1 slow containing nonionic surface active agent
Rush liquid B rinse twice, reuse do not contain nonionic surface active agent buffer solution C rinse twice, and using contain 6 ~ 8M
The buffer solution C soluble proteins of denaturant；
S3, albuminate use ni-sepharose purification, first by the foreign protein that buffer solution C elutes non-specific binding, finally use
Buffer solution D elutes destination protein；
S4, the albuminate of purifying is diluted to into protein concentration less than 1mg/ml using buffer solution D, is placed in elution buffer and protects
It is 4 DEG C to hold dialysis temperature, is dialysed more than 12 hours, and supernatant is collected by centrifugation.
S5, supernatant is concentrated using super filter tube, add FAD, in 4 DEG C of temperature, with reference to more than 30 minutes, then by molecule
Sieve purifies and separates monomeric protein and collectin, obtain homogeneous natural activity albumen, and the destination protein mole is rubbed with FAD
The ratio of your amount is less than 1：3.
Preferably, the buffer A is, 50 mMTris-HCl, 100 mMNaCl, pH 8.0；
The buffer B is, 50 mMTris-HCl, 100 mMNaCl, 0.1 mM EDTA, 0.1 mM DTT, 1% TritonX-
100, pH 8.0；
The buffer solution C is, 50 mMTris-HCl, 200 mMNaCl, 10 ~ 30 mM imidazole, 8 M urea, pH
8.0；
The buffer solution D is, 50 mMTris-HCl, 200 mMNaCl, 300 ~ 500 mM imidazole, 8 M urea, pH
8.0；
The elution buffer is 20mM Na2HPO4, 200mMNaCl, pH 8.0.
Preferably, the nonionic surface active agent used in the S2 is TritonX-100, the thalline and buffer solution
W/v is 1：50.
Preferably, concentration of nonionic surfactant described in the S2 is 0.5%-1%.
Preferably, denaturant is urea in the S2.
Preferably, the albumen after S2 can also be rinsed in the S3 is directly using the buffering containing 300 ~ 500mM imidazoles
The direct soluble protein of liquid, the buffer solution is, 50 mMTris-HCl, 100 mMNaCl, 0.1 mM EDTA, 0.1 mM DTT,
pH 8.0。
Preferably, the L-Aspartic acid oxidase sequence is SEQ ID NO.1.
Preferably, the expression vector is pet vector, and the engineering bacteria abduction delivering temperature is 30 ~ 37 DEG C, induction time
For 4 ~ 6 hours.
During renaturing inclusion bodies, first have to use high concentration denaturant（Such as 8M urea）Or reducing agent makes solubilization of inclusion bodies, peptide
Chain launches, and afterwards by removing unnecessary denaturant or reducing agent, makes albumen slowly be folded into correct structure.Conventional method
Including dilution refolding, dialysis renaturation, chromatogram gel refolding etc..
Beneficial effects of the present invention：The present invention solves the problems, such as L-Aspartic acid oxidizing ferment renaturation, convenient to use
Method carry out the oxidasic inclusion body purification of L-Aspartic acid and renaturation, obtained by the rinsing and ni-sepharose purification of inclusion body
The higher albuminate of purity is obtained, by a step dialysis renaturation process, gradually removes denaturant, protein folding is activated egg
White molecule, annealing efficiency is not less than 10%.
Then activated high-purity enzyme is obtained through step dialysis and molecular sieve purification.
The present invention can effectively save the use of co-factor FAD, added after dialysis, it is to avoid FAD during dialysis
Loss, reduces financial cost.
Description of the drawings
SDS-PAGE detections collection of illustrative plates after Fig. 1 inclusion body purifications.
Native-PAGE detections collection of illustrative plates after Fig. 2 renaturation, 1：Recombinant protein before molecular sieve purification；2nd, it is multiple after molecular sieve purification
Property albumen.
The molecular sieve purification UV collection of illustrative plates of Fig. 3 supernatants soluble protein and recombinant protein.
Specific embodiment
The present invention specifically discloses a kind of effective L-Aspartic acid oxidizing ferment inclusion body purification refolding method.Specifically include
Following steps：
S1, by L-Aspartic acid oxidase gene carry out for e. coli codon optimize, codon optimization after L-
Aspartate oxidase gene order is SEQ ID NO.1.
It is implemented on coli expression carrier by full genome synthesis.By abduction delivering, the expression of high protein amount is obtained
Thalline.Specifically, L-Aspartic acid oxidase gene is carried out optimizing for e. coli codon, mesh is synthesized by full genome
Gene, in synthetic gene include Nco I and Xho I restriction enzyme sites, using Nco I and Xho I endonucleases to purpose base
Because fragment and carrier pET28a carry out digestion, 37 DEG C of h of digestion 1.Mesh is reclaimed using the step QIAquick Gel Extraction Kit of cycle-pure nucleic acid one
Genetic fragment, carrier reclaimed using glue reclaim kit.Fragment and carrier are connected using T4 DNA ligases, is then converted
E.coli DH5α.Correct monoclonal sequencing is selected, correct expression vector conversion BL21 (DE3), 37 DEG C of incubator cultures are sequenced
Grow to bacterium colony.
Picking colony into LB fluid nutrient mediums, 37 DEG C of incubated overnights, according to 1：100 ratio is inoculated in fresh 1L's
In LB culture mediums, it is 0.4 ~ 0.6 that 37 DEG C are cultivated to OD600, adds 1mM IPTG to cultivate 4 ~ 6 hours at 37 DEG C, obtains high egg
White amount expression thalline.
The expression engineered strain can at short notice obtain high yield inclusion body using 37 DEG C of inductions, when induction table
Up to more than 5 hours, the impurity that thalline is contained within can increase.
By buffer solution buffer A（50mMTris-HCl, 100mMNaCl, pH8.0）Wash and hanged, thalline height is crushed
Broken 3 times, with abundant cell lysis.Microscopy crushing efficiency is higher than 98%, and inclusion body precipitation is collected by centrifugation.
S2, add 50ml buffer solution buffer B according to every milligram of inclusion body（50mMTris-HCl, 100mMNaCl,
0.1mM EDTA, 0.1mM DTT, 1% TritonX-100, pH8.0）Ratio, be stirred well to without bulky grain, put on ice
Put 10 minutes, be centrifuged 20 minutes using 15000rpm, supernatant discarded.Said process is repeated once fully to rinse afterwards, with
Fully dissolve esters.
S3, add 50ml buffer solution buffer B according to every milligram of inclusion body（TritonX-100 is not contained）Ratio, fill
Divide stirring to hang, place 10 minutes on ice, tritonX-100 is removed within 20 minutes using 15000rpm centrifugations, remove supernatant.The step
Suddenly it is repeated once.The step removes the surfactant of residual, reduces its impact for protein renaturation.
S4, using buffer solution buffer C（50mM Tris-HCl, 200mM NaCl, 10 ~ 30mM imidazole, 8M
Urea, pH 8.0）Dissolving inclusion body, is centrifuged 40 minutes using 15000rpm, and supernatant is purpose albuminate.
Mix incubation with nickel post, make albumen fully combine nickel post at 4 DEG C.Uncombined component is discarded, using addition 50mM miaows
Buffer C cleaning nickel 3 column volumes of post of azoles, using buffer solution buffer D（50mM Tris-HCl, 200mM NaCl,
400mM imidazole, 8M urea, pH 8.0）Wash-out albuminate.
Inclusion body protein can also directly use buffer solution D（50mM Tris-HCl, 200mM NaCl, 300 ~ 500mM
Imidazole, 8M urea, pH 8.0；）Dissolving inclusion body protein, places 10 minutes on ice, and using 15000rpm 30 points are centrifuged
Clock, takes supernatant i.e. albuminous degeneration solution.
8M urea liquids containing 400mM imidazoles dissolving inclusion body or using containing used in the above S4 buffer solution D
There is inclusion body of the 8M urea liquids elution of bound of 400mM imidazoles on nickel post.Albuminate containing imidazoles dialysis when, on
Clear vigor highest.
S5, using buffer solution D by albuminate concentration dilution to 1mg/ml, take 10ml samples and be placed in elution buffer
（20mM sodium phosphates, 200mMNaCl, pH8.0）It is middle dialysis 18 hours, 15000rpm centrifugation go precipitation, concentrate supernatant, according to mole
Than adding the FAD more than 3 times of protein contents, mix after 4 DEG C of standings, carry out molecular sieve purification.Carry out after the inclusion body purification
SDS-PAGE detects that its collection of illustrative plates is as shown in Figure 1.The elution buffer contains certain salt ion can be effectively promoted albumen
Folding.Compared to other buffer systems, using phosphate buffer annealing efficiency can be effectively improved.
It is typically based on the FAD incubation half an hour that mol ratio adds 5 times of protein contents.It is demonstrated experimentally that adding after dialysis renaturation
FAD can't affect recombinant protein efficiency, consistent with FAD effects are directly added into during dialysis.
Dialysis sample volume is more than 5ml, and concentration is less than 1mg/ml.The protein content of low concentration can be reduced in dialysis
The generation of aggregation, improves annealing efficiency.
Embodiment one
To optimize from the L-Aspartic acid oxidase gene codon of hyperthermophilic archaeon strain Sulfolobustokodaii
Afterwards, genes of interest is synthesized by full genome, is connected on pET-28a carriers, in proceeding to escherichia coli host by molecular cloning
Expression, it is inclusion bodies that product is most of, therefore, the purification renaturation of inclusion body is carried out using this method tested, had
The high-purity L-Aspartic acid oxidizing ferment of activity.Comprise the following steps that：
Step one, by the bacterial strain of abduction delivering using 6000rpm rotating speeds be centrifuged 15 minutes, go supernatant collects thalline.Thalline is made
Use buffer A（50mMTris-HCl, 100mMNaCl, pH8.0）Washing 2 times, then proceedes to hang thalline using buffer A,
It is broken using high pressure, circulate at 4 DEG C and crush three times, it is centrifuged 50 minutes using 15000rpm rotating speeds, collect inclusion body precipitation.
Step 2, every milligram of inclusion body add 50ml buffer solution buffer B（50mMTris-HCl, 100mMNaCl,
0.1mMEDTA, 0.1mM DTT, 1%TritonX-100, pH8.0）Cleaning inclusion body, is centrifuged 20 minutes with 15000rpm, is collected
Precipitation.Afterwards said process is repeated once fully to rinse.
Step 3, every milligram of inclusion body add 50ml buffer solution buffer B（TritonX-100 is not contained）Cleaning is forgiven
Body, 20 minutes are centrifuged to remove tritonX-100 with 15000rpm, remove supernatant.The step is repeated once.
Step 4, using buffer solution buffer D（50mM Tris-HCl, 200mM NaCl, 400mM imidazole, 8M
Urea, pH 8.0）Dissolving inclusion body, is centrifuged 40 minutes using 15000rpm, and supernatant is purpose albuminate.
Step 5, using buffer solution buffer D by albuminate concentration dilution to 1mg/ml, take 10ml load bag filter,
It is placed in elution buffer（20mM sodium phosphates, 200mMNaCl, pH 8.0）Middle dialysed overnight（More than 18 hours）, 15000rpm from
The heart 30 minutes, collects supernatant and concentrates.The FAD of 5 times of moles is added, is combined 30 minutes at 4 DEG C, carried out using molecular sieve pure
Change.
Enzyme activity determination method.
The incrementss of hydrogen peroxide are determined by 4- amino antipyrine phynol methods.The reactant liquor of 1 ml is anti-at 30 DEG C
10 min are answered, the incrementss of its light absorption value per minute under 505 nm are determined.The definition of enzyme-activity unit is：It is per minute at 30 DEG C to release
Put the enzyme amount needed for 1 mol hydrogen peroxide.The reactant liquor system of 1 ml includes the peroxidase of 10 U/mL, 25 mmol/L
Pidolidone, the 4- amino antipyrines of 2 mmol/L, the phenol of 10 mmol/L and appropriate enzyme delay in the phosphate of pH 7.4
In rushing liquid.
As a result, supernatant protein amount is 0.1mg/ml after final dialysis, and annealing efficiency is 10%, after adding FAD, through molecule
Sieve purifying, removes unconjugated FAD and foreign protein, is detected by non denatured glue, and its refolded protein is monomer, obtains pure enzyme
Specific enzyme activity is 0.16 U/mg.
The present invention still has various specific embodiments.All employing equivalents or equivalent transformation and all skills for being formed
Art scheme, all falls within the scope of protection of present invention.
SEQ ID NO.1
ATGATATATATTATTGGCTCAGGCATTGCAGGCCTGTCAGCAGGCGTTGCACTGCGTCGCGCAGGAAAGAAAG
TGACCCTGATTAGTAAACGTATTGATGGCGGCTCTACCCCGATTGCCAAAGGCGGTGTTGCAGCATCAGTGGGTTCA
GATGATAGTCCGGAACTACATGCACAGGATACCATTCGTGTGGGCGATGGTCTGTGTGATGTTAAGACCGTTAATTA
TGTTACCTCAGAAGCAAAGAATGTGATTGAAACCTTTGAATCTTGGGGCTTTGAATTTGAAGAGGACCTGCGCCTGG
AAGGCGGCCATACCAAACGTCGTGTGCTGCATCGTACCGATGAAACCGGTCGCGAAATCTTTAACTTTCTGCTGAAA
CTGGCACGCGAAGAAGGTATTCCGATTATTGAAGATCGCCTGGTGGAAATTCGCGTTAAAGATGGTAAAGTGACCGG
CTTTGTGACCGAGAAACGCGGCCTGGTGGAAGATGTGGATAAACTGGTGCTGGCAACCGGCGGCTATAGCTATCTGT
ATGAATATAGCTCAACCCAGTCTACCAATATTGGGGACGGTATGGCAATTGCCTTCAAAGCAGGTACTATTCTGGCA
GATATGGAATTTGTTCAGTTTCATCCGACCGTTACCTCACTGGATGGCGAAGTGTTTCTGCTGACCGAAACCCTGCG
CGGCGAAGGTGCACAGATTATTAATGAGAATGGCGAACGCTTTCTGTTTAATTATGATAAACGTGGCGAACTGGCAC
CGCGTGATATTCTGAGCCGCGCAATATACATTGAAATGCTGAAAGGCCATAAAGTGTTTATTGATCTGTCTAAGATT
GAAGACTTTGAACGCAAATTTCCGGTTGTTGCCAAATATCTGGCACGTCATGGTCATAATTACAAGGTTAAGATTCC
GATCTTTCCGGCAGCACACTTTGTGGATGGCGGTATTCGCGTTAATATTCGTGGTGAATCAAATATTGTTAATCTGT
ATGCAATTGGCGAAGTGAGCGATAGCGGCCTGCATGGTGCAAATCGCCTGGCATCTAATAGCCTGCTGGAAGGTCTG
GTGTTTGGCATTAATCTGCCGCGCTATGTGGATAGTTCTTGGGAAGGCATTAGTACCGATGATGGTATTGTTCATAG
CGTGCGCATTAGCGGTAATAAGACCCTGTCACTGAAAGAAATTCGCCGCATTAATTGGGAGAATGTGGGCATTATTC
GTAATGAGGAGAAACTGGTTAAAGCAATTAATACCTATAGCTCTAGTACCCAGAACGAGGCAATTATTAGCTATCTG
ACCGCACTGGCAGCAGAAATTCGTAAAGAATCACGCGGTAATCACTTTCGCGAAGATTATCCGTACAAAGACCCGAA
TTGGGAGAAACGCATCTACTTCAAACTGGTTGTG
Claims (9)
1. a kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method, it is characterised in that：Comprise the steps：
The collection of S1, inclusion body precipitation, L-Aspartic acid oxidase gene is carried out to optimize for e. coli codon, is passed through
Full genome synthesis is implemented on coli expression carrier, by abduction delivering, obtains expression thalline；Expression thalline is used
6000rpm is centrifuged 15 minutes collects thallines, is washed using buffer A and has been hanged, and thalline high pressure is crushed, and inclusion body is collected by centrifugation and sinks
Form sediment and obtain inclusion body precipitation；
The abundant washing of S2, inclusion body precipitation, uses the inclusion body precipitation in S1 slow containing nonionic surface active agent
Rush liquid B rinse twice, reuse do not contain nonionic surface active agent buffer solution C rinse twice, and using contain 6 ~ 8M
The buffer solution C soluble proteins of denaturant；
S3, albuminate use ni-sepharose purification, first by the foreign protein that buffer solution C elutes non-specific binding, finally use
Buffer solution D elutes destination protein；
S4, the albuminate of purifying is diluted to into protein concentration less than 1mg/ml using buffer solution D, is placed in elution buffer and protects
It is 4 DEG C to hold dialysis temperature, is dialysed more than 12 hours, and supernatant is collected by centrifugation.
2.S5, supernatant is concentrated using super filter tube, add FAD, in 4 DEG C of temperature, with reference to more than 30 minutes, then by molecular sieve
Purifies and separates monomeric protein and collectin, obtain homogeneous natural activity albumen, the destination protein mole with FAD mole
The ratio of amount is less than 1：3.
3. a kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method as claimed in claim 1, it is characterised in that：It is described
Buffer A is, 50 mMTris-HCl, 100 mMNaCl, pH 8.0；
The buffer B is, 50 mMTris-HCl, 100 mMNaCl, 0.1 mM EDTA, 0.1 mM DTT, 1% TritonX-
100, pH 8.0；
The buffer solution C is, 50 mMTris-HCl, 200 mMNaCl, 10 ~ 30 mM imidazole, 8 M urea, pH
8.0；
The buffer solution D is, 50 mMTris-HCl, 200 mMNaCl, 300 ~ 500 mM imidazole, 8 M urea, pH
8.0；
The elution buffer is 20mM Na2HPO4, 200mMNaCl, pH 8.0.
4. a kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method as claimed in claim 1, it is characterised in that：It is described
Nonionic surface active agent used in S2 is TritonX-100, and the thalline is 1 with buffer solution w/v：50.
5. a kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method as claimed in claim 1, it is characterised in that：It is described
Concentration of nonionic surfactant described in S2 is 0.5%-1%.
6. a kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method as claimed in claim 1, it is characterised in that：It is described
Denaturant is urea in S2.
7. a kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method as claimed in claim 1, it is characterised in that：It is described
Albumen after in S3 S2 can also be rinsed is described directly using the direct soluble protein of buffer solution containing 300 ~ 500mM imidazoles
Buffer solution is, 50 mMTris-HCl, 100 mMNaCl, 0.1 mM EDTA, 0.1 mM DTT, pH 8.0.
8. a kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method as claimed in claim 1, it is characterised in that：It is described
L-Aspartic acid oxidase sequence is SEQ ID NO.1.
9. a kind of L-Aspartic acid oxidizing ferment inclusion body purification refolding method as claimed in claim 1, it is characterised in that：It is described
Expression vector is pet vector, and the engineering bacteria abduction delivering temperature is 30 ~ 37 DEG C, and induction time is 4 ~ 6 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610864870.2A CN106636024A (en) | 2016-09-30 | 2016-09-30 | Purified renaturation method of L-aspartic acid oxidase inclusion bodies |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610864870.2A CN106636024A (en) | 2016-09-30 | 2016-09-30 | Purified renaturation method of L-aspartic acid oxidase inclusion bodies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106636024A true CN106636024A (en) | 2017-05-10 |
Family
ID=58854078
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610864870.2A Pending CN106636024A (en) | 2016-09-30 | 2016-09-30 | Purified renaturation method of L-aspartic acid oxidase inclusion bodies |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106636024A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110437331A (en) * | 2019-07-30 | 2019-11-12 | 四川轻化工大学 | Method for improving inclusion body protein renaturation of smooth turtle shell serine protease inhibitor |
| CN112028975A (en) * | 2020-08-05 | 2020-12-04 | 上海裕隆生物科技有限公司 | 2019 method for preparing novel coronavirus spike protein receptor binding domain protein |
| CN113754754A (en) * | 2020-06-02 | 2021-12-07 | 南京大学 | Method for renaturation of nuclear receptor protein by extracorporeal liquid phase dialysis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161986A (en) * | 2011-03-11 | 2011-08-24 | 西北大学 | Method for purifying and renaturing matrix metalloproteinase 13 |
| CN104160015A (en) * | 2012-01-06 | 2014-11-19 | Cj第一制糖株式会社 | Recombinant microorganism producing quinolinic acid and production method of quinolinic acid using same |
| CN104830885A (en) * | 2015-04-29 | 2015-08-12 | 沈阳药科大学 | Prokaryotic expression method of oncoprotein |
| CN105732820A (en) * | 2016-03-17 | 2016-07-06 | 通化东宝药业股份有限公司 | Renaturation method of restructured human insulin prokaryotic-fusion protein |
-
2016
- 2016-09-30 CN CN201610864870.2A patent/CN106636024A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161986A (en) * | 2011-03-11 | 2011-08-24 | 西北大学 | Method for purifying and renaturing matrix metalloproteinase 13 |
| CN104160015A (en) * | 2012-01-06 | 2014-11-19 | Cj第一制糖株式会社 | Recombinant microorganism producing quinolinic acid and production method of quinolinic acid using same |
| CN104830885A (en) * | 2015-04-29 | 2015-08-12 | 沈阳药科大学 | Prokaryotic expression method of oncoprotein |
| CN105732820A (en) * | 2016-03-17 | 2016-07-06 | 通化东宝药业股份有限公司 | Renaturation method of restructured human insulin prokaryotic-fusion protein |
Non-Patent Citations (1)
| Title |
|---|
| BIFULCO D.等: "Synthetic construct L-aspartate oxidase gene, complete cds", 《GENBANK》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110437331A (en) * | 2019-07-30 | 2019-11-12 | 四川轻化工大学 | Method for improving inclusion body protein renaturation of smooth turtle shell serine protease inhibitor |
| CN110437331B (en) * | 2019-07-30 | 2023-04-07 | 四川轻化工大学 | Method for improving inclusion body protein renaturation of smooth turtle shell serine protease inhibitor |
| CN113754754A (en) * | 2020-06-02 | 2021-12-07 | 南京大学 | Method for renaturation of nuclear receptor protein by extracorporeal liquid phase dialysis |
| CN113754754B (en) * | 2020-06-02 | 2024-04-09 | 南京大学 | Nuclear receptor protein in-vitro liquid phase dialysis renaturation method |
| CN112028975A (en) * | 2020-08-05 | 2020-12-04 | 上海裕隆生物科技有限公司 | 2019 method for preparing novel coronavirus spike protein receptor binding domain protein |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6728294B2 (en) | A new method for protein purification. | |
| CN111423486A (en) | Renaturation method of new type coronavirus recombinant protein inclusion body | |
| CN106636024A (en) | Purified renaturation method of L-aspartic acid oxidase inclusion bodies | |
| CN1537939A (en) | Method for producing recombinaton urokinase | |
| Dong et al. | Lysozyme refolding with immobilized GroEL column chromatography | |
| JPH05504462A (en) | Improvements related to enzymes | |
| EP1668135B1 (en) | A non-denaturing process to purify recombinant proteins from plants | |
| AU624968B2 (en) | Processes for the recovery of microbially produced chymosin | |
| WO2022089340A1 (en) | Method for expressing human chymotrypsinogen and preparing recombinant human chymotrypsin by using genetically engineered rice | |
| CN113025599B (en) | Recombinant clostridium histolyticum type I collagenase as well as preparation method and application thereof | |
| CN1842600B (en) | A process for proteolytic cleavage and purification of recombinant proteins | |
| WO2019175633A1 (en) | Methods for refolding sucrose isomerase | |
| TWI712691B (en) | Dextran affinity tag and application thereof | |
| CN116925208A (en) | Renaturation buffer solution and method for renaturation of recombinant human PAI-1 protein inclusion body | |
| CN117778436A (en) | Process for efficiently preparing soluble RNase inhibitor | |
| JPH10327873A (en) | Plasmid, its preparation, microorganism containing the same plasmid, production of endosialidase using the same microorganism and production of sialic acid trimer using the same endosialidase | |
| Kohda et al. | Development of efficient protein refolding systems using chaperonins | |
| CN107236718A (en) | A kind of low temperature esterase, encoding gene and its application from grand genome | |
| CN106566819A (en) | Gene cloning, expression and application of low-temperature halophilic alpha-amylase | |
| JPS6167483A (en) | Purification of restriction enzyme aatii | |
| JPH03219892A (en) | Method for preparing protein | |
| JPH02227076A (en) | Activation of insoluble protein produced by gene-recombinating fungus | |
| JPS63119497A (en) | Solubilization and activation of foreign protein | |
| JPH03147786A (en) | Method for treating prourokinase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170510 |