CN108504671A - A kind of PTEN-Long protein purifications preparation method - Google Patents
A kind of PTEN-Long protein purifications preparation method Download PDFInfo
- Publication number
- CN108504671A CN108504671A CN201810264951.8A CN201810264951A CN108504671A CN 108504671 A CN108504671 A CN 108504671A CN 201810264951 A CN201810264951 A CN 201810264951A CN 108504671 A CN108504671 A CN 108504671A
- Authority
- CN
- China
- Prior art keywords
- pten
- bacterium
- long
- concentration
- protein
- 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
- 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
- C07K14/4703—Inhibitors; Suppressors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03067—Phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase (3.1.3.67)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
Abstract
A kind of PTEN Long protein purification preparation methods, include the following steps：S100. PTEN Long plasmids are transferred to competent bacteria, obtain the bacterium colony for being transferred to the competent bacteria after plasmid；S200. several described bacterium colonies of picking, to the bacterium in bacterium colony described in several expanded with obtain with target bacteria concentration the second bacterium solution, and to the bacterium in second bacterium solution with target bacteria concentration carry out it is protein induced obtain it is protein induced after thalline；S300. the thalline is cracked, primary PTEN Long protein liquids are obtained；S400. the primary PTEN Long protein liquids described in ni-sepharose purification obtain two level PTEN Long protein liquids；S500. desalination and concentration are carried out to collect the PTEN Long albumen in the two level PTEN Long protein liquids to the two level PTEN Long protein liquids.Using PTEN Long protein purification preparation methods provided by the invention, only need a ni-sepharose purification that can prepare PTEN Long albumen, and the PTEN Long purity of protein highers prepared using the present invention, the expression rate for the PTEN Long albumen prepared is 80% or more.
Description
Technical field
The present invention relates to biomedical sector more particularly to a kind of PTEN-Long protein purifications preparation methods.
Background technology
Phosphatase tensin sample homologue (PTEN) gene also known as a variety of lates cancer are mutated (MMAC1) gene, are a kind of
Classical tumor suppressor gene.PTEN-long is the variation in translation in the areas mRNA 5 '-of PTEN, this variant makes original translation base
173 amino acid are increased in N-terminal on plinth, are pten protein alternative splicing products, and is upper highly conserved evolving.PTEN-
Long can be secreted, and can be found in serum and blood plasma, have apparent exocrine ability.
Compared to pten protein, PTEN-long albumen is found in vitro experiment, is had and is entered cell, and tumour life is inhibited
Long ability.In view of this ability of PTEN-Long, show that PTEN-Long albumen has the ability as drugs against tumor.System
Standby PTEN-Long purifying proteins then have practical significance.
But in the prior art, it is relatively low to prepare PTEN-Long purifying protein purity, and is needed in protein purification procedures.
Invention content
The present invention in view of the above technical problems, provides a kind of PTEN-Long protein purifications preparation method, only needed
Nickel column and the PTEN-Long purity of protein height prepared.
The technical solution that the present invention is used to solve the above technical problem is to provide a kind of PTEN-Long protein purifications preparation
Method includes the following steps：
S100. PTEN-Long plasmids are transferred to competent bacteria, obtain the bacterium colony for being transferred to the competent bacteria after plasmid；
S200. several described bacterium colonies of picking, expanded to the bacterium in bacterium colony described in several has mesh to obtain
The second bacterium solution of bacterial concentration is marked, and protein induced obtain is carried out to the bacterium in second bacterium solution with target bacteria concentration
To the thalline after protein induced；
S300. the thalline is cracked, primary PTEN-Long protein liquids are obtained；
S400. the primary PTEN-Long protein liquids described in ni-sepharose purification obtain two level PTEN-Long protein liquids；
S500. desalination and concentration are carried out to collect the two level PTEN-Long eggs to the two level PTEN-Long protein liquids
PTEN-Long albumen in white liquor.
Optionally, the step S100 includes the following steps：
S110. it takes out the competent bacteria preserved at -80 DEG C and is put into ice water and thaw, extract the sense after 100 microlitres of defrostings
The first centrifuge tube is injected by state bacterium, and 10ng PTEN-Long plasmids will be added in the first centrifuge tube, is uniformly mixed；
S120. by first centrifugation of 100 microlitres of competent bacterias and 10ng PTEN-Long plasmids equipped with mixing
Pipe ice bath 30 minutes is then placed in heat shock 45 seconds in 42 DEG C of water-baths, ice bath 1 minute again after taking-up；
S130. LB liquid medium 1ml are added into first centrifuge tube, first centrifuge tube is placed on shaking table
Interior, at 37 DEG C, under conditions of 200rpm, slowly vibrating culture 90 minutes is transferred to the competent bacteria after plasmid described in formation
First bacterium solution；
S140. first bacterium solution for taking out 100ul is coated in solid medium, and solid medium back-off is placed on
In 37 DEG C of incubator, continue culture 16 hours to form the bacterium colony.
Optionally, step S200 further comprises the steps：
S210. the LB liquid mediums of a concentration of 100ug/ml of 50ml are put into the first conical flask；Picking is individually described
Bacterium colony is inoculated in the LB liquid mediums in first conical flask；First conical flask is placed in shaking table, 37
DEG C, shaken cultivation 7-20 hours under conditions of 150-300rpm, for being expanded to the bacterium in the bacterium colony to obtain
Two bacterium solutions；
S220. 400ml, the LB liquid mediums of a concentration of 100ug/ml in ampicillin and institute are added in the second conical flask
The second bacterium solution 8ml is stated, the second conical flask is put into shaking table, at 37 DEG C, under conditions of 150-300rpm, shaken cultivation 1 hour
30 minutes, for obtaining the second bacterium solution with target bacteria concentration；
S230. the isopropylthiogalactoside of final concentration of 0.1mM-1mM is added into second conical flask again, protects
It holds at 21 DEG C, under conditions of 150-300rpm, second conical flask vibrates induction 3-6 hours on shaking table, for described
Bacterium in the second bacterium solution with target bacteria concentration carries out protein induced to obtain third bacterium solution；
S240. 400mL third bacterium solutions are extracted from the second conical flask, are put into the first centrifuge, and the first centrifuge keeps 4
DEG C, the thalline is collected with the centrifugal speed of 3000xg.
Optionally, the rotating speed of shaking table is 200rpm in step S210, and the time of shaken cultivation is 16 hours；
In step S220, the rotating speed of shaking table is 200rpm；
The rotating speed of the final concentration of 0.1mM of isopropylthiogalactoside in step S230, shaking table are 200rpm, and oscillation lures
It is 4.5 hours to lead the time.
Optionally, the bacterial concentration that the OD values under a concentration of 490nm wavelength of the target bacteria are 0.5；
Optionally, step S300 further comprises：
S310. it takes the thalline that weight in wet base is 2g to be added in the bacterial lysate of 20ml, the bacterium of the thalline will be added
Lysate and the Triton X-100 of 0.1%-2% bacterial lysate volume ratios and the benzyl sulphonyl of final concentration 1mM
Fluorine mixes, and the broken bacterium of ultrasound 3 times, 10 minutes every time, for cracking the thalline, and obtain the 4th bacterium of clear in ice bath
Liquid；
S320. the phenylmethylsulfonyl fluoride of final concentration 1mM is added in the 4th bacterium solution, is put into the second centrifuge, at 4 DEG C
It is centrifuged 30 minutes under conditions of 20000xg centrifugal speeds, extracts supernatant, abandon precipitation, obtain the 5th bacterium solution；
S330. the 5th bacterium solution described in filtration membrane filtration obtains the primary PTEN-Long protein liquids.
Optionally, Triton X-100 and bacterial lysate volume ratio are 2% in step S310；In step S330
Filtration membrane be 0,22um filtration membrane.
Optionally, step S400 further comprises the steps：
S410. respectively with the eluent of 5 times of column volumes, the combination liquid of the deionized water of 5 times of column volumes, 10 times of column volumes is flat
Weigh nickel column；Primary PTEN-Long protein liquids described in speed loading 5mL using 1ml/ minutes are adjusted to 5ml/ after end of the sample
Minute, the primary PTEN-Long protein liquids are continuously added to 10 times of column volumes so that light absorption value is without substantially changeing；
S420. the mixed liquor of the imidazoles of final concentration 50mM-150mM is added into the nickel column, nickel is adsorbed on for removing
Impurity on column balances the nickel column again after the mixed liquor of 10 times of column volumes is added, with elution albumen, after collection
The dithiothreitol (DTT) of final concentration 1mM, the ethylenediamine tetra-acetic acid of final concentration 2mM, the phenylmethylsulfonyl fluoride of final concentration 1mM is added in albumen
Obtain the two level PTEN-Long protein liquids；
Wherein, the mixed liquor of the imidazoles of the final concentration 50mM-150mM is by mixing the combination liquid and the eluent
It obtains.
Optionally, the imidazoles of final concentration 100mM is used in step S420.
Optionally, step S500 further comprises the steps：
S510. desalting column is balanced with the desalinization liquor of 10 times of column volumes, two level PTEN- described in 5ml/ minutes speed loadings
Long protein liquids collect the PTEN-Long albumen after desalination according to the variation of light absorption value；
S520. the PTEN-Long albumen after the desalination is put into super filter tube, the first centrifuge keep 4 DEG C and
Centrifugation 20 minutes is carried out under conditions of 3000xg centrifugal speeds, is concentrated and is collected PTEN-Long albumen.
The present invention provides a kind of PTEN-Long protein purifications preparation methods, it is only necessary to which crossing a nickel column can prepare
PTEN-Long albumen, and the PTEN-Long purity of protein highers prepared using the present invention, the expression rate of PTEN-Long albumen are existed
80% or more.
Description of the drawings
Present invention will be further explained below with reference to the attached drawings and examples, in attached drawing：
Fig. 1 is the step schematic diagram of PTEN-Long protein purifications preparation method provided by the present invention；
Fig. 2 is the step S100 schematic diagrames of PTEN-Long protein purifications preparation method provided by the present invention；
Fig. 3 is the step S200 schematic diagrames of PTEN-Long protein purifications preparation method provided by the present invention；
Fig. 4 is the step S300 schematic diagrames of PTEN-Long protein purifications preparation method provided by the present invention；
Fig. 5 is the step S400 schematic diagrames of PTEN-Long protein purifications preparation method provided by the present invention；
Fig. 6 is the step S500 schematic diagrames of PTEN-Long protein purifications preparation method provided by the present invention.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified
Conventional method.The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
As shown in Figure 1, a kind of PTEN-Long protein purifications preparation method provided by the invention includes the following steps：It is a kind of
PTEN-Long protein purification preparation methods, include the following steps：
S100. PTEN-Long plasmids are transferred to competent bacteria, obtain the bacterium colony for being transferred to the competent bacteria after plasmid；
S200. several described bacterium colonies of picking, expanded to the bacterium in bacterium colony described in several has mesh to obtain
The second bacterium solution of bacterial concentration is marked, and protein induced obtain is carried out to the bacterium in second bacterium solution with target bacteria concentration
To the thalline after protein induced；
S300. the thalline is cracked, primary PTEN-Long protein liquids are obtained；
S400. the primary PTEN-Long protein liquids described in ni-sepharose purification obtain two level PTEN-Long protein liquids；
S500. desalination and concentration are carried out to collect the two level PTEN-Long eggs to the two level PTEN-Long protein liquids
PTEN-Long albumen in white liquor.
By above step, the present invention is during preparing PTEN-Long albumen, it is only necessary to a nickel column was carried out,
And the expression rate of the PTEN-Long albumen prepared is up to 80% or more.Specifically, each step is described as follows.
S100. PTEN-Long plasmids are transferred to competent bacteria, obtain the bacterium colony for being transferred to the competent bacteria after plasmid.
PTEN-Long plasmids in the present embodiment come from addgen, jpExpress404-PTEN-Long-V5/His,
Catalog：49417；Competent bacteria in embodiment is BL21 competent bacterias, is CWBIO products, article No. 03686.
As shown in Fig. 2, step S100 further comprises the steps：
S110. it takes out the competent bacteria preserved at -80 DEG C and is put into ice water and thaw, extract the sense after 100 microlitres of defrostings
The first centrifuge tube is injected by state bacterium, and 10ng PTEN-Long plasmids will be added in the first centrifuge tube, is uniformly mixed.It is preferred that
Ground, first centrifuge tube are 15mL centrifuge tubes.
S120. by first centrifugation of 100 microlitres of competent bacterias and 10ng PTEN-Long plasmids equipped with mixing
Pipe ice bath 30 minutes is then placed in heat shock 45 seconds in 42 DEG C of water-baths, ice bath 1 minute again after taking-up.In the present embodiment to
One centrifuge tube carries out ice bath and the water-bath 100 of water-bath is purchased from the macro experimental facilities Co., Ltd of upper Nereid.
S130. LB liquid medium 1ml are added into first centrifuge tube, first centrifuge tube is placed on shaking table
Interior, at 37 DEG C, under conditions of 200rpm, slowly vibrating culture 90 minutes is transferred to the competent bacteria after plasmid described in formation
First bacterium solution.
Shaking table in the present embodiment is purchased from the macro experimental facilities Co., Ltd of upper Nereid, in the description of following description, if
Not specifically mentioned, the shaking table used is the shaking table.LB liquid mediums in the present embodiment are LB antibiotic-free Liquid Cultures
Liquid configures by the following method：Peptone 10g, yeast extract 5g and NaCl 10g are weighed, 800ml deionized waters are dissolved in
In, with NaOH tune pH to 7.5, adds deionized water to 1 liter of total volume, be put into autoclaving equipment, under high pressure steam sterilization
20 minutes.Wherein, peptone is tryptone, is OXOID products, article No. 1227229；Yeast extract is OXOID products,
Article No. 1844628；NaCl is traditional Chinese medicines product, article No. 7447-40-7；NaOH is traditional Chinese medicines product, article No. 1310-73-2.Deionization
Water comes from deionizing water machine, which is purchased from MILLIPORE companies.
S140. first bacterium solution for taking out 100ul is coated in solid medium, and solid medium back-off is placed on
In 37 DEG C of incubator, continue culture 16 hours to form the bacterium colony.
Solid medium in the present embodiment is LB solid mediums, can be obtained by following configuration method：Weigh egg
White peptone 2g, yeast extract 1g, NaCl 2g and agarose 3g, add deionized water 180ml, are arrived with the NaOH tune PH of 5mol/L
7.0, add deionized water to total volume 200ml, be put into autoclaving equipment, under high pressure steam sterilization 20 minutes.Temperature drops
To 55 degree, ampicillin is added by final concentration 100ug/ml, mixing pours into sterilizing plates in super-clean bench, 4 DEG C are put into after solidification
It is buckled in refrigerator spare.
Wherein, peptone is tryptone, is OXOID products, article No. 1227229；Yeast extract produces for OXOID
Product, article No. 1844628；NaOH is traditional Chinese medicines product, article No. 1310-73-2；Ampicillin is Solarbio companies, article No.
128F036.Deionized water comes from deionizing water machine, which is purchased from MILLIPORE companies.
S200. several described bacterium colonies of picking, expand the bacterium in bacterium colony described in several, and to amplification after
Bacterium carry out it is protein induced obtain it is protein induced after thalline.
As shown in figure 3, step S200 further comprises the steps：
S210. the LB liquid mediums of a concentration of 100ug/ml of 50ml are put into the first conical flask；Picking is individually described
Bacterium colony is inoculated in the LB liquid mediums in first conical flask；First conical flask is placed in shaking table, 37
DEG C, shaken cultivation 7-20 hours under conditions of 150-300rpm, for being expanded to the bacterium in the bacterium colony to obtain
Two bacterium solutions.Preferably, in step S210, the rotating speed of shaking table is 200rpm, and the time of shaken cultivation is 16 hours.LB liquid is trained
Nutrient solution and the LB liquid mediums in step S130.
S220. 400ml, the LB liquid mediums of a concentration of 100ug/ml in ampicillin and institute are added in the second conical flask
The second bacterium solution 8ml is stated, the second conical flask is put into shaking table, at 37 DEG C, under conditions of 150-300rpm, shaken cultivation 1 hour
30 minutes, for obtaining the second bacterium solution with target bacteria concentration.Preferably, which is 1000mL conical flasks.
Preferably, in step S220, the rotating speed of shaking table is 200rpm；OD values under a concentration of 490nm wavelength of target bacteria are
0.5.Wherein, which is measured by microplate reader, extracts 200 microlitre of second bacterium solution to 96 orifice plates, 96 orifice plates are put
Enter microplate reader, and it is that 490nm is measured the target bacteria concentration of second bacterium solution that wavelength, which is arranged,.When bacterial concentration is higher than
When target bacteria concentration, bacterium is excessive, following protein induction can be caused insufficient, and then lead to the PTEN- finally prepared
Long purity of protein is not high, and when bacterial concentration is less than target bacteria concentration, bacterium is very few, the PTEN-Long eggs prepared
White very few, therefore, bacterial concentration ensures in target bacteria concentration, it is ensured that the production of the PTEN-Long albumen prepared
Amount is higher, and the purity of albumen is high.
Bacterium amplification is carried out using step S210 and two steps of step S220, can be expanded to avoid the overlong time of amplification step
The bacterial concentration of increasing is excessive, the less effective for causing following protein to induce.Can be had by step S210 and step S220
There is the second bacterium solution of target bacteria concentration so that following protein inducing effect is preferable.
S230. the isopropylthiogalactoside of final concentration of 0.1mM-1mM is added into second conical flask again, protects
It holds at 21 DEG C, under conditions of 150-300rpm, second conical flask vibrates induction 3-6 hours on shaking table, for having
Bacterium in second bacterium solution of target bacteria concentration carries out protein induced to obtain third bacterium solution.Preferably, different in step S230
The rotating speed of the final concentration of 0.1mM of propyl dithiocarbamate galactoside, shaking table are 200rpm, and oscillation induction time is 4.5 hours.This
Under part, protein induced best results.
Isopropylthiogalactoside (hereinafter referred to as IPTG) in the present embodiment is Solarbio companies, article No.
1213G052。
S240. 400mL third bacterium solutions are extracted from the second conical flask, are put into the first centrifuge, and the first centrifuge keeps 4
DEG C, the thalline is collected with the centrifugal speed of 3000xg.
The first centrifuge in the present embodiment is eppendorf companies, the centrifuge of model centrifuge 5810R,
Second centrifuge is eppendorf companies, the supercentrifuge of model centrifuge 5424R.The two is distinguished as,
The capacity of first centrifuge is more than the second centrifuge, and the centrifugal speed of the second centrifuge is higher than the first centrifuge.
S300. the thalline is cracked, primary PTEN-Long protein liquids are prepared.
As shown in figure 4, step S300 further comprises：
S310. it takes the thalline that weight in wet base is 2g to be added in the bacterial lysate of 20ml, the bacterium of the thalline will be added
Lysate and the Triton X-100 of 0.1%-2% bacterial lysate volume ratios and the benzyl sulphonyl of final concentration 1mM
Fluorine mixes, and the broken bacterium of ultrasound 3 times, 10 minutes every time, for cracking the thalline, and obtain the 4th bacterium of clear in ice bath
Liquid.Above-mentioned bacterial lysate, thalline, Triton X-100 and benzyl sulphonyl add in the second centrifuge tube.It is excellent
Selection of land, Triton X-100 and bacterial lysate volume ratio are 2% in step S310, when this volume ratio, can be subtracted
Few follow-up non-specific binding using in ni-sepharose purification protein process.
Wherein, the phenylmethylsulfonyl fluoride (abbreviation PMSF) in the present embodiment is Solarbio companies, article No. P0100；Poly- second
Glycol octyl phenyl ether (abbreviation Triton-X) is Solarbio companies, article No. 1104B053；Bacterial lysate is：150mM
NaCl, 50mM Tris (trishydroxymethylaminomethane, referred to as " Tris "), wherein trishydroxymethylaminomethane is Solarbio public
Department, article No. 0922s0710.The broken bacterium of ultrasound carries out in ultrasonic cell disruptor, and ultrasonic cell disruptor is new purchased from Ningbo
Sesame biotech inc.
S320. the phenylmethylsulfonyl fluoride of final concentration 1mM is added in the 4th bacterium solution, is put into the second centrifuge, at 4 DEG C
It is centrifuged 30 minutes under conditions of 20000xg centrifugal speeds, extracts supernatant, abandon precipitation, obtain the 5th bacterium solution.
S330. the 5th bacterium solution described in filtration membrane filtration obtains primary PTEN-Long protein liquids.Filtration in the present embodiment
Film MILLIPORE companies, model MILLEX GP 0.22um.Obtained primary PTEN-Long protein liquids are stored temporarily in ice
In water.
S400. the primary PTEN-Long protein liquids described in ni-sepharose purification obtain two level PTEN-Long protein liquids.
As shown in figure 5, step S400 further comprises the steps：
S410. respectively with the eluent of 5 times of column volumes, the combination liquid of the deionized water of 5 times of column volumes, 10 times of column volumes is flat
Weigh nickel column；Primary PTEN-Long protein liquids described in speed loading 5mL using 1ml/ minutes are adjusted to 5ml/ after end of the sample
Minute, the primary PTEN-Long protein liquids are continuously added to 10 times of column volumes so that light absorption value is without substantially changeing.
Wherein, nickel column 400 is General Corporation's product, HISTRAP HP 5ml；It is 500mM NaCl 25mM in conjunction with liquid
Tris pH 7.6,20mM imidazoles；Eluent is 500mM NaCl 25mM Tris pH7.5,500mM imidazoles.Imidazoles is
Solarbio companies, article No. 1102D031.
S420. the mixed liquor of the imidazoles of final concentration 50mM-150mM is added into the nickel column, nickel is adsorbed on for removing
Impurity on column balances the nickel column again after the mixed liquor of 10 times of column volumes is added, with elution albumen, after collection
The dithiothreitol (DTT) of final concentration 1mM, the ethylenediamine tetra-acetic acid of final concentration 2mM, the phenylmethylsulfonyl fluoride of final concentration 1mM is added in albumen
Obtain the two level PTEN-Long protein liquids.Wherein, the mixed liquor of the imidazoles of the final concentration 50mM-150mM is by mixing institute
It states and is obtained in conjunction with liquid and the eluent.The final concentration of imidazoles is less than 50mM, and non-specific binding can be caused excessively high, lead to impurity
It is more；When the final concentration of imidazoles is higher than 150mM, for example, when reaching 200mM, the destination protein that invertibity combines on nickel column can be made
A large amount of elutions, reduce the acquisition amount of albumen.Preferably, using the imidazoles of final concentration 100mM, the purity of protein that is collected at this time compared with
Good and acquisition amount is more.
Wherein, step S420 is carried out in protein purification instrument, and the protein purification instrument in the present embodiment is General Corporation's product,
AKTAprime plus.In the present embodiment, dithiothreitol (DTT) (abbreviation DTT) is Solarbio companies, article No. D1070；Second two
Amine tetraacethyl (hereinafter referred to as EDTA) is Solarbio companies, article No. 1025A065；Phenylmethylsulfonyl fluoride (abbreviation PMSF) is
Solarbio companies, article No. P0100.
S500. desalination and concentration are carried out to collect the two level PTEN-Long eggs to the two level PTEN-Long protein liquids
PTEN-Long albumen in white liquor.
As shown in fig. 6, step S500 further comprises the steps：
S510. desalting column is balanced with the desalinization liquor of 10 times of column volumes, two level PTEN- described in 5ml/ minutes speed loadings
Long protein liquids collect the PTEN-Long albumen after desalination according to the variation of light absorption value.In this step, by two level PTEN-Long
The salt such as imidazoles lock is in desalting column in protein liquid, and therefore, PTEN-Long albumen can first come out from desalting column, then deviate from again
Salt.When desalination, after two level PTEN-Long protein liquids cross column, due to PTEN-Long albumen can make light absorption value rise and salt has conduction
Property, therefore, in the two level PTEN-Long protein liquids after crossing column, light absorption value can first rise, and conducting value is constant, it is meant that at this moment
Only albumen does not have salt, rear light absorption value to decline, and conducting value rises, it is meant that albumen is reduced, and salt gradually flows out.After collecting desalination
PTEN-Long albumen refers to collecting light absorption value in the two level PTEN-Long protein liquids after two level PTEN-Long protein liquids cross column
The part that high and conducting value does not rise, the PTEN-Long albumen that this part includes is high and does not include other salt, the purpose of this step
It is de- imidazoles, imidazoles has harmful effect, salt amount height that PTEN-Long albumen can be made unstable subsequent step.Wherein, light absorption value
It is detected as the prior art, details are not described herein.Therefore, according to the variation of light absorption value, you can what collection came out from desalting column
PTEN-Long albumen.Wherein, desalinization liquor is：50mM NaCl,25mM Tris pH 7.5；Desalting column is General Corporation's product
HiTrap HP 5ml。
S520. the PTEN-Long albumen after the desalination is put into super filter tube, the first centrifuge keep 4 DEG C and
Centrifugation 20 minutes is carried out under conditions of 3000xg centrifugal speeds, is concentrated and is collected PTEN-Long albumen.
Super filter tube in following embodiments is MILLIPORE companies, model Amicon Ultra-50
The albumen being collected into is identified, the coomassie brilliant blue staining after SDS-PAGE system electrophoresis, albumen is carried out
HIS labels detect, and detect PTEN-Long albumen.Testing result shows, the PTEN-Long albumen that this preparation method prepares
Expression rate up to 80% or more, higher than the expression rate of PTEN-Long albumen for preparing of preparation method of the prior art.
In conclusion the present invention provides a kind of PTEN-Long protein purifications preparation methods, it is only necessary to a ni-sepharose purification
PTEN-Long albumen, and the PTEN-Long purity of protein highers prepared using the present invention, the PTEN- prepared can be prepared
The expression rate of Long albumen is 80% or more.
It should be understood that for those of ordinary skills, it can be modified or changed according to the above description,
And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
Claims (10)
1. a kind of PTEN-Long protein purifications preparation method, which is characterized in that include the following steps：
S100. PTEN-Long plasmids are transferred to competent bacteria, obtain the bacterium colony for being transferred to the competent bacteria after plasmid；
S200. several described bacterium colonies of picking, expanded to the bacterium in bacterium colony described in several has target thin to obtain
Second bacterium solution of bacteria concentration, and obtain egg to the bacterium progress in second bacterium solution with target bacteria concentration is protein induced
Thalline after white induction；
S300. the thalline is cracked, primary PTEN-Long protein liquids are obtained；
S400. the primary PTEN-Long protein liquids described in ni-sepharose purification obtain two level PTEN-Long protein liquids；
S500. desalination and concentration are carried out to collect the two level PTEN-Long protein liquids to the two level PTEN-Long protein liquids
In PTEN-Long albumen.
2. preparation method according to claim 1, which is characterized in that the step S100 includes the following steps：
S110. it takes out the competent bacteria preserved at -80 DEG C and is put into ice water and thaw, extract the competence after 100 microlitres of defrostings
Bacterium injects the first centrifuge tube, and 10ngPTEN-Long plasmids will be added in the first centrifuge tube, is uniformly mixed；
S120. by the first centrifuge tube ice of 100 microlitres of competent bacterias and 10ng PTEN-Long plasmids equipped with mixing
Bath 30 minutes, is then placed in heat shock 45 seconds in 42 DEG C of water-baths, ice bath 1 minute again after taking-up；
S130. LB liquid medium 1ml are added into first centrifuge tube, first centrifuge tube is placed in shaking table,
At 37 DEG C, under conditions of 200rpm, slowly vibrating culture 90 minutes is transferred to first of the competent bacteria after plasmid described in formation
Bacterium solution；
S140. first bacterium solution for taking out 100ul is coated in solid medium, and solid medium back-off is placed on 37 DEG C
Incubator in, continue culture 16 hours to form the bacterium colony.
3. preparation method according to claim 2, which is characterized in that step S200 further comprises the steps：
S210. the LB liquid mediums of a concentration of 100ug/ml of 50ml are put into the first conical flask；The single bacterium colony of picking,
It is inoculated in the LB liquid mediums in first conical flask；First conical flask is placed in shaking table, at 37 DEG C,
Shaken cultivation 7-20 hours under conditions of 150-300rpm, for being expanded to the bacterium in the bacterium colony to obtain the second bacterium
Liquid；
S220. 400ml, the LB liquid mediums of a concentration of 100ug/ml in ampicillin and described are added in the second conical flask
Two bacterium solution 8ml, the second conical flask is put into shaking table, at 37 DEG C, under conditions of 150-300rpm, and 30 points of shaken cultivation 1 hour
Clock, for obtaining the second bacterium solution with target bacteria concentration；
S230. the isopropylthiogalactoside of final concentration of 0.1mM-1mM is added into second conical flask again, is maintained at
21 DEG C, under conditions of 150-300rpm, second conical flask vibrates induction 3-6 hours on shaking table, for having to described
Bacterium in second bacterium solution of target bacteria concentration carries out protein induced to obtain third bacterium solution；
S240. 400mL third bacterium solutions are extracted from the second conical flask, are put into the first centrifuge, the first centrifuge is kept for 4 DEG C,
The thalline is collected with the centrifugal speed of 3000xg.
4. preparation method according to claim 3, which is characterized in that the rotating speed of shaking table is 200rpm in step S210, is shaken
The time for swinging culture is 16 hours；
In step S220, the rotating speed of shaking table is 200rpm；
The rotating speed of the final concentration of 0.1mM of isopropylthiogalactoside in step S230, shaking table are 200rpm, when oscillation induces
Between be 4.5 hours.
5. preparation method according to claim 3, which is characterized in that under a concentration of 490nm wavelength of target bacteria
The bacterial concentration that OD values are 0.5.
6. preparation method according to claim 3, which is characterized in that step S300 further comprises：
S310. it takes the thalline that weight in wet base is 2g to be added in the bacterial lysate of 20ml, the bacteria lysis of the thalline will be added
Liquid and the Triton X-100 of 0.1%-2% bacterial lysate volume ratios and the phenylmethylsulfonyl fluoride of final concentration 1mM are mixed
It closes, the broken bacterium of ultrasound 3 times, 10 minutes every time, for cracking the thalline, and obtain the 4th bacterium solution of clear in ice bath；
S320. the phenylmethylsulfonyl fluoride of final concentration 1mM is added in the 4th bacterium solution, is put into the second centrifuge, at 4 DEG C and
It is centrifuged 30 minutes under conditions of 20000xg centrifugal speeds, extracts supernatant, abandon precipitation, obtain the 5th bacterium solution；
S330. the 5th bacterium solution described in filtration membrane filtration obtains the primary PTEN-Long protein liquids.
7. preparation method according to claim 6, which is characterized in that in step S310 Triton X-100 with it is thin
Bacterium lysate volume ratio is 2%；The filtration membrane that filtration membrane in step S330 is 0,22um.
8. preparation method according to claim 6, which is characterized in that step S400 further comprises the steps：
S410. respectively with the eluent of 5 times of column volumes, the deionized water of 5 times of column volumes, the combination liquid balance nickel of 10 times of column volumes
Column；Primary PTEN-Long protein liquids described in speed loading 5mL using 1ml/ minutes are adjusted to 5ml/ minutes after end of the sample,
The primary PTEN-Long protein liquids are continuously added to 10 times of column volumes so that light absorption value is without substantially changeing；
S420. the mixed liquor of the imidazoles of final concentration 50mM-150mM is added into the nickel column, nickel column is adsorbed on for removing
Impurity, be added 10 times of column volumes mixed liquor after balance the nickel column again, with elution albumen, the albumen after collection
The dithiothreitol (DTT) of final concentration 1mM is added, the phenylmethylsulfonyl fluoride of the ethylenediamine tetra-acetic acid of final concentration 2mM, final concentration 1mM obtains
The two level PTEN-Long protein liquids；
Wherein, the mixed liquor of the imidazoles of the final concentration 50mM-150mM is obtained by mixing the combination liquid and the eluent
It arrives.
9. preparation method according to claim 8, which is characterized in that use the imidazoles of final concentration 100mM in step S420.
10. preparation method according to claim 8, which is characterized in that step S500 further comprises the steps：
S510. desalting column is balanced with the desalinization liquor of 10 times of column volumes, two level PTEN-Long eggs described in 5ml/ minutes speed loadings
White liquor collects the PTEN-Long albumen after desalination according to the variation of light absorption value；
S520. the PTEN-Long albumen after the desalination is put into super filter tube, is kept for 4 DEG C and 3000xg in the first centrifuge
Centrifugation 20 minutes is carried out under conditions of centrifugal speed, is concentrated and is collected PTEN-Long albumen.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810264951.8A CN108504671A (en) | 2018-03-28 | 2018-03-28 | A kind of PTEN-Long protein purifications preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810264951.8A CN108504671A (en) | 2018-03-28 | 2018-03-28 | A kind of PTEN-Long protein purifications preparation method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108504671A true CN108504671A (en) | 2018-09-07 |
Family
ID=63379086
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810264951.8A Pending CN108504671A (en) | 2018-03-28 | 2018-03-28 | A kind of PTEN-Long protein purifications preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108504671A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763260A (en) * | 2020-06-11 | 2020-10-13 | 四川大学华西医院 | PTEN-Long polyclonal antibody, preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425645A (en) * | 2002-12-23 | 2003-06-25 | 南京工业大学 | Membrane Technology Extraction Method of L-Phenylalanine |
| CN1844380A (en) * | 2006-05-12 | 2006-10-11 | 黄耀江 | Process for preparing high-purity thrombase |
| CN102387810A (en) * | 2009-02-17 | 2012-03-21 | 纽约哥伦比亚大学理事会 | Identification of extracellular form of PTEN that can be used to treat tumors |
| CN102844043A (en) * | 2010-02-17 | 2012-12-26 | 纽约哥伦比亚大学理事会 | Use of pten-long leader sequence for transmembrane delivery of molecules |
-
2018
- 2018-03-28 CN CN201810264951.8A patent/CN108504671A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1425645A (en) * | 2002-12-23 | 2003-06-25 | 南京工业大学 | Membrane Technology Extraction Method of L-Phenylalanine |
| CN1844380A (en) * | 2006-05-12 | 2006-10-11 | 黄耀江 | Process for preparing high-purity thrombase |
| CN102387810A (en) * | 2009-02-17 | 2012-03-21 | 纽约哥伦比亚大学理事会 | Identification of extracellular form of PTEN that can be used to treat tumors |
| CN102844043A (en) * | 2010-02-17 | 2012-12-26 | 纽约哥伦比亚大学理事会 | Use of pten-long leader sequence for transmembrane delivery of molecules |
Non-Patent Citations (7)
| Title |
|---|
| HUI WANG ET AL.: "Relevance and Therapeutic Possibility of PTEN-Long in Renal Cell Carcinoma", 《PLOS ONE》 * |
| 冯智慧等: "人GST-PTEN融合蛋白的原核表达和纯化", 《医学分子生物学杂志》 * |
| 吕建新等: "《分子诊断学》", 31 August 2004, 中国医药科技出版社 * |
| 张蕾等: "《生物化学实验指导》", 31 August 2011, 武汉大学出版社 * |
| 张谢等: "一个新磷酸酶张力蛋白样同源物亚型的研究", 《中国现代医生》 * |
| 李燕等: "《细胞与分子生物学常用实验技术》", 31 July 2009, 第四军医大学出版社 * |
| 黄山等: "《心脏标志物实验室检测应用指南》", 31 July 2015, 中国科学技术出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763260A (en) * | 2020-06-11 | 2020-10-13 | 四川大学华西医院 | PTEN-Long polyclonal antibody, preparation method and application thereof |
| CN111763260B (en) * | 2020-06-11 | 2022-03-29 | 四川大学华西医院 | PTEN-Long polyclonal antibody, preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Schleif et al. | Practical methods in molecular biology | |
| Elkayam et al. | Enhancing the drug metabolism activities of C3A—a human hepatocyte cell line—by tissue engineering within alginate scaffolds | |
| EP1976975B1 (en) | In vitro expansion of postpartum derived cells in roller bottles | |
| CN101245099A (en) | Amino acid sequence of recombined human papilloma virus L1 capsid protein and uses thereof | |
| CN107501405B (en) | Autophagy inhibiting polypeptide | |
| CN108342362A (en) | A kind of stable cell lines MDCK and its construction method for expanding recombination hepatitis infectiosa canis virus CAV2 | |
| CN108504671A (en) | A kind of PTEN-Long protein purifications preparation method | |
| CN101481408A (en) | Modification sequence of recombinant human mammilla tumor virus L1 capsid protein for preventing high polymerization | |
| US9868762B2 (en) | Method for purifying virus-like particles (VLP) | |
| GB2042554A (en) | Method for preparation of recombinant dna | |
| CN111793604A (en) | Human non-small cell lung cancer cell strain H1975/OR resistant to oxitinib and application thereof | |
| MX2008014763A (en) | Process for separating and determining the viral load in a pancreatin sample. | |
| CN101481407A (en) | Modification sequence of recombinant human mammilla tumor virus L1 capsid protein | |
| CN102212535A (en) | Construction and identification of over-expression lentivirus vector of rat GSK-3 beta (Glycogen Synthase Kinase-3 beta) target gene | |
| US20060292678A1 (en) | Gluconobacter oxydans 2-ketoreductase enzyme and applications thereof | |
| CN108486156B (en) | Immortalized tree shrew small intestine epithelial cell line and construction method and application thereof | |
| Fedoroff et al. | Factor requirement of the bacteriophage f2 replicase | |
| CN106084030B (en) | A method of improving interleukins IL-34 thermal stability | |
| Castellino et al. | trans-Dominant and non-trans-dominant mutant simian virus 40 large T antigens show distinct responses to ATP | |
| Cardi et al. | Heterologous expression and affinity purification of eukaryotic membrane proteins in view of functional and structural studies: The example of the sarcoplasmic reticulum Ca 2+-ATPase | |
| CN106367360A (en) | Gene transformation method for agrobacterium-mediated paecilomyces cicadae | |
| CN104450781B (en) | A kind of cell line of overexpression CIAPIN1 albumen and its preparation method and application | |
| CN107828789A (en) | Application of the inhibitor and its application and KAP1 of targeted therapy of lung cancer as drug targets in the medicine of screening anti-lung cancer | |
| CN107253988A (en) | A kind of preparation method of people's source program death factors part PD L2 albumen | |
| CN112494661A (en) | Application of Dvl3-DEP peptide segment in preparation of drug for repairing support cell damage in testis |
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: 20180907 |