CN105085607A - Method for separating specific metal-binding proteins by using protein denaturation - Google Patents
Method for separating specific metal-binding proteins by using protein denaturation Download PDFInfo
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Abstract
The invention discloses a method for separating specific metal-binding proteins by using protein denaturation, and relates to the technical field of protein chemistry. The method comprises the following steps: firstly, taking a biological tissue or cell, after fully grinding with liquid nitrogen, adding protein extracting liquid, and after uniformly mixing, carrying out low-temperature centrifugation, so as to obtain a denatured protein solution; replacing Ni in Ni-NTA agarose with Cu, and putting into a chromatographic column; incubating the denatured protein solution with Cu-NTA agarose, so as to ensure that the denatured proteins are fully combined with the Cu-NTA agarose; then under the low-temperature condition, injecting eluant 1, so as to renature the proteins and elute non-specific copper-binding proteins; injecting eluant 2, so as to separate specific copper-binding proteins. According to the method, the copper-binding proteins under the denaturing condition are separated by using agarose-NTA, so that the obtaining efficiency of the copper-binding proteins is greatly improved; a technology suitable for separating the specific metal-binding proteins from the biological tissue or cell under the excess heavy metal treatment condition is established.
Description
Technical field
The present invention relates to protein chemistry art field, one utilizes protein denaturation to be separated the protein-bonded method of special metal specifically.
Background technology
Whole nation Survey of contaminating status publication display, the total exceeding standard rate of national soil pollution is 16.1%, and wherein heavy metal contamination accounts for more than 80%.Heavy metal great majority in soil easily at plant interior accumulation, and pass through the health of food chain harm humans.。On the other hand, because the halfcystine of protein, methionine(Met) and histidine residues have very high avidity to divalent-metal ion, therefore excessive in cell heavy metal may disturb the combination of essential metal element and albumen, causes protein denaturation or enzyme deactivation, disturbs normal metabolic process.
Immobilized metal affinity chromatography (IMAC) and mass spectrum coupling are the important technologies identifying metal binding protein.IMAC is some amino acid utilized in albumen, as halfcystine, Histidine and tryptophane etc., and can with the upper metallic cation adsorbed of solid column (as agarose-IDA) (as Zn
2+, Cu
2+, Ni
2+and Cd
2+deng) occur combine characteristic, be separated the protein with this metal-binding sites.But in Heavy-metal Polluted Environment, the metal-binding sites of protein in plant cells may be entered intracellular metal ion in advance and be occupied, make protein not have unnecessary metal-binding sites fixing metal on affinity column to be combined, thus cause metal binding protein can not be separated or be separated not exclusively.The more important thing is, the metal-binding sites of most albumen is present in intramolecule, therefore, under state of nature, we are difficult to be separated to special metal binding protein by IMAC method, but all do not take in this in the Research Literature of metal binding protein before.
Summary of the invention
The present invention seeks to the deficiency for solving the problems of the technologies described above, one is provided to utilize protein denaturation to be separated the protein-bonded method of special metal, utilize agarose-NTA(nitrilotriacetic acid) be separated copper-binding protein under Denaturing, substantially increase the acquisition efficiency of copper-binding protein, establish a set of isolation technique being suitable for metalloprotein group in excessive heavy metal treatment condition undertissue or cell.
One utilizes protein denaturation to be separated the protein-bonded method of special metal, comprises the following steps:
(1), get after biological tissue or cell liquid nitrogen fully grind, add protein extract, after mixing, low-temperature centrifugation, obtains the protein solution of sex change; The solid-to-liquid ratio of grinding artifact tissue or cell and protein extract is: 1:4; The composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, and mass percent is the TritonX-100 of 0.1-1%, 8mol/L urea, and surplus is water;
(2), by Ni in the Ni-NTA agarose of purchase
2+replace to Cu
2+load in chromatography column; The protein solution of sex change above-mentioned steps (1) obtained injects Cu-NTA agarose with 0.25ml/min flow velocity, hatches 15-40min, metaprotein is fully combined with it under 37 DEG C of conditions; Then under cold condition, slowly inject elutriant 1 with 0.25ml/min flow velocity, renaturation is carried out to albumen, by nonspecific copper-binding protein wash-out out; And then with 0.5ml/min flow velocity injection elutriant 2, specific copper-binding protein is separated;
Described elutriant 1 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 1,300mmol/L sodium-chlor, and 10-50mmol/L surplus is water; The pH value of elutriant 1 is 6.5;
Described elutriant 2 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 2,300mmol/L sodium-chlor, 40-100mmol/L imidazoles; The pH value of elutriant 2 is 5.8.
Low-temperature centrifugation described in step (1), its method is preferably, under 4 DEG C of low temperature, the centrifugal 30min of 15000g.
Described in step (2), Ni in Ni-NTA agarose (GEHealthcare) is replaced to Cu, its method is: loaded by the Ni-NTA agarose of 2ml in chromatography column, injects the solution removal Ni containing 0.05mol/LEDTA and 0.5mol/L sodium-chlor of 10 times of volumes
2+, then remove EDTA with the 0.5mol/L sodium chloride aqueous solution of 3 times of volumes, the 0.2mol/L copper sulfate solution ambient temperatare of 1 times of volume uses level pad after putting 30min subsequently, and wash-out 30min, until ultraviolet detection baseline stability; Flow velocity is 0.5ml/min.The composition of described level pad is: containing 50mmol/L sodium phosphate in often liter of level pad, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.1-1%, and surplus is water.
Described in step (2), protein solution in (1) and Cu-NTA agarose 37 DEG C are hatched 30min, metaprotein is fully combined with Cu-NTA agarose.
Step adds elutriant 1 described in (2), with the wash-out of 0.25ml/min flow velocity, until ultraviolet detection baseline reaches stable; Describedly add elutriant 2, with the wash-out of 0.5ml/min flow velocity, until ultraviolet detection baseline reaches stable, collect specificity copper-binding protein.
Described in step (1), the composition of protein extract is: the composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.2%, 8mol/L urea, surplus is water.
Beneficial effect is:
The protein denaturation that utilizes provided by the invention is separated the protein-bonded method of special metal, program is simple, substantially increase the acquisition efficiency of copper-binding protein, establish a set of isolation technique being suitable for metalloprotein group in biological tissue under excessive heavy metal treatment condition or cell.Denaturing in the present invention can make protein polypeptide chain be in extended configuration, be more conducive to the affine adsorption with fixing metal ions, and removing the protein renaturation process of Denaturing owing to carrying out in magnetic bead surfaces, avoid the aggregate and precipitate of metaprotein, and the title complex formed is more stable, ensure to be separated to copper-binding protein as much as possible.
Accompanying drawing explanation
Fig. 1 is the ultraviolet detection figure of non-specific copper-binding protein (I) and specificity copper-binding protein (II);
Fig. 2 is that the 2-DE of paddy rice copper-binding protein contrasts collection of illustrative plates;
Fig. 3 is the 2-DE collection of illustrative plates of paddy rice copper-binding protein;
Fig. 4 identifies the mass spectrum of copper-binding protein (GST);
Mark in figure: con is contrast; Cu is copper.
Embodiment
One utilizes protein denaturation to be separated the protein-bonded method of special metal, comprises the following steps:
(1), get after biological tissue or cell liquid nitrogen fully grind, add protein extract, after mixing, low-temperature centrifugation, obtains the protein solution of sex change; The solid-to-liquid ratio of grinding artifact tissue or cell and protein extract is: 1:4; The composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, and mass percent is the TritonX-100 of 0.1-1%, 8mol/L urea, and surplus is water;
(2) the middle Ni of Ni-NTA agarose (GEHealthcare), will bought
2+replace to Cu
2+load in chromatography column; The protein solution of sex change above-mentioned steps (1) obtained injects Cu-NTA agarose with 0.25ml/min flow velocity, hatches 15-40min, metaprotein is fully combined with it under 37 DEG C of conditions; Then under cold condition, slowly inject elutriant 1 with 0.25ml/min flow velocity, renaturation is carried out to albumen, by nonspecific copper-binding protein wash-out out; And then with 0.5ml/min flow velocity injection elutriant 2, specific copper-binding protein is separated;
Described elutriant 1 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 1,300mmol/L sodium-chlor, and 10-50mmol/L surplus is water; The pH value of elutriant 1 is 6.5;
Described elutriant 2 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 2,300mmol/L sodium-chlor, 40-100mmol/L imidazoles; The pH value of elutriant 2 is 5.8.
Low-temperature centrifugation described in step (1), its method is preferably, under 4 DEG C of low temperature, the centrifugal 30min of 15000g.
Described in step (2), Ni2+ in Ni-NTA agarose (buying in GEHealthcare company) is replaced to Cu2+, its method is: loaded by the Ni-NTA agarose of 2ml in chromatography column, injects the solution removal Ni containing 0.05mol/LEDTA and 0.5mol/L sodium-chlor of 10 times of volumes
2+, then remove EDTA with the 0.5mol/L sodium chloride aqueous solution of 3 times of volumes, the 0.2mol/L copper sulfate solution ambient temperatare of 1 times of volume uses level pad after putting 30min subsequently, and wash-out 30min, until ultraviolet detection baseline stability; Flow velocity is 0.5ml/min.The composition of described level pad is: containing 50mmol/L sodium phosphate in often liter of level pad, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.1-1%, and surplus is water.
Described in step (2), protein solution in (1) and Cu-NTA agarose 37 DEG C are hatched 30min, metaprotein is fully combined with Cu-NTA agarose.
Step adds elutriant 1 described in (2), with the wash-out of 0.25ml/min flow velocity, until ultraviolet detection baseline reaches stable; Describedly add elutriant 2, with the wash-out of 0.5ml/min flow velocity, until ultraviolet detection baseline reaches stable, collect specificity copper-binding protein.
Described in step (1), the composition of protein extract is: the composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.2%, 8mol/L urea, surplus is water.
Cu in the Cu-NTA agarose mentioned in the present invention
2+also Ni can be replaced with
2+zn
2+cd
2+deng other divalent-metal ions, thus obtain corresponding metal binding protein.Following examples, for explaining explanation the present invention, do not have any type of restriction to protection scope of the present invention.
embodiment 1
One, the extraction of vegetable-protein
With 100 μm of olL
-1the rice embryo of the sprouting of copper sulfate process, Copper treatment got fresh rice embryo after 5 days, fully ground with liquid nitrogen, then 30min is extracted at being placed on 4 DEG C after the protein extract adding 4 times of volumes fully mixes, at 4 DEG C, 15,000 × g centrifugal 30min, collect supernatant.Carry out quantitatively, using chicken egg white as standard protein by the Bradford method (Ramagli, 1999) of amendment to metaprotein.The composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.1-1%, 8mol/L urea, and surplus is water.
Two, Cu-NTA agarose affinity chromatography process
(1) fill post: join in Glass tubing by the Ni-NTA agarose (GEHealthcare) of 2ml bed volume along tube wall, after glue precipitation, shift piston onto suitable position.The mixing solutions adding 0.05mol/LEDTA and the 0.5mol/L sodium-chlor of 10 times of volumes removes Ni
2+, then remove EDTA with the 0.5mol/L sodium-chlor of 3 times of volumes, then with distillation washing, flow velocity is 0.5ml/min, until ultraviolet detection reaches steady baseline.
(2) in conjunction with Cu
2+: the 0.2mol/L copper-bath adding 1 times of bed volume, ambient temperatare puts 30min, makes Cu
2+fully be attached on filler.Wash pillar 30min with level pad, flow velocity is 0.5ml/min, ultraviolet detection baseline stability.
(3) combination is hatched: be that protein solution and the Cu-NTAsepharose of 25mg hatches 20min under 37 DEG C of conditions by protein content, metaprotein is fully combined with fixation of C u ion; Wash 30min with level pad subsequently, flow velocity is 0.5ml/min.
(4) renaturation and non-specific wash-out: with consisting of of elutriant 1(elutriant 1: 50mmolL sodium phosphate, pH6.5,300mmol/L sodium-chlor, 20mmol/L imidazoles) wash-out, flow velocity is 0.25ml/min, ensures that protein renaturation carries out smoothly, be eluted to about 30min, ultraviolet detection registration starts to increase, and starts to gather image, collects non-specific elution peak.When IMAQ is to 48min, ultraviolet detection baseline has reached stable.
(5) specificity wash-out: with consisting of of elutriant 2(elutriant 2: 50mmol/L sodium phosphate, pH5.8,300mmol/L sodium-chlor l, 60mmol/L imidazoles) carry out specificity wash-out, flow velocity is 0.5ml/min.Start after 10min to occur specificity elution peak, start to collect sample, after 15min, reach ultraviolet detection baseline, stop collecting sample.Fig. 1 is the ultraviolet detection result of specificity and non-specific copper-binding protein.
Three, the 2-DE of specificity copper-binding protein analyzes and Mass Spectrometric Identification
4 times of volume ice acetone (containing 10% trichoroacetic acid(TCA)) will be added in the specificity copper-binding protein of collecting above, precipitates overnight at-20 DEG C, then precipitation is washed 2 ~ 3 times with 80% ice acetone, after vacuum-freeze-dry, add lysate and fully dissolve, consisting of of lysate: 8mol/L urea, 4%CHAPS, 65mmol/LDTT and 0.2% (w/v) amphotericeledrolyte, 12,000 × g centrifugal 15min at 4 DEG C.By the Bradford method (Ramagli, 1999) of amendment, metaprotein is carried out quantitatively subsequently.Get 100 μ g total proteins, be diluted to 300 μ l with lysate, adopt PROTEANIEFCELL(Bio-Rad, USA) isoelectrofocusing system, the linear IPG adhesive tape (Bio-Rad, USA) in 17cmpH4 ~ 7.Isoelectrofocusing parameter: active aquation 12h at 20 DEG C; 20 DEG C of isoelectrofocusing, maximum current 50mA/strip, focusing on total V h is 60,000.Second to SDS-PAGE at ProteanPlusDodecacellapparatus(Bio-Rad, USA) carry out in vertical slab electrophoresis groove.With Colloidal Coomassie Brilliant Blue dyeing process, glue is dyeed.Examine glue UMAXPowerlookIII(UMAXTechnologies, USA after dye) scanner carries out transmission surface sweeping, and resolving power is 300dpi.Finally get that differential protein spot carries out decolouring, after enzymolysis and desalination supervisor, with MALDI-TOF-TOP(ReflexIII, Bruker-Daltonics) mass spectrograph identifies.
Find after 2-DE collection of illustrative plates (Fig. 2 and Fig. 3) is analyzed, the 2-DE collection of illustrative plates of contrast (Con) and Copper treatment (Cu) identifies 467 ± 7.05 and 452 ± 9.13 copper-binding protein points respectively, than the copper-binding protein identified under non denatured condition add to contrast under 36.2% and 43.1%(non denatured condition and Copper treatment 2-DE collection of illustrative plates on identify 298 ± 4.15 and 257 ± 3.33 copper-binding protein points respectively).Contrast the discrepancy reaching 2 times between Copper treatment and have 65, differential protein spot reaches 2 times between contrast with Copper treatment discrepancy under adding 46%(non denatured condition than the copper-binding protein identified under non denatured condition has 35).The mass spectrum of the copper-binding protein (GST) obtained by the present invention is shown in Fig. 4.
embodiment 2
One utilizes protein denaturation to be separated the protein-bonded method of special metal, comprises the following steps:
(1), get after biological tissue or cell liquid nitrogen fully grind, add protein extract, after mixing, low-temperature centrifugation, obtains the protein solution of sex change; The add-on of protein extract is, the solid-to-liquid ratio of grinding artifact tissue or cell and protein extract is: 1:4; The composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, and mass percent is the TritonX-100 of 0.1-1%, 8mol/L urea, and surplus is water;
(2) the middle Ni of Ni-NTA agarose (GEHealthcare), will bought
2+replace to Cu
2+load in chromatography column; The protein solution of sex change above-mentioned steps (1) obtained injects Cu-NTA agarose with 0.25ml/min flow velocity, hatches 15-40min, metaprotein is fully combined with it under 37 DEG C of conditions; Then under cold condition, slowly inject elutriant 1 with 0.25ml/min flow velocity, renaturation is carried out to albumen, by nonspecific copper-binding protein wash-out out; And then with 0.5ml/min flow velocity injection elutriant 2, specific copper-binding protein is separated;
Described elutriant 1 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 1,300mmol/L sodium-chlor, and 10-50mmol/L surplus is water; The pH value of elutriant 1 is 6.5;
Described elutriant 2 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 2,300mmol/L sodium-chlor, 40-100mmol/L imidazoles; The pH value of elutriant 2 is 5.8.
Low-temperature centrifugation described in step (1), its method is preferably, under 4 DEG C of low temperature, the centrifugal 30min of 15000g.
Described in step (2), Ni2+ in Ni-NTA agarose (buying in GEHealthcare company) is replaced to Cu2+, its method is: loaded by the Ni-NTA agarose of 2ml in chromatography column, injects the solution removal Ni containing 0.05mol/LEDTA and 0.5mol/L sodium-chlor of 10 times of volumes
2+, then remove EDTA with the 0.5mol/L sodium chloride aqueous solution of 3 times of volumes, the 0.2mol/L copper sulfate solution ambient temperatare of 1 times of volume uses level pad after putting 30min subsequently, and wash-out 30min, until ultraviolet detection baseline stability; Flow velocity is 0.5ml/min.The composition of described level pad is: containing 50mmol/L sodium phosphate in often liter of level pad, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.1-1%, and surplus is water.
Described in step (2), protein solution in (1) and Cu-NTA agarose 37 DEG C are hatched 30min, metaprotein is fully combined with Cu-NTA agarose.
Step adds elutriant 1 described in (2), with the wash-out of 0.25ml/min flow velocity, until ultraviolet detection baseline reaches stable; Describedly add elutriant 2, with the wash-out of 0.5ml/min flow velocity, until ultraviolet detection baseline reaches stable, collect specificity copper-binding protein.
Described in step (1), the composition of protein extract is: the composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.2%, 8mol/L urea, surplus is water.
embodiment 3
One utilizes protein denaturation to be separated the protein-bonded method of special metal, comprises the following steps:
(1), get after biological tissue or cell liquid nitrogen fully grind, add protein extract, after mixing, low-temperature centrifugation, obtains the protein solution of sex change; The add-on of protein extract is, the solid-to-liquid ratio of grinding artifact tissue or cell and protein extract is: 1:4; The composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, and mass percent is the TritonX-100 of 0.2%, 8mol/L urea, and surplus is water;
(2), Ni in the Ni-NTA agarose (GEHealthcare) bought being replaced to Cu loads in chromatography column; The protein solution of sex change above-mentioned steps (1) obtained injects Cu-NTA agarose with 0.25ml/min flow velocity, hatches 15-40min, metaprotein is fully combined with it under 37 DEG C of conditions; Then under cold condition, slowly inject elutriant 1 with 0.25ml/min flow velocity, renaturation is carried out to albumen, by nonspecific copper-binding protein wash-out out; And then with 0.5ml/min flow velocity injection elutriant 2, specific copper-binding protein is separated;
Described elutriant 1 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 1,300mmol/L sodium-chlor, and 20mmol/L surplus is water; The pH value of elutriant 1 is 6.5;
Described elutriant 2 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 2,300mmol/L sodium-chlor, 60mmol/L imidazoles; The pH value of elutriant 2 is 5.8.
Low-temperature centrifugation described in step (1), its method is preferably, under 4 DEG C of low temperature, the centrifugal 30min of 15000g.
Described in step (2), Ni in Ni-NTA agarose (buying in GEHealthcare company) is replaced to Cu, its method is: loaded by the Ni-NTA agarose of 2ml in chromatography column, injects the solution removal Ni containing 0.05mol/LEDTA and 0.5mol/L sodium-chlor of 10 times of volumes
2+, then remove EDTA with the 0.5mol/L sodium chloride aqueous solution of 3 times of volumes, the 0.2mol/L copper sulfate solution ambient temperatare of 1 times of volume uses level pad after putting 30min subsequently, and wash-out 30min, until ultraviolet detection baseline stability; Flow velocity is 0.5ml/min.The composition of described level pad is: containing 50mmol/L sodium phosphate in often liter of level pad, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.1-1%, and surplus is water.
Described in step (2), protein solution in (1) and Cu-NTA agarose 37 DEG C are hatched 30min, metaprotein is fully combined with Cu-NTA agarose.
Step adds elutriant 1 described in (2), with the wash-out of 0.25ml/min flow velocity, until ultraviolet detection baseline reaches stable; Describedly add elutriant 2, with the wash-out of 0.5ml/min flow velocity, until ultraviolet detection baseline reaches stable, collect specificity copper-binding protein.
Described in step (1), the composition of protein extract is: the composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.2%, 8mol/L urea, surplus is water.
Claims (7)
1. utilize protein denaturation to be separated the protein-bonded method of special metal, it is characterized in that: comprise the following steps:
(1), get after biological tissue or cell liquid nitrogen fully grind, add protein extract, after mixing, low-temperature centrifugation, obtains the protein solution of sex change; The solid-to-liquid ratio of grinding artifact tissue or cell and protein extract is: 1:4; The composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, and mass percent is the TritonX-100 of 0.1-1%, 8mol/L urea, and surplus is water;
(2), by Ni in Ni-NTA agarose
2+replace to Cu
2+load in chromatography column; The protein solution of sex change above-mentioned steps (1) obtained injects Cu-NTA agarose with 0.25ml/min flow velocity, hatches 15-40min, metaprotein is fully combined with it under 37 DEG C of conditions; Then under cold condition, slowly inject elutriant 1 with 0.25ml/min flow velocity, renaturation is carried out to albumen, and by nonspecific copper-binding protein wash-out out; And then with 0.5ml/min flow velocity injection elutriant 2, specific copper-binding protein is separated;
Described elutriant 1 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 1,300mmol/L sodium-chlor, and 10-50mmol/L surplus is water; The pH value of elutriant 1 is 6.5;
Described elutriant 2 composition is: containing 50mmol/L sodium phosphate in often liter of elutriant 2,300mmol/L sodium-chlor, 40-100mmol/L imidazoles; The pH value of elutriant 2 is 5.8.
2. utilize protein denaturation to be separated the protein-bonded method of special metal as claimed in claim 1, it is characterized in that: low-temperature centrifugation described in step (1), under 4 DEG C of low temperature, the centrifugal 30min of 15000g.
3. utilize protein denaturation to be separated the protein-bonded method of special metal as claimed in claim 1, it is characterized in that: described in step (2), Ni in Ni-NTA agarose (GEHealthcare) is replaced to Cu, its method is: loaded by the Ni-NTA agarose of 2ml in chromatography column, injects the solution removal Ni containing 0.05mol/LEDTA and 0.5mol/L sodium-chlor of 10 times of volumes
2+, then remove EDTA with the 0.5mol/L sodium chloride aqueous solution of 3 times of volumes, the 0.2mol/L copper sulfate solution ambient temperatare of 1 times of volume uses level pad after putting 30min subsequently, and wash-out 30min, until ultraviolet detection baseline stability; Flow velocity is 0.5ml/min.
4. utilize protein denaturation to be separated the protein-bonded method of special metal as claimed in claim 3, it is characterized in that: the composition of described level pad is: containing 50mmol/L sodium phosphate in often liter of level pad, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.1-1%, and surplus is water.
5. utilize protein denaturation to be separated the protein-bonded method of special metal as claimed in claim 1, it is characterized in that: described in step (2), protein solution in (1) and Cu-NTA agarose 37 DEG C are hatched 30min, metaprotein is fully combined with Cu-NTA agarose.
6. utilize protein denaturation to be separated the protein-bonded method of special metal as claimed in claim 1, it is characterized in that: step adds elutriant 1 described in (2), with the wash-out of 0.25ml/min flow velocity, until ultraviolet detection baseline reaches stable; Describedly add elutriant 2, with the wash-out of 0.5ml/min flow velocity, until ultraviolet detection baseline reaches stable, collect specificity copper-binding protein.
7. utilize protein denaturation to be separated the protein-bonded method of special metal as claimed in claim 1, it is characterized in that: described in step (1), the composition of protein extract is: the composition of described protein extract is: containing 50mmol/L sodium phosphate in often liter of protein solution, pH7.8,300mmol/L sodium-chlor, mass percent is the TritonX-100 of 0.2%, 8mol/L urea, surplus is water.
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| YUFENG SONG等: "Proteomic analysis of copper-binding proteins in excess copper-stressed rice roots by immobilized metal affinity chromatography and two-dimensional electrophoresis", 《BIOMETALS》 * |
| 李淑娟: "金属螯合亲和层析技术及其应用", 《科技经济市场》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116174044A (en) * | 2023-02-21 | 2023-05-30 | 集美大学 | New preparation method and application of artificial metalloenzyme with protein framework |
| CN116174044B (en) * | 2023-02-21 | 2024-07-02 | 集美大学 | New preparation method and application of artificial metalloenzyme with protein framework |
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