CN114150010A - Expression and purification method of human BAF45D fusion protein and application thereof - Google Patents
Expression and purification method of human BAF45D fusion protein and application thereof Download PDFInfo
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Abstract
The invention discloses a method for expressing and purifying a human BAF45D fusion protein and application thereof, comprising the following steps: constructing a prokaryotic expression vector of human BAF45D to obtain a recombinant plasmid pGEX-5X-1-BAF 45D; prokaryotic expression of GST-BAF45D fusion protein: transferring the recombinant plasmid pGEX-5X-1-BAF45D into Rosetta (DE3) escherichia coli, and carrying out IPTG induced expression to obtain a thallus precipitate; breaking bacteria and collecting supernatant; purification of GST-BAF45D fusion protein: the supernatant obtained by prokaryotic expression was purified by centrifugation with GST Resin. The method can realize the high-efficiency expression of the thalli, and obtains the GST-BAF45D fusion protein with high activity, thereby being capable of combining and incubating the fusion protein with the human embryonic stem cell lysis total protein, being capable of simply screening the protein from the human embryonic stem cell combined with the target protein, and having practical application value.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a method for expressing and purifying a human BAF45D fusion protein, and application of the human BAF45D fusion protein in searching and identifying a binding protein of a eukaryotic protein derived from a human embryonic stem cell.
Background
BAF45D, also known as DPF2, double PHD fingers 2 or di-zinc finger proteins 2, belongs to the new Kr ü ppel-type zinc finger protein d4 protein family. BAF45D gene was localized to mouse Chr 19, which is known to be expressed in mouse embryonic brain tissue; in addition, BAF45D is localized to human 11q13.1, and is located in the long arm 13 region of human chromosome 11 as one of the important gene expression regulatory factors in cells, and such a region is closely related to the occurrence of various tumors.
The human embryonic stem cell has the characteristics of self-renewal and totipotency, can be differentiated into any cell of a human body, and has very important application value in regenerative medicine. The discovery of new interactions of human embryonic stem cell-related proteins can provide important clues for functional studies. BAF45D is used as a protein which is expressed abundantly in human embryonic stem cells, the biological function of the BAF45D is possibly related to the induced differentiation of the human embryonic stem cells, and the stable screening and identification of unknown protein interacting with known protein have important significance for the research on molecular mechanism in the biological and medical fields. However, at present, a method for expressing and purifying human BAF45D fusion protein with high efficiency and high activity is lacked in the world and the country, and particularly, a research for searching and identifying the binding protein derived from the human embryonic stem cells by using the BAF45D purified protein or similar research results are lacked.
Disclosure of Invention
In view of the above, the present invention needs to provide a method for expressing and purifying human BAF45D fusion protein, which improves the efficiency of expressing fusion protein by bacteria, maintains higher protein activity, and provides a basis for searching and identifying binding protein of eukaryotic protein derived from human embryonic stem cells by using the human BAF45D fusion protein.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for expressing and purifying a human BAF45D fusion protein, which comprises the following steps:
constructing a prokaryotic expression vector of human BAF45D to obtain a recombinant plasmid pGEX-5X-1-BAF 45D;
prokaryotic expression of GST-BAF45D fusion protein: transferring the recombinant plasmid pGEX-5X-1-BAF45D into Rosetta (DE3) escherichia coli, and carrying out IPTG induced expression to obtain a thallus precipitate; breaking bacteria and collecting supernatant;
purification of GST-BAF45D fusion protein: the supernatant obtained by prokaryotic expression was purified by centrifugation with GST Resin.
In a further embodiment, the IPTG inducible expression parameters are: the final concentration is 0.3mM IPTG, the temperature is 30 ℃, and the shaking culture is carried out for inducing expression for 5h at 250 rpm.
In a further scheme, the IPTG induction expression steps specifically comprise:
selecting a plasmid-transferred escherichia coli strain GST-BAF45D-Rosseta, inoculating the strain in an LB liquid culture medium, and shaking overnight to obtain an overnight culture;
inoculating the overnight culture into LB liquid medium, and performing shake culture to OD600After the culture medium was incubated at 30 ℃ and 250rpm for 5 hours with 100mM IPTG to a final concentration of 0.3mM after changing to 0.4 to 0.8, the culture was centrifuged, and the supernatant was discarded to obtain a pellet.
In a further aspect, the step of disrupting the bacteria specifically comprises: precooling the bacterial lysate, suspending the thallus precipitate according to the ratio of 100mL bacterial liquid precipitate to 6-8mL bacterial lysate, collecting, carrying out ice bath ultrasonic disruption on bacteria, and collecting supernatant.
In a further embodiment, the bacterial lysate consists of 50mM Tris, 1mM EDTA, 1% triton x100, 5mM DTT, 2mM PMSF, pH 8.
Further, the parameters of the ultrasound are specifically as follows: 40-60% of power 425W, ultrasonic treatment for 4s, stopping for 8s, and working for 4-6 min.
In a further scheme, the GST Resin centrifugal purification step specifically comprises the following steps:
30-50 μ L GST Resin and 1500-2000 μ L GST-BAF45D-Rosseta bacterium lysed GST-BAF45D total protein were incubated at 4 ℃ for 3 hours, and the precipitate was collected; washing the impurity protein with 1000-2000 μ L of bacterial lysate, shaking for 5min on ice, and collecting the precipitate; and repeatedly washing the impurity protein for 3 times, and collecting the precipitate to obtain the purified GST-BAF45D fusion protein.
The invention further provides a method for searching and identifying the binding protein of eukaryotic protein derived from human embryonic stem cells, which is characterized by comprising the following steps:
providing a purified GST-BAF45D fusion protein obtained using the method of human BAF45D fusion protein expression and purification as claimed in any one of claims 1-7;
and (3) combining the GST-BAF45D purified protein with eukaryotic total protein from human embryonic stem cells by using a GST-pull down method, and searching and identifying the binding protein from the human embryonic stem cells.
In a further embodiment, the eukaryotic total protein derived from human embryonic stem cells is selected from H9 cell protein, NCCIT cell protein or purified His-SMAD3 protein.
In a further scheme, the GST-pull down method specifically comprises the following steps:
carrying out rotary incubation on the GST-BAF45D fusion protein and 1000 mu L of eukaryotic total protein derived from the human embryonic stem cells at 4 ℃ overnight, and collecting precipitates; slowly adding 1-2mL NP-40 lysate along the wall to wash the beads, collecting the precipitate, and repeatedly washing the beads for 5-6 times; adding SDS loading buffer solution into the collected precipitate, and boiling for 4-6min at 100 ℃ together with Input; IB validation after SDS-PAGE treatment.
Compared with the prior art, the invention has the following beneficial effects:
the expression and purification method can realize high-efficiency expression of thalli, extracts recombinant protein with thalli activity, has high activity, and can be used for searching and identifying binding protein of eukaryotic protein from human embryonic stem cells.
The obtained recombinant protein adopts GST-pull down experiment, thereby searching and identifying the binding protein of eukaryotic protein from human embryonic stem cells, reducing the difficulty of the GST-pull down experiment in the field of molecular biology, being capable of stably screening and identifying unknown protein interacting with known protein, and having important significance for the research on the aspect of molecular mechanism in the fields of biology and medicine.
Drawings
FIG. 1 is a schematic diagram of electrophoresis of induced expression and purification of plasmid-transferred Escherichia coli strains GST-BL21 and GST-BAF 45D-Rosseta;
FIG. 2 is a schematic diagram of the electrophoresis of fusion proteins GST, GST-BAF45D binding to H9 cell proteins, respectively;
FIG. 3 is a schematic diagram of the electrophoresis of the binding of the fusion proteins GST and GST-BAF45D to NCCIT cell proteins, respectively;
FIG. 4 is an electrophoretic representation of the binding of the fusion proteins GST, GST-BAF45D, respectively, to the purified His-SMAD3 protein.
Detailed Description
In order that the invention may be more fully understood, reference will now be made to the specific embodiments illustrated. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The invention provides a method for expressing and purifying a human BAF45D fusion protein in a first aspect, which comprises the following steps:
firstly, constructing a prokaryotic expression vector of human BAF45D to obtain a recombinant plasmid pGEX-5X-1-BAF45D, wherein the construction of the recombinant plasmid adopts the prior art in the field, and specifically, the protein sequence of wild type BAF45D is shown as SEQ ID NO. 1.
BAF45D cDNA was amplified from pCMV-SPORT6-DPF2(Open Biosystems, Huntsville, AL) vector using an upstream primer 5'-CGGAATTCCCATGGAAGATGGCGGCTGTGGTGGAG-3' (SEQ ID NO.2) and a downstream primer 5'-ACGCGTCGACCAAGAGGAGTTCTGGTTCTGGTAGA-3' (SEQ ID NO.3), and cloned into pGEX-5X-1 vector (GST sequence contained in the entire sequence) through EcoR I/Sal I cleavage site to obtain recombinant plasmid pGEX-5X-1-BAF 45D.
Prokaryotic expression of the GST-BAF45D fusion protein was then performed: transferring the recombinant plasmid pGEX-5X-1-BAF45D into Rosetta (DE3) escherichia coli, selecting a strain GST-BAF45D-Rosetta transferred into the plasmid escherichia coli, inoculating the strain GST-BAF45D-Rosetta into an LB liquid culture medium, and performing constant-temperature shaking culture overnight to obtain an overnight culture, wherein the LB liquid culture medium is a conventional bacterial basal culture medium in the field, and specifically adopts 10g of Tryptone, 5g of yest extract, 10g of NaCl, 10M of NaOH to adjust the pH to be 7, and ddH2O is constant volume to 1L, 1mL of Amp is added for preparation after autoclaving, and in one or more embodiments of the invention, the shake culture parameters are 37 ℃ and 270 rpm;
the overnight cultures obtained were grown according to 1: inoculating 100 vol% into LB liquid culture medium, culturing at 37 deg.C and 270rpm with shaking to OD600After the concentration is 0.4-0.8, preferably 0.6, adding 100mM IPTG to the final concentration of 0.3mM, inducing expression for 5 hours at 30 ℃ and 250rpm by shaking, and centrifuging to obtain thalli sediment;
precooling a bacterial lysate (50mM Tris, 1mM EDTA, 1% TritonX100, 5mM DTT, 2mM PMSF, adjusting the pH value to 8 by concentrated HCl), suspending the obtained thallus precipitate according to the proportion of 100mL to 6-8mL of the bacterial lysate, collecting and carrying out ice bath ultrasound, wherein the ultrasound process is 40-60% of power (preferably 50% of power 425W), carrying out ultrasound for 4s, stopping for 8s, working for 4-6min, and centrifuging to obtain the precipitate, namely GST-BAF45D fusion total protein.
Finally, purification of the GST-BAF45D fusion protein was performed: and (3) centrifuging and purifying the supernatant obtained by prokaryotic expression by GST Resin, specifically, taking 30-50 mu L of GST Resin into a 2mL EP tube, adding 2000 mu L of GST-BAF45D fusion total protein 1500-protein, incubating for 3 hours at 4 ℃, centrifuging for 5min at 4 ℃ and 12000rpm, and discarding the supernatant. The contaminating proteins were washed with 1000-. mu.L of bacterial lysate (50mM Tris, 1mM EDTA, 1% TritonX100, 5mM DTT, 2mM PMSF, pH 8 in concentrated HCl), shaken on ice for 5min, centrifuged at 12000rpm for 1min at 4 ℃ and the supernatant discarded. Repeating the steps for 3 times, discarding the supernatant and reserving the precipitate to obtain a large amount of purified GST-BAF45D fusion protein, wherein the protein sequence of the fusion protein is shown as SEQ ID NO. 4.
In the method, the final concentration of 0.3mM IPTG is induced for 5 hours at 30 ℃, so that the generation of inclusion bodies can be reduced, the expression efficiency of thalli is improved, and the thalli can be efficiently induced to express the fusion protein; further, a centrifugal purification method is matched with a disc rotating mixer to incubate for 3 hours at the temperature of 4 ℃, so that the total bacterial lytic protein is combined with GST Resin more fully, the purification efficiency is ensured, and the purified fusion protein with high activity is obtained. Provides support for subsequent incubation in combination with human embryonic stem cells and screening of proteins interacting with BAF45D, uses human embryonic stem cells as target cells for screening of cells interacting with BAF45D, uses human cells as tools, and has good practical value.
The second aspect of the invention provides a method for searching and identifying the binding protein of eukaryotic protein derived from human embryonic stem cells, wherein the GST-BAF45D fusion protein obtained by the first aspect of the invention adopts a GST-pull down method to search and identify the binding protein of eukaryotic protein derived from human embryonic stem cells, and the method specifically comprises the following steps:
providing a purified GST-BAF45D fusion protein obtained using a method of expression and purification of a human BAF45D fusion protein according to the first aspect of the invention;
combining the GST-BAF45D fusion protein with eukaryotic total protein from human embryonic stem cells by adopting a GST-pull down method, searching and identifying the binding protein of the eukaryotic protein from the human embryonic stem cells, specifically, rotationally incubating the GST-BAF45D fusion protein and 1000 mu L of eukaryotic total protein from human at 4 ℃ overnight, and collecting precipitates; slowly adding 1-2mL NP-40 lysate along the wall to wash the beads, collecting the precipitate, and repeatedly washing the beads for 5-6 times; adding SDS loading buffer solution into the collected precipitate, and boiling for 4-6min at 100 ℃ together with Input; IB validation after SDS-PAGE treatment.
In a further embodiment, the eukaryotic total protein derived from human embryonic stem cells is selected from H9 cell protein, NCCIT cell protein or purified His-SMAD3 protein.
As the GST-Pull down method adopts GST-BAF45D fusion protein with high efficiency expression and higher protein activity, the GST-Pull down method can simply screen the protein from the human embryonic stem cell combined with the target protein by combining and incubating the fusion protein with the human embryonic stem cell cracked total protein, and can reduce the experimental difficulty of the GST-Pull down method.
The technical solution of the present invention will be more clearly and completely described below with reference to specific embodiments.
Example 1 inducible expression of GST fusion protein of BL21 and Rosseta strains
pGEX-5X-1-BAF45D recombinant positive plasmid is transformed into E.coli Rosetta (DE3) strain, and pGEX-5X-1 plasmid is transformed into E.coli BL21 strain (as negative control), so as to obtain transformed plasmid Escherichia coli strains GST-BL21 and GST-BAF45D-Rosseta (which are all prepared and stored in the laboratory);
respectively picking escherichia coli strains (GST-BL21, GST-BAF45D-Rosseta) to inoculate in 8mL LB liquid culture medium, shaking overnight in a constant temperature shaking incubator at 37 ℃ and 270rpm to obtain overnight culture;
the overnight cultures were incubated at 1: inoculating 100 vol% into LB liquid culture medium, culturing at 37 deg.C and 270rpm with shaking for 2-4 hr to OD600About 0.6 (detection by absorption of light at 600nm in a 96-well plate of a microplate reader); 100mM IPTG were added to a final concentration of 0.3mM each; performing shake culture at 30 deg.C and 250rpm for 5 hr to obtain centrifugal culture and control before induction, centrifuging at 12000r at room temperature for 10min, and discarding supernatant; suspending thallus precipitates according to the proportion of the precipitation of 100mL of bacteria liquid to 6mL of lysate (the composition is 50mM Tris, 1mM EDTA, 1% TritonX100, 5mM DTT, 2mM PMSF, and the pH value is 8), collecting samples before and after thallus induction into 50mL centrifuge tubes respectively, and carrying out ice bath ultrasonic crushing, wherein the ultrasonic power is 50% (425W), the ultrasonic is started for 4s and stopped for 8s, and the work is 5 min; centrifuging at 4 deg.C and 12000r for 10min, discarding the precipitate, storing the precipitate at-80 deg.C, and identifying by SDS-PAGE and Coomassie blue staining (see the result in FIG. 1).
FIG. 1 shows that after the plasmid-transferred Escherichia coli GST-BL21 strain induces expression, a large amount of protein is expressed at the molecular weight of 26kDa in total protein, and after the plasmid-transferred Escherichia coli GST-BAF45D-Rosseta strain induces expression, a large amount of protein is expressed at the molecular weight of 70kDa in total protein.
Example 2 purification of GST protein and GST-BAF45D fusion protein
GST Resin (Transgen, Code # DP201-01) reagent bottles were removed and gently shaken to form a complete suspension. Cutting off a small amount of the front end of the liquid transfer suction head, enlarging an opening, and taking 40 mu L of each group in an EP tube; washing the magnetic beads of Glutathione-Sepharose 4B with 1 XPBS (pH7.4) at 1000. mu.L each, mixing, centrifuging at 12000rpm at 4 ℃ for 3min, discarding the supernatant, and washing three times;
taking the supernatant of the thalli in the example 1, namely, 1000 microlitres of GST total protein cracked by GST-BL21, GST-BAF45D-Rosseta and GST-BAF45D total protein respectively, adding the supernatant into a washed GST Resin reagent bottle, incubating the mixture on a 4 ℃ disc rotating mixer for 3 hours, then centrifuging the mixture at 4 ℃ and 12000rpm for 5min, and discarding the supernatant; washing the impurity proteins with 1000. mu.L of sonication (50mM Tris, 1mM EDTA, 1% Triton X100, 5mM DTT, 2mM PMSF, concentrated HCl pH 8), shaking on ice for 5min, centrifuging at 12000rpm for 1min at 4 ℃ and discarding the supernatant; repeatedly washing impurity protein for 3 times, adding 100 μ L of eluent into the precipitate, sealing with sealing film, and rotary incubating at 4 deg.C for 1 hr. Adding 6X loading buffer solution, water bathing at 100 deg.C for 5min, and SDS-PAGE identifying purification effect, the results are shown in FIG. 1.
FIG. 1 shows the in vitro purification of inducible expression of GST-BAF45D protein using GST Resin EP, resulting in a fusion protein of approximately 72kDa in size and a purified GST protein of approximately 26 kDa.
Example 3GS-pull down H9 cell and NCCIT cell protein methods
Preparing a Matrigel (Corning, REF356234) coated six-well cell culture plate, sterilizing, irradiating an ice box with ultraviolet light, 160 microliters of Matrigel gel (thawing on ice), a six-well plate, a 15mL centrifuge tube, a 10mL pipette, a pipettor, and a waste liquid tank for more than thirty minutes;
turning on a super-clean bench fluorescent lamp and a fan, and igniting an alcohol lamp;
DMEM/F-12, 1mL and 200 mu L sterile suction heads are sterilized by alcohol spraying and placed in a super clean bench, 12mL of DMEM/F-12 is sucked in a 15mL centrifuge tube by a pipette after burning the bottle mouth by an alcohol lamp, and the centrifuge tube is placed on ice. 160 microliter Matrigel gel was pipetted and transferred to 12ml DMEM/F-12 and mixed quickly. Transfer to six well plates, 1ml per well, gently shake to distribute the liquid evenly. Standing at room temperature for 1h or sealing with sealing film and standing at 4 deg.C.
H9 cell resuscitation passage and induction
Disinfection of Pre-warmed DMEM/F-12(Hyclone, SH30023.01) (Medium), mTeSRTM1(Stem Cell Technologies, #85850) (human embryonic Stem Cell culture medium); rapidly and gently and continuously shaking the freezing tube in a water bath kettle at 37 ℃ to unfreeze H9 cells until only one small frozen mass is left, taking out the frozen mass from the freezing tube, and disinfecting with alcohol; transferring the liquid in the cryopreserved tube to a 15mL centrifuge tube by using a 2mL pipette; dropwise adding 7mL of DMEM/F-12 into the centrifuge tube, and centrifuging the cells at room temperature and 500rpm for 5 min; the supernatant was discarded and the cells were resuspended in 2mL of mTeSR by gentle pipettingTM1, blowing and beating once to keep the cell aggregation state; gently tilt the plate, aspirate excess Matrigel solution with pipette, and mix mTeSR with cell aggregatesTM1 transfer of culture broth to Matrigel gel coated plates (1 ml/well); 1ml of mTeSR was added per wellTM1 culture solution, adding 5% CO at 37 deg.C2In the incubator, the culture plate is quickly shaken front, back, left and right to disperse cell clusters uniformly. Changing culture solution mTeSR every dayTM1(2 ml/well); when passaging (1: 3) was performed when 70% of the cells had grown, H9 cells were passaged through mTeSR at a final concentration of 10uM Y27623TM1 culture broth was incubated at 37 ℃ for 1h in an incubator. The cells were digested with Accutase (1 ml/well) at 37 ℃ for 6-8 min, the digestion was stopped with DMEM/F12, and the cells were pipetted out and transferred to a 15ml centrifuge tube. Centrifuging at room temperature at 500rpm for 5min, discarding the supernatant, resuspending the cells in mTeSR1 culture medium containing 10uM Y27623, air-blowing to disperse the cells into single cells, transferring the cells to a Matrigel-coated six-well culture plate (2 ml/well), and shaking the culture plate back and forth and left and right to disperse the cell clusters uniformly. The next day is changed into mTeSRTM1 and a culture solution (2 ml/well) of mTeSRtm1 containing 5. mu.M tretinoin (RA). The culture was carried out for five days, and the culture medium was changed every day. Cells were harvested on the fifth day.
Resuscitation passage and induction of NCCIT (NCCIT cell activating and inducing)
Sterilizing and preheating RPMI-1640(Hyclone, SH30809.01) complete culture solution and PBS, ultraviolet irradiating 150mm cell culture dish, 15ml centrifuge tube, sterile liquid-transferring suction head, liquid-transferring machine and waste liquid cylinder thirtyFor more than minutes. And turning on the super-clean bench fluorescent lamp and the fan, and igniting the alcohol lamp. Frozen NCCIT cells are taken out from liquid nitrogen, quickly placed in a water bath kettle at 37 ℃ and shaken until only one small frozen mass is left, the cells are taken out of a frozen tube, sterilized by alcohol, taken into an ultra-clean bench, transferred into a 15ml centrifuge tube, added with 5ml PBS and centrifuged at room temperature and 1000rpm for 5min, and the supernatant is discarded. The cells were resuspended in RPMI-1640 medium, transferred to a 150mm cell culture dish, and the RPMI-1640 medium was changed every other day. And (3) when the growth density reaches 75%, carrying out warm digestion for 2min by using a 0.5% pancreatin digestive juice chamber, adding PBS to stop digestion, blowing cells by using a pipette to shed the cells, transferring the cells into a 15ml centrifuge tube, and centrifuging the cells for 5min at the room temperature of 1000 rpm. Resuspend cells in RPMI-1640 Medium at 1.5X106Inoculating in a dish (100mm), and adding Retinoic Acid (RA) after 48h of growth. Induction: retinoic acid 10 μmol/L (1:1000) was dissolved in RPMI-1640 medium and the medium was changed by 10 mL/dish daily. Cells were harvested at 8d induction.
H9/NCCIT cell protein extraction
Taking out the cells with the cell density of more than 90 percent from the culture box; precooling 1mL of sterile suction head and PBS, sucking the culture solution on ice and discarding, and then adding the precooled PBS to wash the culture plate for 1-2 times. Adding 1 mL/hole PBS, scraping the bottom cell of the dish by using a cell scraper, collecting the bottom cell into a 1.5mLEP tube, centrifuging at 12000rpm at 4 ℃ for 2 min; discarding the supernatant, and keeping the precipitate; adding 200 μ L of NP-40 lysate to each tube, performing ice lysis for 15-30min, blowing or flicking the EP tube with a gun head at 4 deg.C and 12000rpm, centrifuging for 20min, collecting supernatant, transferring to new EP tube, and determining protein concentration by BCA method.
GST-pull down
Purified GST fusion proteins (GST, GST-BAF45D) obtained in example 2 were added to 800. mu. L H9 cell protein and NCCIT cell protein, respectively, and incubated overnight at 4 ℃ for rotation; centrifuging at 4 ℃ and 3000rpm for 5min, discarding the supernatant, and slowly adding 1mL NP-40 lysate along the wall to clean the beads; centrifuging at 4 deg.C and 3000rpm for 5min, discarding the supernatant, and repeatedly cleaning the beads for 5-6 times; the supernatant was discarded, and SD loading buffer was added along with Input, and boiled at 100 ℃ for 5 min.
Western blot was performed after SDS-PAGE. And (3) loading sequence: input, GST-beads pull-down, and GST-BAF45D beads pull-down. The results are shown in FIGS. 2 and 3.
In FIG. 2A, GST and GST-BAF45D fusion proteins are shown to be incubated and precipitated with H9 cell lysates before (0d) and after (5d) induction, respectively. After repeated washing, SDS-PAGE and Western blot analysis are carried out to identify, and antibody screening is used to identify the protein which interacts with GST-BAF45D to phosphorylate SMAD3(pSMAD 3); it was shown that H9 did not induce interaction between GST-BAF45D and Psmad3 in normal cultured cells, and that H9 cells induced by Retinoic Acid (RA) increased Psmad3 and also interacted with GST-BAF 45D; OCT4 is a characteristic protein of human embryonic stem cells, and the expression is reduced almost completely after differentiation is induced. FIG. 2B shows that the PVDF membrane obtained in experiment A was stained with ponceau, showing GST and GST-BAF45D purified proteins.
FIG. 3A shows GST and GST-BAF45D purified proteins were incubated and precipitated with NCCIT cell lysates before (0d) and after (8d) induction, respectively. After repeated washing, SDS-PAGE, membrane transfer and Western blot analysis were performed to identify proteins that interact with GST-BAF45D, phosphorylate SMAD3(pSMAD3) by antibody screening. OCT4 is a characteristic protein of human embryonic stem cells, and the expression is reduced almost completely after differentiation is induced. FIG. 3B shows that the PVDF membrane obtained in experiment A was stained with ponceau, showing GST and GST-BAF45D purified proteins. NCCIT cell results were consistent with those of H9 cells.
Example 4 GS-pull down method for purifying recombinant protein His-SMAD3
The purified fusion proteins of example 2 (GST, GST-BAF45D) were added to 10. mu.g of purified recombinant human SMAD3 protein (ab89353), supplemented with PBS to 2mL, and incubated overnight at 4 ℃ with rotation; centrifuging at 4 deg.C and 3000rpm for 5min, and removing supernatant; slowly adding 1mL of RIPA cleaning beads along the wall; centrifuging at 4 deg.C and 3000rpm for 5min, discarding the supernatant, and repeatedly cleaning the beads for 5-6 times; the supernatant was discarded, SDS loading buffer was added, and the mixture was boiled at 100 ℃ for 10min together with Input (0.25. mu.g Smad 3). Western blot analysis, sample loading sequence: input, GST-beads pull-down, GST-BAF45D beads pull-down.
As a result, as shown in FIG. 4, it can be seen that GST-BAF45D has an in vitro binding phenomenon with His-SMAD3, indicating that GST-BAF45D has a direct interaction with His-SMAD 3.
The above examples show that the expression and purification method of the human BAF45D fusion protein can obtain the GST-BAF45D fusion protein with high efficiency and high activity, the GST-BAF45D fusion protein can be used for searching and identifying the binding protein of the eukaryotic protein from the human embryonic stem cell, and the difficulty of the GST-pull down method is reduced.
The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
SEQUENCE LISTING
<110> university of medical in Anhui
<120> expression and purification method of human BAF45D fusion protein and application
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<170> PatentIn version 3.3
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Thr Gly Ala Thr Gly Ala Cys Gly Ala Cys Ala Ala Ala Thr Gly
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420 425 430
Gly Thr Thr Gly Cys Gly Cys Ala Cys Thr Gly Ala Cys Cys Cys Cys
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Gly Gly Cys Gly Ala Gly Thr Thr Thr Cys Cys Thr Gly Thr Gly Ala
500 505 510
Cys Cys Ala Ala Cys Ala Gly Thr Cys Gly Ala Gly Cys Gly Cys Gly
515 520 525
Ala Ala Ala Gly Cys Gly Gly Ala Thr Cys Cys Thr Ala Gly Ala Ala
530 535 540
Cys Cys Ala Gly Ala Thr Gly Ala Cys Thr Thr Cys Cys Thr Gly Gly
545 550 555 560
Ala Thr Gly Ala Cys Cys Thr Cys Gly Ala Thr Gly Ala Thr Gly Ala
565 570 575
Ala Gly Ala Cys Thr Ala Thr Gly Ala Ala Gly Ala Ala Gly Ala Thr
580 585 590
Ala Cys Thr Cys Cys Cys Ala Ala Gly Cys Gly Thr Cys Gly Gly Gly
595 600 605
Gly Ala Ala Ala Gly Gly Gly Gly Ala Ala Ala Thr Cys Cys Ala Ala
610 615 620
Gly Gly Gly Thr Ala Ala Gly Gly Gly Thr Gly Thr Gly Gly Gly Cys
625 630 635 640
Ala Gly Thr Gly Cys Cys Cys Gly Thr Ala Ala Gly Ala Ala Gly Cys
645 650 655
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660 665 670
Gly Gly Ala Gly Gly Ala Cys Cys Gly Gly Gly Ala Thr Ala Ala Gly
675 680 685
Cys Cys Cys Thr Ala Thr Gly Cys Cys Thr Gly Thr Gly Ala Cys Ala
690 695 700
Thr Thr Thr Gly Thr Gly Gly Ala Ala Ala Ala Cys Gly Thr Thr Ala
705 710 715 720
Cys Ala Ala Gly Ala Ala Cys Cys Gly Ala Cys Cys Ala Gly Gly Cys
725 730 735
Cys Thr Cys Ala Gly Thr Thr Ala Cys Cys Ala Cys Thr Ala Thr Gly
740 745 750
Cys Cys Cys Ala Cys Thr Cys Cys Cys Ala Cys Thr Thr Gly Gly Cys
755 760 765
Thr Gly Ala Gly Gly Ala Gly Gly Ala Gly Gly Gly Cys Gly Ala Gly
770 775 780
Gly Ala Cys Ala Ala Gly Gly Ala Ala Gly Ala Cys Thr Cys Thr Cys
785 790 795 800
Ala Ala Cys Cys Ala Cys Cys Cys Ala Cys Thr Cys Cys Thr Gly Thr
805 810 815
Thr Thr Cys Cys Cys Ala Gly Ala Gly Gly Thr Cys Thr Gly Ala Gly
820 825 830
Gly Ala Gly Cys Ala Gly Ala Ala Ala Thr Cys Cys Ala Ala Ala Ala
835 840 845
Ala Gly Gly Gly Thr Cys Cys Thr Gly Ala Thr Gly Gly Ala Thr Thr
850 855 860
Gly Gly Cys Cys Thr Thr Gly Cys Cys Cys Ala Ala Cys Ala Ala Cys
865 870 875 880
Thr Ala Cys Thr Gly Thr Gly Ala Cys Thr Thr Cys Thr Gly Cys Cys
885 890 895
Thr Gly Gly Gly Gly Gly Ala Cys Thr Cys Ala Ala Ala Gly Ala Thr
900 905 910
Thr Ala Ala Cys Ala Ala Gly Ala Ala Gly Ala Cys Gly Gly Gly Ala
915 920 925
Cys Ala Ala Cys Cys Cys Gly Ala Gly Gly Ala Gly Cys Thr Gly Gly
930 935 940
Thr Gly Thr Cys Cys Thr Gly Thr Thr Cys Thr Gly Ala Cys Thr Gly
945 950 955 960
Thr Gly Gly Cys Cys Gly Cys Thr Cys Ala Gly Gly Gly Cys Ala Thr
965 970 975
Cys Cys Ala Thr Cys Thr Thr Gly Cys Cys Thr Cys Cys Ala Ala Thr
980 985 990
Thr Thr Ala Cys Cys Cys Cys Cys Gly Thr Gly Ala Thr Gly Ala Thr
995 1000 1005
Gly Gly Cys Gly Gly Cys Ala Gly Thr Gly Ala Ala Gly Ala Cys
1010 1015 1020
Ala Thr Ala Cys Cys Gly Cys Thr Gly Gly Cys Ala Gly Thr Gly
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Cys Ala Thr Cys Gly Ala Gly Thr Gly Cys Ala Ala Ala Thr Gly
1040 1045 1050
Thr Thr Gly Cys Ala Ala Thr Ala Thr Cys Thr Gly Cys Gly Gly
1055 1060 1065
Cys Ala Cys Cys Thr Cys Cys Gly Ala Gly Ala Ala Thr Gly Ala
1070 1075 1080
Cys Gly Ala Cys Cys Ala Gly Thr Thr Gly Cys Thr Cys Thr Thr
1085 1090 1095
Cys Thr Gly Thr Gly Ala Thr Gly Ala Cys Thr Gly Cys Gly Ala
1100 1105 1110
Thr Cys Gly Thr Gly Gly Cys Thr Ala Cys Cys Ala Cys Ala Thr
1115 1120 1125
Gly Thr Ala Cys Thr Gly Thr Cys Thr Cys Ala Cys Cys Cys Cys
1130 1135 1140
Gly Thr Cys Cys Ala Thr Gly Thr Cys Thr Gly Ala Gly Cys Cys
1145 1150 1155
Cys Cys Cys Thr Gly Ala Ala Gly Gly Ala Ala Gly Thr Thr Gly
1160 1165 1170
Gly Ala Gly Cys Thr Gly Cys Cys Ala Cys Cys Thr Gly Thr Gly
1175 1180 1185
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1190 1195 1200
Ala Gly Ala Gly Ala Ala Ala Gly Cys Thr Thr Cys Cys Ala Thr
1205 1210 1215
Cys Thr Ala Cys Cys Ala Gly Ala Ala Cys Cys Ala Gly Ala Ala
1220 1225 1230
Cys Thr Cys Cys Thr Cys Thr Ala Cys Cys Gly Gly Thr Ala Thr
1235 1240 1245
Gly Gly Ala Cys Thr Ala Cys Ala Ala Gly Gly Ala Thr Gly Ala
1250 1255 1260
Cys Gly Ala Thr Gly Ala Cys Ala Ala Gly Gly Ala Thr Thr Ala
1265 1270 1275
Cys Ala Ala Ala Gly Ala Cys Gly Ala Cys Gly Ala Thr Gly Ala
1280 1285 1290
Thr Ala Ala Gly Gly Ala Cys Thr Ala Thr Ala Ala Gly Gly Ala
1295 1300 1305
Thr Gly Ala Thr Gly Ala Cys Gly Ala Cys Ala Ala Ala Thr Gly
1310 1315 1320
Ala Gly Cys Thr Ala Gly Cys Cys Thr Gly Thr Gly Gly
1325 1330 1335
Claims (10)
1. A method for expressing and purifying a human BAF45D fusion protein, which is characterized by comprising the following steps:
constructing a prokaryotic expression vector of human BAF45D to obtain a recombinant plasmid pGEX-5X-1-BAF 45D;
prokaryotic expression of GST-BAF45D fusion protein: transferring the recombinant plasmid pGEX-5X-1-BAF45D into Rosetta (DE3) escherichia coli, and carrying out IPTG induced expression to obtain a thallus precipitate; breaking bacteria and collecting supernatant;
purification of GST-BAF45D fusion protein: the supernatant obtained by prokaryotic expression was purified by centrifugation with GST Resin.
2. The method of claim 1, wherein the parameters for IPTG-induced expression are: the final concentration is 0.3mM IPTG, the temperature is 30 ℃, and the shaking culture is carried out for inducing expression for 5h at 250 rpm.
3. The method for expression and purification of human BAF45D fusion protein according to claim 1, wherein the step of IPTG-induced expression is in particular:
selecting a plasmid-transferred escherichia coli strain GST-BAF45D-Rosseta, inoculating the strain in an LB liquid culture medium, and shaking overnight to obtain an overnight culture;
inoculating the overnight culture into LB liquid medium, and performing shake culture to OD600After the culture medium was incubated at 30 ℃ and 250rpm for 5 hours with 100mM IPTG to a final concentration of 0.3mM after changing to 0.4 to 0.8, the culture was centrifuged, and the supernatant was discarded to obtain a pellet.
4. The method for expression and purification of human BAF45D fusion protein according to claim 1, wherein the step of disrupting the bacteria is specifically: precooling the bacterial lysate, suspending the thallus precipitate according to the ratio of 100mL bacterial liquid precipitate to 6-8mL bacterial lysate, collecting, carrying out ice bath ultrasonic disruption on bacteria, and collecting supernatant.
5. The method of claim 4, wherein the bacterial lysate consists of 50mM Tris, 1mM EDTA, 1% TritonX100, 5mM DTT, 2mM PMSF, pH 8.
6. The method for expression and purification of human BAF45D fusion protein according to claim 4, wherein the parameters of the ultrasound are specifically: 40-60% of power 425W, ultrasonic treatment for 4s, stopping for 8s, and working for 4-6 min.
7. The method for expression and purification of human BAF45D fusion protein according to claim 1, wherein the step of GST Resin centrifugation purification comprises:
30-50 μ L GST Resin and 1500-2000 μ L GST-BAF45D-Rosseta bacterium lysed GST-BAF45D total protein were incubated at 4 ℃ for 3 hours, and the precipitate was collected; washing the impurity protein with 1000-2000 μ L of bacterial lysate, shaking for 5min on ice, and collecting the precipitate; and repeatedly washing the impurity protein for 3 times, and collecting the precipitate to obtain the purified GST-BAF45D fusion protein.
8. A method for finding and identifying binding proteins to eukaryotic proteins derived from human embryonic stem cells, comprising the steps of:
providing a purified GST-BAF45D fusion protein obtained using the method of human BAF45D fusion protein expression and purification as claimed in any one of claims 1-7;
and (3) combining the GST-BAF45D purified protein with eukaryotic total protein from human embryonic stem cells by using a GST-pull down method, and searching and identifying the binding protein from the human embryonic stem cells.
9. The method for finding and identifying binding proteins to eukaryotic proteins derived from human embryonic stem cells according to claim 8, wherein said eukaryotic proteins derived from human embryonic stem cells are selected from the group consisting of H9 cellular protein, NCCIT cellular protein and purified His-SMAD3 protein.
10. The method for searching and identifying binding proteins of eukaryotic proteins derived from human embryonic stem cells according to claim 8, wherein the GST-pull down method comprises the following specific steps:
carrying out rotary incubation on the GST-BAF45D fusion protein and 1000 mu L of eukaryotic total protein derived from the human embryonic stem cells at 4 ℃ overnight, and collecting precipitates; slowly adding 1-2mL NP-40 lysate along the wall to wash the beads, collecting the precipitate, and repeatedly washing the beads for 5-6 times; adding SDS loading buffer solution into the collected precipitate, and boiling for 4-6min at 100 ℃ together with Input; IB validation after SDS-PAGE treatment.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1490332A (en) * | 2003-07-17 | 2004-04-21 | 上海市内分泌代谢病研究所 | Recombinant human IA-2 antigen preparing method |
| CN101270359A (en) * | 2008-04-30 | 2008-09-24 | 江苏省原子医学研究所 | Method for preparing recombined human amyloid A beta 42 and application thereof |
| CN104404010A (en) * | 2014-10-27 | 2015-03-11 | 西北工业大学 | Methicillin-resistant staphylococcus aureus (MRSA) PBP2a transpeptidase region prokaryotic expression and purification method |
| CN105504067A (en) * | 2016-01-20 | 2016-04-20 | 中国医学科学院医学生物学研究所 | Expression and purification methods of fusion protein containing recombinant human fibroblast growth factor 21 |
| CN105755030A (en) * | 2016-03-08 | 2016-07-13 | 中国水产科学研究院南海水产研究所 | Preparation method of Pinctada fucata meat antioxidant peptides |
| CN107043764A (en) * | 2016-12-26 | 2017-08-15 | 扬州大学 | A kind of method that the yellow chicken interaction of genes albumen in Rugao is found based on GST Pull Down and analytical technique of mass spectrum |
| CN108822200A (en) * | 2018-06-19 | 2018-11-16 | 侯峰 | A kind of vaccine and preparation method thereof for preventing canine arch insect infection |
-
2021
- 2021-11-02 CN CN202111289460.7A patent/CN114150010A/en active Pending
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1490332A (en) * | 2003-07-17 | 2004-04-21 | 上海市内分泌代谢病研究所 | Recombinant human IA-2 antigen preparing method |
| CN101270359A (en) * | 2008-04-30 | 2008-09-24 | 江苏省原子医学研究所 | Method for preparing recombined human amyloid A beta 42 and application thereof |
| CN104404010A (en) * | 2014-10-27 | 2015-03-11 | 西北工业大学 | Methicillin-resistant staphylococcus aureus (MRSA) PBP2a transpeptidase region prokaryotic expression and purification method |
| CN105504067A (en) * | 2016-01-20 | 2016-04-20 | 中国医学科学院医学生物学研究所 | Expression and purification methods of fusion protein containing recombinant human fibroblast growth factor 21 |
| CN105755030A (en) * | 2016-03-08 | 2016-07-13 | 中国水产科学研究院南海水产研究所 | Preparation method of Pinctada fucata meat antioxidant peptides |
| CN107043764A (en) * | 2016-12-26 | 2017-08-15 | 扬州大学 | A kind of method that the yellow chicken interaction of genes albumen in Rugao is found based on GST Pull Down and analytical technique of mass spectrum |
| CN108822200A (en) * | 2018-06-19 | 2018-11-16 | 侯峰 | A kind of vaccine and preparation method thereof for preventing canine arch insect infection |
Non-Patent Citations (6)
| Title |
|---|
| GEORGIA VASILEIOU等: "Mutations in the BAF-Complex Subunit DPF2 Are Associated with Coffin-Siris Syndrome", AM J HUM GENET, vol. 102, no. 3, pages 468 - 479 * |
| REIKO MATSUYAMA等: "Double PHD Fingers Protein DPF2 Recognizes Acetylated Histones and Suppresses the Function of Estrogen-related Receptor α through Histone Deacetylase 1", J BIOL CHEM, vol. 285, no. 24, pages 18166 * |
| 安徽医科大学，基础医学院: "2021年新增科研课题", HTTPS://JC.AHMU.EDU.CN/INDEX/ARTICLEINFO/80/689.HTML, pages 1 - 7 * |
| 张文胜;CONSTANTINOS CHRONIS;陈曦;HEYAO ZHANG;RAPOLAS SPALINSKAS;MERCEDES PARDO;LIANGLIANG CHEN;吴光明;朱哲鑫;余勇;LU YU;JYOTI CHOUDHARY;JENNIFE: "BAF和PRC2复合物亚基Dpf2和Eed拮抗调控Tbx3控制胚胎干细胞分化", 科学新闻, no. 02, pages 1 - 2 * |
| 张涤娟: "DPF2下调OCT4蛋白水平促进胚胎干细胞向神经外胚层分化", 基础科学;医药卫生科技, no. 2, pages 1 - 61 * |
| 陈雪莹: "BAF45D协同SMAD3诱导PAX6的表达促成人胚胎干细胞分化为脊髓神经干细胞", 医药卫生科技, pages 1 - 71 * |
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