CN109609532A - A kind of production method of anti-oxidant fusion protein - Google Patents

A kind of production method of anti-oxidant fusion protein Download PDF

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Publication number
CN109609532A
CN109609532A CN201910027485.6A CN201910027485A CN109609532A CN 109609532 A CN109609532 A CN 109609532A CN 201910027485 A CN201910027485 A CN 201910027485A CN 109609532 A CN109609532 A CN 109609532A
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oxidant
fusion protein
culture
ala
production method
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王学庆
李鹏云
王楠
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Xuchang Baike Protein And Gene Engineering Research Institute Co Ltd
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Xuchang Baike Protein And Gene Engineering Research Institute Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/825—Metallothioneins
    • 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
    • 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.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The present invention provides a kind of production methods of anti-oxidant fusion protein, belong to anti-oxidation tech field, including anti-oxidant fusion is transferred to Escherichia coli, after inoculating, cultivate, inducing, mixes, incubates with T4 Lysozyme after the thallus that centrifugation obtains is resuspended with buffer, carry out water-bath after obtained incubation object is mixed with Tween 20, the temperature of water-bath is 60~70 DEG C, the time of water-bath is 0.5~1.5h, and obtained water-bath object is centrifuged, anti-oxidant fusion protein is obtained；Most of thallus can not only be cracked using method provided by the invention, but also can guarantee the activity of anti-oxidant fusion protein, additionally it is possible to so that other albuminous degenerations, are suitble to large-scale production.

Description

A kind of production method of anti-oxidant fusion protein
Technical field
The present invention relates to anti-oxidation tech fields, and in particular to a kind of production method of anti-oxidant fusion protein.
Background technique
Superoxide dismutase SOD is the first line of defence to free radical resisting in vivo, the gold combined according to its activated centre Belong to the difference of ion, SOD is broadly divided into three classes: copper-zinc superoxide dismutase (Cu/Zn-SOD, also referred to as SOD1), iron super oxygen Compound mutase (Fe-SOD) and manganese superoxide dismutase (Mn-SOD).Wherein, Fe-SOD is primarily present in prokaryotic cell In matrix.When our body oxygen intake progress metabolism, ultra-oxygen anion free radical will be generated, if not eliminated, Chain reaction can be generated in vivo and destroy cell.Under normal circumstances, the generation and removing of interior free yl are in dynamic equilibrium. SOD is many in the blood of young man, and people crossed after 30 years old, and the intracorporal SOD of people is generated with the aging of tissue Rate gradually decreases.With the increase at age and decrescence due to intracorporal SOD, along with the deterioration of environment, a large amount of free radical is super The degree that body can be dealt with is crossed, therefore is required by external supplement.
The source of SOD is extremely wide, at present from bacterium, fungi, algae, fish, insect, plant and mammal etc. Various organisms are isolated.Domestic developed SOD resource has: animal blood (ox, pig etc.), and mushroom fermentation is (saccharomycete, big Enterobacteria etc.), higher plant (algae, Rosa roxburghii Tratt etc.) etc..The primary raw material that animal extracts SOD can be various mammal blood Liquid, liver, brain etc., however the reason of some zoonosis, forbids many countries in fields such as food, cosmetics, medical treatment Use the SOD of animal origin.However the extracted generally existing enzymatic activity of SOD is lower from above-mentioned source, scavenging activated oxygen Ability it is poor the disadvantages of.
Metallothionein (Metallothionein, MT), also known as sulfydryl metal-binding protein, the entitled metal sulphur group of chemistry Propylhomoserin hercynine is a kind of to be widely present in human body, animal, plant and the intracorporal albumen for having specific function of microorganism Matter.Mankind MT is divided into four subgroups: MT1, MT2, MT3, MT4 by characterization of molecules and distribution.MT1 divides 9 subclass again, and MT2 divides again MT-2a and MT-2b.MT1 and MT2 is distributed whole body, and MT3 is distributed in brain, and MT4 is distributed mainly on skin.It is micro in vivo with participating in Element metabolism, heavy metal detoxification remove free radical, participate in DNA replication dna and transcription, influence protein and energetic supersession, antagonism electricity From radiation, enhancing immunity of organisms and it the biological functions such as stress protect.Wherein, it removes free radical and bind metal ion is MT Most important two functions.Although the current domestic research and development starting to MT is more early, the clone of gene is all concentrated on mostly In the discussion of biological function, there is no large-scale production recombination metallothionein fusion proteins.
Summary of the invention
The purpose of the present invention is to provide a kind of production methods of anti-oxidant fusion protein, using producer of the invention Anti-oxidant fusion protein can be mass produced for method.
The present invention provides a kind of production methods of anti-oxidant fusion protein, comprising the following steps:
1) it is transferred to Escherichia coli after connecting anti-oxidant fusion with carrier, obtains containing the big of anti-oxidant fusion Enterobacteria；
The anti-oxidant fusion is obtained after connecting MT gene with sod gene；
The MT gene has nucleotide sequence shown in SEQ ID No.1；
The sod gene has nucleotide sequence shown in SEQ ID No.2；
2) Escherichia coli containing anti-oxidant fusion for obtaining the step 1) are inoculated in mould containing ammonia benzyl and chlorine The first culture is carried out in the LB liquid medium of element, obtains the first culture；
The time of first culture is 12~16h；
3) the first culture for obtaining the step 2) is inoculated in the TB fluid nutrient medium containing ammonia benzyl and chloramphenicol The second culture is carried out, the second culture is obtained；
The time of second culture is 3.5~4.5h；
4) the second culture for obtaining the step 3) lures after mixing with isopropylthiogalactoside, zinc chloride Culture is led, inducer is obtained；
5) inducer that the step 4) obtains is centrifuged, after obtained thallus is resuspended with buffer with T4 bacteriolyze Enzyme mixing incubates, and carries out water-bath after obtained incubation object is mixed with Tween20, obtained water-bath object is centrifuged, antioxygen is obtained Change fusion protein；
The temperature of the water-bath is 60~70 DEG C；
The time of the water-bath is 0.5~1.5h.
Preferably, the amino acid sequence of the anti-oxidant fusion protein of the step 1) is as shown in SEQ ID No.3.
Preferably, the amino acid sequence of the anti-oxidant fusion protein of the step 1) is as shown in SEQ ID No.4.
Preferably, the amino acid sequence of the anti-oxidant fusion protein of the step 1) is as shown in SEQ ID No.5.
Preferably, the amino acid sequence of the anti-oxidant fusion protein of the step 1) is as shown in SEQ ID No.6.
Preferably, in the step 2) LB liquid medium, the content of the Adenosine acid is 90~110 μ G/ml, the content of the chloramphenicol are 30~40 μ g/ml.
Preferably, in step 3) the TB fluid nutrient medium, the content of the Adenosine acid is 90~110 μ G/ml, the content of the chloramphenicol are 30~40 μ g/ml.
Preferably, the time of the step 4) Fiber differentiation is 12~20h, and the temperature of the Fiber differentiation is 18~22 ℃。
Preferably, the time that the step 5) incubates is 25~35min, and the temperature of the incubation is 18~22 DEG C.
Preferably, after step 5) the water-bath object centrifugation further include: by obtained supernatant after Ni column purification, resisted Aoxidize fusion protein.
The present invention provides a kind of production method of anti-oxidant fusion protein, anti-oxidant fusion is transformed by the present invention It is expressed after in Escherichia coli, Escherichia coli are after T4 Lysozyme cracks, and 0.5~1.5h of water-bath at 60~70 DEG C can Escherichia coli are enough cracked, and anti-oxidant fusion protein can be discharged, and can guarantee the activity of anti-oxidant fusion protein.
The embodiment of the present invention is as the result is shown: can not only crack most of thallus, but also energy using method provided by the invention Enough guarantee the activity of anti-oxidant fusion protein, additionally it is possible to so that other albuminous degenerations, are suitble to large-scale production.
Detailed description of the invention
Fig. 1 is that Coomassie brilliant blue detects albumen result.
Specific embodiment
The present invention provides a kind of production methods of anti-oxidant fusion protein, which comprises the following steps:
1) it is transferred to Escherichia coli after connecting anti-oxidant fusion with carrier, obtains containing the big of anti-oxidant fusion Enterobacteria；
The anti-oxidant fusion is obtained after connecting MT gene with sod gene；
The MT gene has nucleotide sequence shown in SEQ ID No.1；
The sod gene has nucleotide sequence shown in SEQ ID No.2；
2) Escherichia coli containing anti-oxidant fusion for obtaining the step 1) are inoculated in mould containing ammonia benzyl and chlorine The first culture is carried out in the LB liquid medium of element, obtains the first culture；
The time of first culture is 12~16h；
3) the first culture for obtaining the step 2) is inoculated in the TB fluid nutrient medium containing ammonia benzyl and chloramphenicol The second culture is carried out, the second culture is obtained；
The time of second culture is 3.5~4.5h；
4) the second culture for obtaining the step 3) lures after mixing with isopropylthiogalactoside, zinc chloride Culture is led, inducer is obtained；
5) inducer that the step 4) obtains is centrifuged, by obtained thallus with pMSF buffer be resuspended after with T4 Lysozyme mixing incubates, and carries out water-bath after obtained incubation object is mixed with Tween20, obtained water-bath object is centrifuged, is obtained Anti-oxidant fusion protein；
The temperature of the water-bath is 60~70 DEG C；
The time of the water-bath is 0.5~1.5h.
The present invention is transferred to Escherichia coli after connecting anti-oxidant fusion with carrier, obtains containing anti-oxidant fusion Escherichia coli；The anti-oxidant fusion is obtained after connecting MT gene with sod gene；The MT gene has SEQ Nucleotide sequence shown in ID No.1；The sod gene has nucleotide sequence shown in SEQ ID No.2.
In the present invention, the anti-oxidant fusion is obtained after connecting MT gene with sod gene, the MT gene With nucleotide sequence shown in SEQ ID No.1, particular sequence is as follows:
ATGGATCCGAACTGCAGCTGCGCGGCGGGCGATAGCTGCACCTGCGCGGGCAGCTGCAAATGCAAAGA ATGCAAATGCACCAGCTGCAAGAAAAGCTGCTGCAGCTGCTGCCCGGTGGGCTGCGCGAAATGCGCGCAGGGCTGC ATTTGCAAAGGCGCGAGCGATAAATGCAGCTGCTGCGCG。
In the present invention, the sod gene has nucleotide sequence shown in SEQ ID No.2, and particular sequence is as follows:
ATGGCGTTTGTGCAGGAACCTCTGCCTTTTGATCCGGGAGCGCTGGAACCGTATGGCATGAGCGCGAA AACCCTGGAATTTCACTATGGCAAACATCATAAAGGCTATGTGGATAATCTGAATAAACTGACCCAGGATACCGAA CTGGCGGATAAAAGCCTGGAAGATGTGATTCGTACCACCTATGGGGACGCTGCCAAGGTGGGCATTTTCAATAATG CGGCGCAGGTGTGGAATCATACCTTTTTTTGGAATAGCCTGAAACCGGGAGGTGGCGGCGTGCCGACCGGGGATGT GGCCGCTCGTATAAACAGCGCGTTTGGGAGCTATGACGAATTCAAGGCGCAGTTTAAGAATGCCGCAGCCACCCAG TTTGGCTCTGGCTGGGCGTGGCTGGTGCTGGAAGCGGGCACCCTGAAGGTGACCAAAACCGCGAACGCGGAGAATC CGCTTGTTCATGGCCAAGTGCCACTGCTGACCATTGATGTGTGGGAACATGCGTATTATCTGGATTATCAGAATCG TCGTCCGGATTTCATAGACAATTTTCTGAATCAGCTGGTGAATTGGGATTTTGTGGCGAAAAACTTAGCAGCGGCG。
The method that the present invention connects the MT gene with sod gene is not particularly limited, using conventional method.
Escherichia coli containing anti-oxidant fusion are inoculated in and train containing the LB liquid of ammonia benzyl and chloramphenicol by the present invention It supports and carries out the first culture in base, obtain the first culture；The time of first culture is 12~16h.
In the present invention, in the LB liquid medium, the content of the Adenosine acid is preferably 90~110 μ g/ml, more preferably 100 μ g/ml；The content of the chloramphenicol is preferably 30~40 μ g/ml, more preferably 34 μ g/ml.This hair The bright effect in the first culture addition Adenosine acid and chloramphenicol is to provide resistance for engineering bacteria growth.
In the present invention, the time of first culture is 12~16h, preferably 13~15h, more preferably 14h；It is described The temperature of culture is preferably 37 DEG C.The present invention to the Escherichia coli containing anti-oxidant fusion to connect bacterium amount not special It limits, connects bacterium amount using conventional.
First culture is inoculated in the TB fluid nutrient medium containing ammonia benzyl and chloramphenicol and carries out second by the present invention Culture, obtains the second culture；The time of second culture is 3.5~4.5h.
In the present invention, the volume ratio of first culture and TB fluid nutrient medium is preferably 1:100.
In the present invention, in the TB fluid nutrient medium, the content of the Adenosine acid is preferably 90~110 μ g/ml, more preferably 100 μ g/ml；The content of the chloramphenicol is preferably 30~40 μ g/ml, more preferably 34 μ g/ml.This hair The bright effect in the second culture addition ammonia benzyl and chloramphenicol is to provide resistance for engineering bacteria growth.The present invention is by the second training After supporting, engineering bacteria expands culture, increases bacterial number, makes Escherichia coli Growth to logarithmic growth phase.
In the present invention, the time of second culture is preferably 3.5~4.5h, more preferably 4h；Second culture Temperature be preferably 37 DEG C.In the present invention, the OD value of second culture is preferably 0.5~1.
The present invention carries out Fiber differentiation after mixing the second culture with isopropylthiogalactoside, zinc chloride, obtains Inducer.
In the present invention, after second culture is mixed with isopropylthiogalactoside, zinc chloride, the isopropyl The final concentration of thiogalactoside is preferably 0.2mM, and the final concentration of the zinc chloride is preferably 0.2mM.The present invention is trained in induction The effect that isopropylthiogalactoside and zinc chloride are added in supporting is: IPTG is inducer, and inducible protein synthesizes, and zinc chloride lures Zn-MT is given birth in artificial delivery, and the MT is metallothionein gene.
In the present invention, the time of the Fiber differentiation is preferably 12~20h, more preferably 13~18h, most preferably 16h；The temperature of the Fiber differentiation is preferably 18~22 DEG C, and more preferably 20 DEG C.
Obtained inducer is centrifuged by the present invention, is mixed after obtained thallus is resuspended with buffer with T4 Lysozyme It closes, incubate, carry out water-bath after obtained incubation object is mixed with Tween20, obtained water-bath object is centrifuged, anti-oxidant melt is obtained Hop protein；The temperature of the water-bath is 60~70 DEG C；The time of the water-bath is 0.5~1.5h.
The centrifugal condition that the present invention is centrifuged the inducer is not particularly limited, and obtains thallus using conventional centrifugal Condition.
In the present invention, the buffer preferably includes: 20mM Tris, 300mM NaCl, 1mM EDTA, 0.5mM PMSF buffer.
In the present invention, the thallus mixes after being resuspended with buffer with T4 Lysozyme, the final concentration of the T4 Lysozyme Preferably 0.2mg/ml.In the present invention, the T4 Lysozyme can crack thallus.
In the present invention, the time of the incubation is preferably 25~35min, more preferably 30min；The temperature of the incubation Preferably 18~22 DEG C, more preferably 20 DEG C.
In the present invention, after the incubation object is mixed with Tween20, the whole mass percentage of the Tween20 is preferred It is 0.5%.In the present invention, the Tween20 stable protein promotes Escherichia coli cracking.
State in the present invention, the temperature of the water-bath are 60~70 DEG C, preferably 65 DEG C；The time of the water-bath is 0.5 ~1.5h, preferably 1h.In the present invention, the temperature and time of the water-bath can either can retain anti-oxidant fusion protein Activity, and engineering bacteria can be cracked.
In the present invention, the revolving speed of the water-bath object centrifugation is preferably 8000~12000rpm, more preferably 10000rpm； The time of the centrifugation is preferably 25~35min, more preferably 30min.
In the present invention, further preferably include: the supernatant that will obtain after water-bath object centrifugation after Ni column purification, obtain Anti-oxidant fusion protein.The present invention is not particularly limited the condition of the Ni column purification.
In the present invention, the amino acid sequence of the anti-oxidant fusion protein is as shown in SEQ ID No.3, institute specific as follows Show:
MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCAGGGGSGG GGSGGGGSMAFVQEPLPFDPGALEPYGMSAKTLEFHYGKHHKGYVDNLNKLTQDTELADKSLEDVIRTTYGDAAKV GIFNNAAQVWNHTFFWNSLKPGGGGVPTGDVAARINSAFGSYDEFKAQFKNAAATQFGSGWAWLVLEAGTLKVTKT ANAENPLVHGQVPLLTIDVWEHAYYLDYQNRRPDFIDNFLNQLVNWDFVAKNLAAAHHHHHH.Wherein GGGGSGGGGSGGGGS is link peptide, and HHHHHH is his label.
In the present invention, the amino acid sequence of the anti-oxidant fusion protein is as shown in SEQ ID No.4, institute specific as follows Show:
MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCAGGGGSGG GGSGGGGSMAFVQEPLPFDPGALEPYGMSAKTLEFHYGKHHKGYVDNLNKLTQDTELADKSLEDVIRTTYGDAAKV GIFNNAAQVWNHTFFWNSLKPGGGGVPTGDVAARINSAFGSYDEFKAQFKNAAATQFGSGWAWLVLEAGTLKVTKT ANAENPLVHGQVPLLTIDVWEHAYYLDYQNRRPDFIDNFLNQLVNWDFVAKNLAAA.Wherein, GGGGSGGGGSGGGGS For link peptide.
In the present invention, the amino acid sequence of the anti-oxidant fusion protein is as shown in SEQ ID No.5, institute specific as follows Show:
HHHHHHMAFVQEPLPFDPGALEPYGMSAKTLEFHYGKHHKGYVDNLNKLTQDTELADKSLEDVIRTTY GDAAKVGIFNNAAQVWNHTFFWNSLKPGGGGVPTGDVAARINSAFGSYDEFKAQFKNAAATQFGSGWAWLVLEAGT LKVTKTANAENPLVHGQVPLLTIDVWEHAYYLDYQNRRPDFIDNFLNQLVNWDFVAKNLAAAGGGGSGGGGSGGGG SMDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA.Wherein, HHHHHH is His label, GGGGSGGGGSGGGGS are link peptide.
In the present invention, the amino acid sequence of the anti-oxidant fusion protein is as shown in SEQ ID No.6, institute specific as follows Show:
MAFVQEPLPFDPGALEPYGMSAKTLEFHYGKHHKGYVDNLNKLTQDTELADKSLEDVIRTTYGDAAKV GIFNNAAQVWNHTFFWNSLKPGGGGVPTGDVAARINSAFGSYDEFKAQFKNAAATQFGSGWAWLVLEAGTLKVTKT ANAENPLVHGQVPLLTIDVWEHAYYLDYQNRRPDFIDNFLNQLVNWDFVAKNLAAAGGGGSGGGGSGGGGSMDPNC SCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA.Wherein GGGGSGGGGSGGGGS For link peptide.
A kind of production method of anti-oxidant fusion protein of the present invention is done further combined with specific embodiments below Detailed introduction, technical solution of the present invention include but is not limited to following embodiment.
Embodiment 1
The production of MT-SOD-His fusion protein:
The amplification of MT-SOD-his segment:
(1) with the MT-2a complete genome sequence of synthesis (SEQQ ID No.1) for template, PCR expansion is carried out using following primer Increase, obtain MT-2a segment:
The system of PCR amplification: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
PCR response procedures are as follows:
95℃2min；95 DEG C, 30s, 57 DEG C, 30s, 72 DEG C, 10s, 30 circulations；72 DEG C, 5min (SEQ ID No.7) MT-Fw-2:
CTTTAAGAAGGAGATATACATATGGATCCGAACTGCAGCTG；
(SEQ ID No.8) MT-Rev-2:
CCTCCACTTCCGCCACCGCCGCTGCCGCCACCTCCCGCGCAGCAGCTGCATTTATC。
(2) with the FeSOD complete genome sequence of synthesis (SEQ ID No.2) for template, PCR expansion is carried out using following primer Increase, obtain FeSOD segment:
The system of PCR amplification: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
PCR response procedures are as follows:
95℃2min；95 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 20s, 30 circulations；72 DEG C, 5min
(SEQ ID No.9) SOD-Fw-2:
GGCGGTGGCGGAAGTGGAGGCGGTGGCAGCATGGCGTTTGTGCAGGAAC；
(SEQ ID No.10)SOD-Rev-2:
GGATCCGTTATCCACTTTTAATGATGATGATGATGGTGCGCCGCTGCTAAGTTTTTC。
(3) after mixing above-mentioned two sections of PCR fragments, PCR pre-reaction 10 are recycled, and reaction condition is as follows:
Amplification system: every 20uL includes: 2 μ l, dNTP (10mM each) 0.4 of PFU ultra II Fusion buffer μ l, FW primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, MT-2a 5 μ l, FeSOD 5ul, Sterile 6.4 μ l, pFU ultra IIfusion enzyme of miliQ water, 0.4 μ l.
Amplification program are as follows: 95 DEG C of 2 minutes；95 DEG C of 20s, 50 DEG C of 20s, 72 DEG C of 30s, 10 circulations；72℃5min.
(4) using product after step (3) reaction as template, primer MT-FW-2 and SOD-Rev-2 carry out PCR, obtain MT- SOD-His segment；
Amplification system: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
Amplification program: 95 DEG C of 2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72 DEG C, 5min.
(5) clone of MT-SOD-His segment
MT-SOD-His segment is connected respectively on carrier pet15b, convert escherichia coli DH5a, be coated on containing In the LB solid medium tablets of 100ug/ml Amp, 37 DEG C are cultivated 16 hours, are selected monoclonal colonies respectively and are identified.Mirror The DH5a for being set to positive colony is correct through DNA sequencing verifying gene order.Plasmid containing recombination fusion protein is converted respectively To in Escherichia coli Rosseta (DE3), the expression bacterial strain of fusion protein is obtained.
(6) expression and purification of fusion protein
A. the expression of fusion protein
Above-mentioned fusion protein monoclonal is inoculated into the LB liquid medium containing 100ug/ml Amp, 34ug chloramphenicol In, 37 DEG C after culture 12 hours, culture according to 1:100 ratio is inoculated into 500ml, and to contain 100ug/ml Amp, 34ug chlorine mould In the fresh TB culture medium of element, when 37 DEG C of culture 4h to OD600 reach 0.8~1, IPTG final concentration is added to 0.2mM, ZnCl2It is dense It spends to 0.2mM.20 DEG C of 12~16h of induction.
B. bacterium solution cracks
Thalline were collected by centrifugation, is resuspended in 20mM Tris, in 300mM NaCl, 1mM EDTA, 0.5nM PMSF buffer, The T4 Lysozyme of final concentration 0.2mg/ml is added, 20 DEG C of incubation 30min are added Tween20 to final concentration 0.5%, after mixing, set It in 65 DEG C of water-bath 1h, stirs for several times, adds PMSF buffer 1 time during heating, by the solution after water-bath at 4 DEG C, 10000rpm It is centrifuged 30min, takes supernatant, crosses ni-sepharose purification.
C.Ni column purification
(1) Ni column first balances 5 times of column volumes with the sample-loading buffer of the imidazoles containing 10mM.
(2) by crude enzyme liquid by 0.45um membrane filtration, filtered supernatant passes through the Ni column after balance.
(3) contain 10mM imidazoles, 0.1%Triton X-114 buffer rinses 10 times of column volumes.
(4) 5 times of column volumes of 20mM imidazoles flushing are washed miscellaneous.
(5) 300mM imidazoles elutes destination protein, obtains the anti-oxidant fusion protein of MT-SOD-His.
Through detection it is found that the purity of anti-oxidant fusion protein can reach 95% or more, amino acid sequence such as SEQ ID No.3 It is shown, specific as follows:
MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCAGGGGSGG GGSGGGGSMAFVQEPLPFDPGALEPYGMSAKTLEFHYGKHHKGYVDNLNKLTQDTELADKSLEDVIRTTYGDAAKV GIFNNAAQVWNHTFFWNSLKPGGGGVPTGDVAARINSAFGSYDEFKAQFKNAAATQFGSGWAWLVLEAGTLKVTKT ANAENPLVHGQVPLLTIDVWEHAYYLDYQNRRPDFIDNFLNQLVNWDFVAKNLAAAHHHHHH.Wherein GGGGSGGGGSGGGGS is link peptide, and HHHHHH is his label.
Embodiment 2
The production of MT-SOD fusion protein:
The amplification of his-MT-SOD segment:
(1) with the MT-2a complete genome sequence of synthesis (SEQQ ID No.1) for template, PCR expansion is carried out using following primer Increase, obtain MT-2a segment:
The system of PCR amplification: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
PCR response procedures are as follows:
95℃2min；95 DEG C, 30s, 57 DEG C, 20s, 72 DEG C, 10s, 30 circulations；72 DEG C, 5min (SEQ ID No.11) MT-Fw-1:
CTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATGAGAATC；
(SEQ ID No.12) MT-Rev-1:
CCTCCACTTCCGCCACCGCCGCTGCCGCCACCTCCCGCGCAGCAGCTGCATTTATC。
(2) with the FeSOD complete genome sequence of synthesis (SEQ ID No.2) for template, PCR expansion is carried out using following primer Increase, obtain FeSOD segment:
The system of PCR amplification: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
PCR response procedures are as follows:
95℃2min；95 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 20s, 30 circulations；72 DEG C, 5min
(SEQ ID No.13) SOD-Fw-1:
GGCGGTGGCGGAAGTGGAGGCGGTGGCAGCATGGCGTTTGTGCAGGAAC；
(SEQ ID No.14)SOD-Rev-1:
GGATCCGTTATCCACTTTTACGCCGCTGCTAAGTTTTTC。
(3) after mixing above-mentioned two sections of PCR fragments, PCR pre-reaction 10 are recycled, and reaction condition is as follows:
Amplification system: every 20uL includes: 2 μ l, dNTP (10mM each) 0.4 of PFU ultra II Fusion buffer μ l, FW primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, MT-2a 5 μ l, FeSOD 5ul, Sterile 6.4 μ l, pFU ultra IIfusion enzyme of miliQ water, 0.4 μ l.
Amplification program are as follows: 95 DEG C of 2min；95 DEG C of 20s, 50 DEG C of 20s, 72 DEG C of 30s, 10 circulations；72℃5min.
(4) using product after step (3) reaction as template, primer MT-FW-1 and SOD-Rev-1 carry out PCR, obtain His- MT-SOD segment；
Amplification system: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
Amplification program: 95 DEG C of 2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72 DEG C, 5min.
(5) clone of His-MT-SOD segment
His-MT-SOD segment is connected respectively on carrier pet15b, convert escherichia coli DH5a, be coated on containing In the LB solid medium tablets of 100ug/ml Amp, 37 DEG C are cultivated 16 hours, are selected monoclonal colonies respectively and are identified.Mirror The DH5a for being set to positive colony is correct through DNA sequencing verifying gene order.Plasmid containing recombination fusion protein is converted respectively To in Escherichia coli Rosseta (DE3), the expression bacterial strain of fusion protein is obtained.
(6) expression and purification of fusion protein
A. the expression of fusion protein
Above-mentioned fusion protein monoclonal is inoculated into the LB liquid medium containing 100ug/ml Amp, 34ug chloramphenicol In, 37 DEG C after culture 12 hours, culture according to 1:100 ratio is inoculated into 500ml, and to contain 100ug/ml Amp, 34ug chlorine mould In the fresh TB culture medium of element, when 37 DEG C of culture 4h to OD600 reach 0.8~1, IPTG final concentration is added to 0.2mM, ZnCl2It is dense It spends to 0.2mM.20 DEG C of 12~16h of induction.
B. bacterium solution cracks
Thalline were collected by centrifugation, is resuspended in 20mM Tris, in 300mM NaCl, 1mM EDTA, 0.5nM PMSF buffer, The T4 Lysozyme of final concentration 0.2mg/ml is added, 20 DEG C of incubation 30min are added Tween20 to final concentration 0.5%, after mixing, set It in 65 DEG C of water-bath 1h, stirs for several times, adds PMSF buffer 1 time during heating, by the solution after water-bath at 4 DEG C, 10000rpm It is centrifuged 30min, takes supernatant, crosses ni-sepharose purification.
C.Ni column purification
Ni column first balances 5 times of column volumes with the sample-loading buffer of the imidazoles containing 10mM；
By crude enzyme liquid by 0.45um membrane filtration, filtered supernatant passes through the Ni column after balance；
Containing 10mM imidazoles, 0.1%TritonX-114 buffer rinses 10 times of column volumes；
20mM imidazoles 5 times of column volumes of flushing are washed miscellaneous；
300mM imidazoles elutes destination protein, obtains the anti-oxidant fusion protein of His-MT-SOD.
Through detecting it is found that the purity of anti-oxidant fusion protein can reach 95% or more.
(7) excision of His label
Contain one section of TEV protease recognition site behind the His-tag of His-MT-SOD fusion protein, TEV can be passed through Albumen enzyme effect removes label.
After fusion protein after Ni column purification measures protein concentration, TEV protease, i.e. 1 μ gTEV is added in 50:1 in mass ratio Protease acts on 50 μ g destination proteins.4 DEG C of digestions are stayed overnight.
(8) it is concentrated by ultrafiltration, changes the removing of liquid and label
Albumen after digestion changes liquid to 10mM imidazoles after 10kd super filter tube is concentrated into suitable volumes, with desalting column G25 It in sample-loading buffer, reuses Ni column and is purified, at this point, being existed by the label under cutting and the protein adsorption for not cutting off label On Ni column, the fusion protein of no his label passes through pillar, collects across liquid, as destination protein, obtains that MT-SOD is anti-oxidant to be melted Hop protein.
The amino acid sequence of the anti-oxidant fusion protein of MT-SOD is specific as follows as shown in SEQ ID No.4:
MDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCAGGGGSGG GGSGGGGSMAFVQEPLPFDPGALEPYGMSAKTLEFHYGKHHKGYVDNLNKLTQDTELADKSLEDVIRTTYGDAAKV GIFNNAAQVWNHTFFWNSLKPGGGGVPTGDVAARINSAFGSYDEFKAQFKNAAATQFGSGWAWLVLEAGTLKVTKT ANAENPLVHGQVPLLTIDVWEHAYYLDYQNRRPDFIDNFLNQLVNWDFVAKNLAAA.Wherein, GGGGSGGGGSGGGGS For link peptide.
Embodiment 3
The production of His-SOD-MT fusion protein:
The amplification of His-SOD-MT segment:
(1) with the MT-2a complete genome sequence of synthesis (SEQQ ID No.1) for template, PCR expansion is carried out using following primer Increase, obtain MT-2a segment:
The system of PCR amplification: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
PCR response procedures are as follows:
95℃2min；95 DEG C, 30s, 57 DEG C, 30s, 72 DEG C, 10s, 30 circulations；72 DEG C, 5min (SEQ ID No.15) MT-Fw-3:
GGCGGTGGCGGAAGTGGAGGCGGTGGCAGCATGGATCCGAACTGCAGCTG；
(SEQ ID No.16) MT-Rev-3:
GGATCCGTTATCCACTTTTACGCGCAGCAGCTGCATTTATC。
(2) with the FeSOD complete genome sequence of synthesis (SEQ ID No.2) for template, PCR expansion is carried out using following primer Increase, obtain FeSOD segment:
The system of PCR amplification: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
PCR response procedures are as follows:
95℃2min；95 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 20s, 30 circulations；72 DEG C, 5min
(SEQ ID No.17) SOD-Fw-3:
CTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATGAGAATC；
(SEQ ID No.18)SOD-Rev-3:
CCTCCACTTCCGCCACCGCCGCTGCCGCCACCTCCCGCCGCTGCTAAGTTTTTC。
(3) after mixing above-mentioned two sections of PCR fragments, PCR pre-reaction 10 are recycled, and reaction condition is as follows:
Amplification system: every 20uL includes: 2 μ l, dNTP (10mM each) 0.4 of PFU ultra II Fusion buffer μ l, FW primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, MT-2a 5 μ l, FeSOD 5ul, Sterile 6.4 μ l, pFU ultra IIfusion enzyme of miliQ water, 0.4 μ l.
Amplification program are as follows: 95 DEG C of 2minutes；95 DEG C of 20s, 50 DEG C of 20s, 72 DEG C of 30s, 10 circulations；72℃5min.
(4) using product after step (3) reaction as template, primer MT-FW-2 and SOD-Rev-2 carry out PCR, obtain His- SOD-MT segment；
Amplification system: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
Amplification program: 95 DEG C of 2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72 DEG C, 5min.
(5) clone of His-SOD-MT segment
His-SOD-MT segment is connected respectively on carrier pet15b, convert escherichia coli DH5a, be coated on containing In the LB solid medium tablets of 100ug/ml Amp, 37 DEG C are cultivated 16 hours, are selected monoclonal colonies respectively and are identified.Mirror The DH5a for being set to positive colony is correct through DNA sequencing verifying gene order.Plasmid containing recombination fusion protein is converted respectively To in Escherichia coli Rosseta (DE3), the expression bacterial strain of fusion protein is obtained.
(6) expression and purification of fusion protein
A. the expression of fusion protein
Above-mentioned fusion protein monoclonal is inoculated into the LB liquid medium containing 100ug/ml Amp, 34ug chloramphenicol In, 37 DEG C after culture 12 hours, culture according to 1:100 ratio is inoculated into 500ml, and to contain 100ug/ml Amp, 34ug chlorine mould In the fresh TB culture medium of element, when 37 DEG C of culture 4h to OD600 reach 0.8~1, IPTG final concentration is added to 0.2mM, ZnCl2It is dense It spends to 0.2mM.20 DEG C of 12~16h of induction.
B. bacterium solution cracks
Thalline were collected by centrifugation, is resuspended in 20mM Tris, in 300mM NaCl, 1mM EDTA, 0.5nM PMSF buffer, The T4 Lysozyme of final concentration 0.2mg/ml is added, 20 DEG C of incubation 30min are added Tween20 to final concentration 0.5%, after mixing, set It in 65 DEG C of water-bath 1h, stirs for several times, adds PMSF buffer 1 time during heating, by the solution after water-bath at 4 DEG C, 10000rpm It is centrifuged 30min, takes supernatant, crosses ni-sepharose purification.
C.Ni column purification
(1) Ni column first balances 5 times of column volumes with the sample-loading buffer of the imidazoles containing 10mM.
(2) by crude enzyme liquid by 0.45um membrane filtration, filtered supernatant passes through the Ni column after balance.
(3) contain 10mM imidazoles, 0.1%TritonX-114 buffer rinses 10 times of column volumes.
(4) 5 times of column volumes of 20mM imidazoles flushing are washed miscellaneous.
(5) 300mM imidazoles elutes destination protein, obtains the anti-oxidant fusion protein of His-SOD-MT.
Through detecting it is found that the purity of anti-oxidant fusion protein can reach 95% or more.
For the amino acid sequence of the anti-oxidant fusion protein of His-SOD-MT as shown in SEQ ID No.5, particular sequence is as follows:
HHHHHHMAFVQEPLPFDPGALEPYGMSAKTLEFHYGKHHKGYVDNLNKLTQDTELADKSLEDVIRTTY GDAAKVGIFNNAAQVWNHTFFWNSLKPGGGGVPTGDVAARINSAFGSYDEFKAQFKNAAATQFGSGWAWLVLEAGT LKVTKTANAENPLVHGQVPLLTIDVWEHAYYLDYQNRRPDFIDNFLNQLVNWDFVAKNLAAAGGGGSGGGGSGGGG SMDPNCSCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA.Wherein, HHHHHH is His label, GGGGSGGGGSGGGGS are link peptide.
Embodiment 4
The production of SOD-MT fusion protein:
The amplification of SOD-MT-his segment:
(1) with the MT-2a complete genome sequence of synthesis (SEQQ ID No.1) for template, PCR expansion is carried out using following primer Increase, obtain MT-2a segment:
The system of PCR amplification: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
PCR response procedures are as follows:
95℃2min；95 DEG C, 30s, 57 DEG C, 20s, 72 DEG C, 10s, 30 circulations；72 DEG C, 5min
(SEQ ID No.19)MT-Fw-4:
GGCGGTGGCGGAAGTGGAGGCGGTGGCAGCATGGATCCGAACTGCAGCTG；
(SEQ ID No.20) MT-Rev-4:
GGATCCGTTATCCACTTTTACGCGCAGCAGCTGCATTTATC。
(2) with the FeSOD complete genome sequence of synthesis (SEQ ID No.2) for template, PCR expansion is carried out using following primer Increase, obtain FeSOD segment:
The system of PCR amplification: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
PCR response procedures are as follows:
95℃2min；95 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 20s, 30 circulations；72 DEG C, 5min
(SEQ ID No.21) SOD-Fw-4:
CTTTAAGAAGGAGATATACATATGCACCATCATCATCATCATATGGCGTTTGTGCAGGAAC；
(SEQ ID No.22)SOD-Rev-4:
CCTCCACTTCCGCCACCGCCGCTGCCGCCACCTCCCGCCGCTGCTAAGTTTTTC。
(3) after mixing above-mentioned two sections of PCR fragments, PCR pre-reaction 10 are recycled, and reaction condition is as follows:
Amplification system: every 20uL includes: 2 μ l, dNTP (10mM each) 0.4 of PFU ultra II Fusion buffer μ l, FW primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, MT-2a 5 μ l, FeSOD 5ul, Sterile 6.4 μ l, pFU ultra IIfusion enzyme of miliQ water, 0.4 μ l.
Amplification program are as follows: 95 DEG C of 2min；95 DEG C of 20s, 50 DEG C of 20s, 72 DEG C of 30s, 10 circulations；72℃5min.
(4) using product after step (3) reaction as template, primer MT-FW-4 and SOD-Rev-4 carry out PCR, obtain His- MT-SOD segment；
Amplification system: PFU ultra II Fusion buffer 2 μ l, dNTP (10mM each) 0.4 μ l, FW 0.1 μ l, Sterile miliQ water of primer (20 μM) 0.4 μ l, REV primer (20 μM) 0.4 μ l, Template 16.3 μ l, pFU ultra IIfusion enzyme, 0.4 μ l.
Amplification program: 95 DEG C of 2min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72 DEG C, 5min.
(5) clone of SOD-MT-his segment
SOD-MT-his segment is connected respectively on carrier pet15b, convert escherichia coli DH5a, be coated on containing In the LB solid medium tablets of 100ug/ml Amp, 37 DEG C are cultivated 16 hours, are selected monoclonal colonies respectively and are identified.Mirror The DH5a for being set to positive colony is correct through DNA sequencing verifying gene order.Plasmid containing recombination fusion protein is converted respectively To in Escherichia coli Rosseta (DE3), the expression bacterial strain of fusion protein is obtained.
(6) expression and purification of fusion protein
A. the expression of fusion protein
Above-mentioned fusion protein monoclonal is inoculated into the LB liquid medium containing 100ug/ml Amp, 34ug chloramphenicol In, 37 DEG C after culture 12 hours, culture according to 1:100 ratio is inoculated into 500ml, and to contain 100ug/ml Amp, 34ug chlorine mould In the fresh TB culture medium of element, when 37 DEG C of culture 4h to OD600 reach 0.8~1, IPTG final concentration is added to 0.2mM, ZnCl2It is dense It spends to 0.2mM.20 DEG C of 12~16h of induction.
B. bacterium solution cracks
Thalline were collected by centrifugation, is resuspended in 20mM Tris, in 300mM NaCl, 1mM EDTA, 0.5nM PMSF buffer, The T4 Lysozyme of final concentration 0.2mg/ml is added, 20 DEG C of incubation 30min are added Tween20 to final concentration 0.5%, after mixing, set It in 65 DEG C of water-bath 1h, stirs for several times, adds PMSF buffer 1 time during heating, by the solution after water-bath at 4 DEG C, 10000rpm It is centrifuged 30min, takes supernatant, crosses ni-sepharose purification.
C.Ni column purification
Ni column first balances 5 times of column volumes with the sample-loading buffer of the imidazoles containing 10mM；
By crude enzyme liquid by 0.45um membrane filtration, filtered supernatant passes through the Ni column after balance；
Containing 10mM imidazoles, 0.1%TritonX-114 buffer rinses 10 times of column volumes；
20mM imidazoles 5 times of column volumes of flushing are washed miscellaneous；
300mM imidazoles elutes destination protein, obtains the anti-oxidant fusion protein of SOD-MT-his.
Through detecting it is found that the purity of anti-oxidant fusion protein can reach 95% or more.
(7) excision of His label
Contain one section of TEV protease recognition site behind the His-tag of SOD-MT-his fusion protein, TEV can be passed through Albumen enzyme effect removes label.
After fusion protein after Ni column purification measures protein concentration, TEV protease, i.e. 1 μ gTEV is added in 50:1 in mass ratio Protease acts on 50 μ g destination proteins.4 DEG C of digestions are stayed overnight.
(8) it is concentrated by ultrafiltration, changes the removing of liquid and label
Albumen after digestion changes liquid to 10mM imidazoles after 10kd super filter tube is concentrated into suitable volumes, with desalting column G25 It in sample-loading buffer, reuses Ni column and is purified, at this point, being existed by the label under cutting and the protein adsorption for not cutting off label On Ni column, the fusion protein of no his label passes through pillar, collects across liquid, as destination protein, obtains that SOD-MT is anti-oxidant to be melted Hop protein.
The amino acid sequence of the anti-oxidant fusion protein of SOD-MT is specific as follows as shown in SEQ ID No.6:
MAFVQEPLPFDPGALEPYGMSAKTLEFHYGKHHKGYVDNLNKLTQDTELADKSLEDVIRTTYGDAAKV GIFNNAAQVWNHTFFWNSLKPGGGGVPTGDVAARINSAFGSYDEFKAQFKNAAATQFGSGWAWLVLEAGTLKVTKT ANAENPLVHGQVPLLTIDVWEHAYYLDYQNRRPDFIDNFLNQLVNWDFVAKNLAAAGGGGSGGGGSGGGGSMDPNC SCAAGDSCTCAGSCKCKECKCTSCKKSCCSCCPVGCAKCAQGCICKGASDKCSCCA.Wherein GGGGSGGGGSGGGGS For link peptide.
Embodiment 5
The anti-oxidant fusion protein and use conventional method that the embodiment of the present invention 1~2 is obtained (adopt without heating by bacterium It is crushed with ultrasound or high pressure) obtained anti-oxidant albumen carries out survey enzyme activity, and it is control with SOD albumen.It the results are shown in Table 1.
1 enzyme activity result of table
Conventional method purifies U/mg Industrialized purification U/mg
SOD 36 34
MT-SOD 46 45
MT-SOD-his 46 46
As can be drawn from Table 1, using the anti-oxidant fusion protein of production method production of the invention and using routine side The enzyme activity difference for the antioxidant activity albumen that method obtains is little, compared with the control, significant difference.Comparative example 1
In the cracking of B. bacterium solution, thalline were collected by centrifugation, is resuspended in 20mM Tris, 300mM NaCl, 1mM EDTA, 0.5nM In PMSF buffer, the T4 Lysozyme of final concentration 0.2mg/ml is added, Tween20 is added to final concentration in 20 DEG C of incubation 30min 0.5%, after mixing, 70,90 DEG C of water-baths 20min, 30min, 40min, 1h, 3h and 4h are placed in, stirs for several times, adds during heating PMSF buffer 1 time, by the solution after water-bath at 4 DEG C, 10000rpm is centrifuged 30min, takes supernatant, crosses ni-sepharose purification.Remaining Part is identical as the condition in embodiment 2, detects the enzyme activity of albumen.It is control with SOD albumen, the results are shown in Table 2 and Fig. 1.
2 enzyme activity result of table
It can be concluded that, albumen retains most of activity at 70 DEG C, and most of bacterium can be made to split from table 2 and Fig. 1 Solution, but the temperature used is higher；At 90 DEG C.The loss of activity of albumen is too many.Therefore the present invention is according to experiment experience, by water-bath Temperature setting is 65 DEG C, can both stimulate cellular lysate, also make other albuminous degenerations.
Most of bacterium it can be concluded that, can both be cracked using method provided by the invention by above embodiments and comparative example Body, and can guarantee the activity of anti-oxidant fusion protein, additionally it is possible to so that other albuminous degenerations, are suitble to large-scale production.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.
Sequence table
<110>one hundred Ke's albumen of Xuchang and Co., Ltd, genetic engineering research institute
<120>a kind of production method of anti-oxidant fusion protein
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 183
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
atggatccga actgcagctg cgcggcgggc gatagctgca cctgcgcggg cagctgcaaa 60
tgcaaagaat gcaaatgcac cagctgcaag aaaagctgct gcagctgctg cccggtgggc 120
tgcgcgaaat gcgcgcaggg ctgcatttgc aaaggcgcga gcgataaatg cagctgctgc 180
gcg 183
<210> 2
<211> 600
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atggcgtttg tgcaggaacc tctgcctttt gatccgggag cgctggaacc gtatggcatg 60
agcgcgaaaa ccctggaatt tcactatggc aaacatcata aaggctatgt ggataatctg 120
aataaactga cccaggatac cgaactggcg gataaaagcc tggaagatgt gattcgtacc 180
acctatgggg acgctgccaa ggtgggcatt ttcaataatg cggcgcaggt gtggaatcat 240
accttttttt ggaatagcct gaaaccggga ggtggcggcg tgccgaccgg ggatgtggcc 300
gctcgtataa acagcgcgtt tgggagctat gacgaattca aggcgcagtt taagaatgcc 360
gcagccaccc agtttggctc tggctgggcg tggctggtgc tggaagcggg caccctgaag 420
gtgaccaaaa ccgcgaacgc ggagaatccg cttgttcatg gccaagtgcc actgctgacc 480
attgatgtgt gggaacatgc gtattatctg gattatcaga atcgtcgtcc ggatttcata 540
gacaattttc tgaatcagct ggtgaattgg gattttgtgg cgaaaaactt agcagcggcg 600
<210> 3
<211> 282
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 3
Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys Thr Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Ile Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala Gly Gly Gly
50 55 60
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala Phe Val
65 70 75 80
Gln Glu Pro Leu Pro Phe Asp Pro Gly Ala Leu Glu Pro Tyr Gly Met
85 90 95
Ser Ala Lys Thr Leu Glu Phe His Tyr Gly Lys His His Lys Gly Tyr
100 105 110
Val Asp Asn Leu Asn Lys Leu Thr Gln Asp Thr Glu Leu Ala Asp Lys
115 120 125
Ser Leu Glu Asp Val Ile Arg Thr Thr Tyr Gly Asp Ala Ala Lys Val
130 135 140
Gly Ile Phe Asn Asn Ala Ala Gln Val Trp Asn His Thr Phe Phe Trp
145 150 155 160
Asn Ser Leu Lys Pro Gly Gly Gly Gly Val Pro Thr Gly Asp Val Ala
165 170 175
Ala Arg Ile Asn Ser Ala Phe Gly Ser Tyr Asp Glu Phe Lys Ala Gln
180 185 190
Phe Lys Asn Ala Ala Ala Thr Gln Phe Gly Ser Gly Trp Ala Trp Leu
195 200 205
Val Leu Glu Ala Gly Thr Leu Lys Val Thr Lys Thr Ala Asn Ala Glu
210 215 220
Asn Pro Leu Val His Gly Gln Val Pro Leu Leu Thr Ile Asp Val Trp
225 230 235 240
Glu His Ala Tyr Tyr Leu Asp Tyr Gln Asn Arg Arg Pro Asp Phe Ile
245 250 255
Asp Asn Phe Leu Asn Gln Leu Val Asn Trp Asp Phe Val Ala Lys Asn
260 265 270
Leu Ala Ala Ala His His His His His His
275 280
<210> 4
<211> 276
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 4
Met Asp Pro Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys Thr Cys Ala
1 5 10 15
Gly Ser Cys Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser
20 25 30
Cys Cys Ser Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys
35 40 45
Ile Cys Lys Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala Gly Gly Gly
50 55 60
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Ala Phe Val
65 70 75 80
Gln Glu Pro Leu Pro Phe Asp Pro Gly Ala Leu Glu Pro Tyr Gly Met
85 90 95
Ser Ala Lys Thr Leu Glu Phe His Tyr Gly Lys His His Lys Gly Tyr
100 105 110
Val Asp Asn Leu Asn Lys Leu Thr Gln Asp Thr Glu Leu Ala Asp Lys
115 120 125
Ser Leu Glu Asp Val Ile Arg Thr Thr Tyr Gly Asp Ala Ala Lys Val
130 135 140
Gly Ile Phe Asn Asn Ala Ala Gln Val Trp Asn His Thr Phe Phe Trp
145 150 155 160
Asn Ser Leu Lys Pro Gly Gly Gly Gly Val Pro Thr Gly Asp Val Ala
165 170 175
Ala Arg Ile Asn Ser Ala Phe Gly Ser Tyr Asp Glu Phe Lys Ala Gln
180 185 190
Phe Lys Asn Ala Ala Ala Thr Gln Phe Gly Ser Gly Trp Ala Trp Leu
195 200 205
Val Leu Glu Ala Gly Thr Leu Lys Val Thr Lys Thr Ala Asn Ala Glu
210 215 220
Asn Pro Leu Val His Gly Gln Val Pro Leu Leu Thr Ile Asp Val Trp
225 230 235 240
Glu His Ala Tyr Tyr Leu Asp Tyr Gln Asn Arg Arg Pro Asp Phe Ile
245 250 255
Asp Asn Phe Leu Asn Gln Leu Val Asn Trp Asp Phe Val Ala Lys Asn
260 265 270
Leu Ala Ala Ala
275
<210> 5
<211> 282
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 5
His His His His His His Met Ala Phe Val Gln Glu Pro Leu Pro Phe
1 5 10 15
Asp Pro Gly Ala Leu Glu Pro Tyr Gly Met Ser Ala Lys Thr Leu Glu
20 25 30
Phe His Tyr Gly Lys His His Lys Gly Tyr Val Asp Asn Leu Asn Lys
35 40 45
Leu Thr Gln Asp Thr Glu Leu Ala Asp Lys Ser Leu Glu Asp Val Ile
50 55 60
Arg Thr Thr Tyr Gly Asp Ala Ala Lys Val Gly Ile Phe Asn Asn Ala
65 70 75 80
Ala Gln Val Trp Asn His Thr Phe Phe Trp Asn Ser Leu Lys Pro Gly
85 90 95
Gly Gly Gly Val Pro Thr Gly Asp Val Ala Ala Arg Ile Asn Ser Ala
100 105 110
Phe Gly Ser Tyr Asp Glu Phe Lys Ala Gln Phe Lys Asn Ala Ala Ala
115 120 125
Thr Gln Phe Gly Ser Gly Trp Ala Trp Leu Val Leu Glu Ala Gly Thr
130 135 140
Leu Lys Val Thr Lys Thr Ala Asn Ala Glu Asn Pro Leu Val His Gly
145 150 155 160
Gln Val Pro Leu Leu Thr Ile Asp Val Trp Glu His Ala Tyr Tyr Leu
165 170 175
Asp Tyr Gln Asn Arg Arg Pro Asp Phe Ile Asp Asn Phe Leu Asn Gln
180 185 190
Leu Val Asn Trp Asp Phe Val Ala Lys Asn Leu Ala Ala Ala Gly Gly
195 200 205
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Asp Pro
210 215 220
Asn Cys Ser Cys Ala Ala Gly Asp Ser Cys Thr Cys Ala Gly Ser Cys
225 230 235 240
Lys Cys Lys Glu Cys Lys Cys Thr Ser Cys Lys Lys Ser Cys Cys Ser
245 250 255
Cys Cys Pro Val Gly Cys Ala Lys Cys Ala Gln Gly Cys Ile Cys Lys
260 265 270
Gly Ala Ser Asp Lys Cys Ser Cys Cys Ala
275 280
<210> 6
<211> 276
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 6
Met Ala Phe Val Gln Glu Pro Leu Pro Phe Asp Pro Gly Ala Leu Glu
1 5 10 15
Pro Tyr Gly Met Ser Ala Lys Thr Leu Glu Phe His Tyr Gly Lys His
20 25 30
His Lys Gly Tyr Val Asp Asn Leu Asn Lys Leu Thr Gln Asp Thr Glu
35 40 45
Leu Ala Asp Lys Ser Leu Glu Asp Val Ile Arg Thr Thr Tyr Gly Asp
50 55 60
Ala Ala Lys Val Gly Ile Phe Asn Asn Ala Ala Gln Val Trp Asn His
65 70 75 80
Thr Phe Phe Trp Asn Ser Leu Lys Pro Gly Gly Gly Gly Val Pro Thr
85 90 95
Gly Asp Val Ala Ala Arg Ile Asn Ser Ala Phe Gly Ser Tyr Asp Glu
100 105 110
Phe Lys Ala Gln Phe Lys Asn Ala Ala Ala Thr Gln Phe Gly Ser Gly
115 120 125
Trp Ala Trp Leu Val Leu Glu Ala Gly Thr Leu Lys Val Thr Lys Thr
130 135 140
Ala Asn Ala Glu Asn Pro Leu Val His Gly Gln Val Pro Leu Leu Thr
145 150 155 160
Ile Asp Val Trp Glu His Ala Tyr Tyr Leu Asp Tyr Gln Asn Arg Arg
165 170 175
Pro Asp Phe Ile Asp Asn Phe Leu Asn Gln Leu Val Asn Trp Asp Phe
180 185 190
Val Ala Lys Asn Leu Ala Ala Ala Gly Gly Gly Gly Ser Gly Gly Gly
195 200 205
Gly Ser Gly Gly Gly Gly Ser Met Asp Pro Asn Cys Ser Cys Ala Ala
210 215 220
Gly Asp Ser Cys Thr Cys Ala Gly Ser Cys Lys Cys Lys Glu Cys Lys
225 230 235 240
Cys Thr Ser Cys Lys Lys Ser Cys Cys Ser Cys Cys Pro Val Gly Cys
245 250 255
Ala Lys Cys Ala Gln Gly Cys Ile Cys Lys Gly Ala Ser Asp Lys Cys
260 265 270
Ser Cys Cys Ala
275
<210> 7
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ctttaagaag gagatataca tatggatccg aactgcagct g 41
<210> 8
<211> 56
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cctccacttc cgccaccgcc gctgccgcca cctcccgcgc agcagctgca tttatc 56
<210> 9
<211> 49
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ggcggtggcg gaagtggagg cggtggcagc atggcgtttg tgcaggaac 49
<210> 10
<211> 57
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
ggatccgtta tccactttta atgatgatga tgatggtgcg ccgctgctaa gtttttc 57
<210> 11
<211> 49
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
ctttaagaag gagatataca tatgcaccat catcatcatc atgagaatc 49
<210> 12
<211> 56
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cctccacttc cgccaccgcc gctgccgcca cctcccgcgc agcagctgca tttatc 56
<210> 13
<211> 49
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
ggcggtggcg gaagtggagg cggtggcagc atggcgtttg tgcaggaac 49
<210> 14
<211> 39
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
ggatccgtta tccactttta cgccgctgct aagtttttc 39
<210> 15
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
ggcggtggcg gaagtggagg cggtggcagc atggatccga actgcagctg 50
<210> 16
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
ggatccgtta tccactttta cgcgcagcag ctgcatttat c 41
<210> 17
<211> 49
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
ctttaagaag gagatataca tatgcaccat catcatcatc atgagaatc 49
<210> 18
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
cctccacttc cgccaccgcc gctgccgcca cctcccgccg ctgctaagtt tttc 54
<210> 19
<211> 50
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
ggcggtggcg gaagtggagg cggtggcagc atggatccga actgcagctg 50
<210> 20
<211> 41
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
ggatccgtta tccactttta cgcgcagcag ctgcatttat c 41
<210> 21
<211> 61
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 21
ctttaagaag gagatataca tatgcaccat catcatcatc atatggcgtt tgtgcaggaa 60
c 61
<210> 22
<211> 54
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 22
cctccacttc cgccaccgcc gctgccgcca cctcccgccg ctgctaagtt tttc 54

Claims (10)

1. a kind of production method of anti-oxidant fusion protein, which comprises the following steps:
1) it is transferred to Escherichia coli after connecting anti-oxidant fusion with carrier, obtains the large intestine bar containing anti-oxidant fusion Bacterium；
The anti-oxidant fusion is obtained after connecting MT gene with sod gene；
The MT gene has nucleotide sequence shown in SEQ ID No.1；
The sod gene has nucleotide sequence shown in SEQ ID No.2；
2) Escherichia coli containing anti-oxidant fusion for obtaining the step 1) are inoculated in containing ammonia benzyl and chloramphenicol The first culture is carried out in LB liquid medium, obtains the first culture；
The time of first culture is 12~16h；
3) the first culture for obtaining the step 2) is inoculated in the TB fluid nutrient medium containing ammonia benzyl and chloramphenicol and carries out Second culture, obtains the second culture；
The time of second culture is 3.5~4.5h；
4) the second culture for obtaining the step 3) carries out induction training after mixing with isopropylthiogalactoside, zinc chloride It supports, obtains inducer；
5) inducer that the step 4) obtains is centrifuged, is mixed after obtained thallus is resuspended with buffer with T4 Lysozyme It closes, incubate, carry out water-bath after obtained incubation object is mixed with Tween 20, obtained water-bath object is centrifuged, is obtained anti-oxidant Fusion protein；
The temperature of the water-bath is 60~70 DEG C；
The time of the water-bath is 0.5~1.5h.
2. production method according to claim 1, which is characterized in that the amino acid of the anti-oxidant fusion protein of step 1) Sequence is as shown in SEQ ID No.3.
3. production method according to claim 1, which is characterized in that the amino acid of the anti-oxidant fusion protein of step 1) Sequence is as shown in SEQ ID No.4.
4. production method according to claim 1, which is characterized in that the amino acid of the anti-oxidant fusion protein of step 1) Sequence is as shown in SEQ ID No.5.
5. production method according to claim 1, which is characterized in that the amino acid of the anti-oxidant fusion protein of step 1) Sequence is as shown in SEQ ID No.6.
6. production method according to claim 1, which is characterized in that in the step 2) LB liquid medium, the ammonia The content of benzyl is 90~110 μ g/ml, and the content of the chloramphenicol is 30~40 μ g/ml.
7. production method according to claim 1, which is characterized in that in step 3) the TB fluid nutrient medium, the ammonia The content of benzyl is 90~110 μ g/ml, and the content of the chloramphenicol is 30~40 μ g/ml.
8. production method according to claim 1, which is characterized in that the time of the step 4) Fiber differentiation be 12~ 20h, the temperature of the Fiber differentiation are 18~22 DEG C.
9. production method according to claim 1, which is characterized in that the time that the step 5) incubates is 25~35min, The temperature of the incubation is 18~22 DEG C.
10. production method according to claim 1, which is characterized in that after step 5) the water-bath object centrifugation further include: will Obtained supernatant obtains anti-oxidant fusion protein after Ni column purification.
CN201910027485.6A 2019-01-11 2019-01-11 A kind of production method of anti-oxidant fusion protein Pending CN109609532A (en)

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Application publication date: 20190412