CN104844684A - Protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier - Google Patents
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Abstract
The invention refers to a protein refolding method using metal ion chelate affinity chromatography column as solid phase carrier. The invention uses the specificity affinity relation between rFGF with His or SUMO labels and metal ion chelate affinity chromatography column, attaches the denatured and reduced His-rFGF or SUMO-rFGF to the metal ion chelate affinity chromatography column, replaces denatured buffer solution with refolding buffer solution by gradually changing the composition of mobile phase, so as to prevent the refolding protein folding intermediates from gathering together, and obtain highly effective protein refold while finishing the purification of target protein. Compared to the traditional refolding by dialysis, the method not only can greatly reduce the time needed for protein refolding, but also can make the denatured His-rFGF or SUMO-rFGF obtain refolding protein with high purity and activity in the high concentration(1-10mg/mL), further providing a new process for accomplishing highly effective protein refolding and purifying.
Description
Technical field
The invention belongs to the protein renaturation method of field of biological pharmacy, be specifically related to a kind of protein renaturation method being solid phase carrier with metal ion-chelant affinity chromatographic column.
Background technology
Fibroblast growth factor (fibroblast growth factor, FGF) is the peptide matters that a class has extensive biologic activity, containing 18 members (FGF1-10 and FGF16-23).According to the difference of sequence and structure, FGF can be divided into five paracrine subfamilies (FGF1, FGF3, FGF4, FGF8 and FGF18 subfamily) and an internal secretion subfamily (FGF19 subfamily), participates in the physiological processs such as the growth of tissue and organ, vascularization, trauma repair and endocrine regulation respectively.These biological functions above-mentioned have highlighted FGF in the wide application prospect of clinical medicine domain and promotional value, become the research and development focus of field of biological pharmacy.
Reassemble into the recombination engineering method preparation also large-scale production of fibroblast growth factor (recombinant fibroblast growth factor, rFGF) mainly through taking intestinal bacteria as Host Strains.But as foreign protein, the great expression of rFGF in intestinal bacteria, often form inactive protein aggregate, i.e. inclusion body, as FGF9, FGF19, FGF20 and FGF21 etc.RFGF inclusion body must through bacterial cell disruption be separated, inclusion body washing and the step such as sex change and protein renaturation, high purity and highly active target protein could be prepared; Wherein, the renaturation of protein has become one of key factor of restriction rFGF preparation and mass-producing.At present, the protein renaturation of rFGF inclusion body mainly adopts dialysis renaturation, and the rFGF that namely sex change is dissolved is placed in dialysis tubing, is carried out the removal speed of controlled denaturation agent by the concentration reducing denaturing agent in outer transparent liquid gradually, makes rFGF recover its biological structure gradually with active.The method is simple to operate, protein solution constancy of volume, but has its limitation, and as length consuming time, process volume is little, consumption damping fluid is large, cannot be applied to large-scale production.
The comparative maturity that chromatographic technology has developed as the means of separation and purification protein, but to be used in protein renaturation process be the technology just grown up in recent years, is also referred to as chromatographic column protein renaturation.The method is adsorbed in all kinds of chromatographic column by the inclusion body protein that sex change is dissolved, then with wash-out not by the denaturing agent that adsorbs and heteroproteins etc., finally with renaturation buffer, the albumen wash-out of absorption is realized renaturation.Compared with traditional refolding method, its advantage is: (1) provides metaprotein one to form continually varying, alternative folding environment, and constantly revises the folding intermediate containing wrong three-dimensional structure; (2) eliminate the molecular aggregates after metaprotein molecule disengaging denaturing agent environment completely, avoid the generation precipitated, improve quality and the activity recovery of protein renaturation; (3) target protein can be made while protein renaturation to obtain purifying to a certain extent.Chromatographic column Solid phase protein renaturation mainly adopts ion-exchange chromatography, and other chromatograms report is less.The protein renaturation that chromatographic column Solid phase protein renaturation is applied to rFGF is not yet reported so far.
Summary of the invention
The object of the invention is to provide to overcome the deficiencies in the prior art a kind of protein renaturation method that recombinant protein purity is high and activity is high that protein renaturation time is short, obtain.
In order to achieve the above object, the invention discloses a kind of Solid phase protein renaturation technique being carrier with metal ion-chelant affinity chromatographic column, it is characterized in that comprising the following steps: (1) will His or SUMO label be carried and with inclusion bodies express reassemble into fibroblast growth factor albumen, namely His-rFGF or SUMO-rFGF is dissolved in denaturation buffer, makes it to be in the state of reducing denaturation; 2) the above-mentioned protein adsorption of the state of reducing denaturation will be in metal ion-chelant affinity chromatographic column; (3) with renaturation buffer, with the flow velocity of 0.1 milliliter of per minute, described denaturation buffer is replaced; (4) rinse chromatographic column with store buffer liquid A and displace renaturation buffer wherein, and with store buffer liquid B, renaturation His-rFGF or SUMO-rFGF is eluted from chromatographic column.
The present invention utilizes the rFGF(carrying His or SUMO label as His-FGF9, His-FGF15, SUMO-FGF15, His-FGF19, SUMO-FGF19, His-FGF20, SUMO-FGF20, His-FGF21 and His-FGF23) and metal ion-chelant affinity chromatographic column between specificity affinity interaction, His-rFGF or SUMO-rFGF reduced denaturation is adsorbed onto on metal ion-chelant affinity chromatographic column, denaturation buffer in chromatographic column is replaced into renaturation buffer by the composition then by changing moving phase gradually, make while completing target protein purifying, avoid recombinant protein folding intermediate mutually to assemble, obtain efficient protein renaturation.Compared to traditional dialysis renaturation, the method is adopted not only to substantially reduce the time of protein renaturation, sex change His-rFGF or SUMO-rFGF can also be made under high density (1-10mg/mL) to obtain high purity and highly active recombinant protein, realize efficient protein renaturation for a step and purifying provides novel process.
Below in conjunction with the drawings and specific embodiments, the invention will be further described.
Accompanying drawing explanation
fig. 1cleer and peaceful deposit sample before induction for SDS-PAGE analysis SUMO-FGF19 in the fermentation expression of SUMO-FGF19 in the present invention, after induction, on cellular lysate.Wherein M is standard protein molecular weight (170kD, 130kD, 100kD, 70kD, 55kD, 40kD, 35kD, 25kD from top to bottom, 15kD, 10kD); A/C/E is thalline before induction; B/D/F is thalline after induction; G be thalline through broken and centrifugal after supernatant liquor; H be thalline through broken and centrifugal after precipitation.
fig. 2for SDS-PAGE in the embodiment of the present invention 2 analyzes SUMO-FGF19 metal ion-chelant affinity chromatographic column solid-phase refolding products therefrom.Wherein M is standard protein molecular weight (116.0kD, 66.2kD, 45kD, 35kD, 25kD from top to bottom, 18.4kD, 14.4kD); A is sex change SUMO-FGF19 before renaturation; B and C is renaturation SUMO-FGF19.
fig. 3for protein immunoblot in the embodiment of the present invention 2 (Western blotting) identifies metal ion-chelant affinity chromatographic column solid-phase refolding gained SUMO-FGF19.
fig. 4for Western blotting in the embodiment of the present invention 2 detects solid-phase refolding gained SUMO-FGF19 to the impact of ERK1/2 tyrosine phosphorylation degree in HepG2 cell.
fig. 5for polymerase chain reaction (PCR) method in the embodiment of the present invention 2 detects renaturation gained SUMO-FGF19 to the impact of mouse liver cholesterol 7-hydroxylase (cholesterol 7-alpha hydroxyl-lase, Cyp7a1) mRNA level in-site.
Embodiment
The fermentation expression of SUMO-FGF19
By SUMO-FGF19 plasmid transformation escherichia coli BL21 competent cell correct for order-checking, choose mono-clonal bacterium colony, 37 degrees Celsius, 200 rpms shake cultivation 6 hours.According to 5% switching fresh LB, 37 degrees Celsius, cultivate 1.5 hours for 200 rpms, 37 degrees Celsius add 1 mmole and often rise isopropylthiogalactoside (IPTG), bacterium is got after 1,2,4 and 8 hours respectively in induction, collected by centrifugation thalline, SDS-PAGE analyzing proteins expression, and preserve bacterial classification.Choose high expression level inoculation in ammonia benzyl (100 milligrams per liter) LB substratum, 37 degrees Celsius, 160 rpms of concussion overnight incubation.According to 5% switching fresh LB, 37 degrees Celsius, cultivate 1.5 hours, get pre-induction sample for 200 rpms; 37 degrees Celsius add 1 mmole and often rise IPTG, induce after 4 hours, get postinduction sample, collect thalline.SDS-PAGE analyzes SUMO-FGF19 protein expression situation before and after IPTG induction.
4 grams of SUMO-FGF19 thalline are put into 200 ml beakers, adds lysate (25mM Tris-HCl damping fluid, pH=8.0,10% glycerine, 150mM NaCl) to 100 milliliters, then add 200 microlitre phenylmethylsulfonyl fluoride (PMSF), glass cylinder stirs and makes it become suspension.The broken thalline of low temperature ultrasonic, 10 minutes each, repeats 6 times.4 degrees Celsius, 18000 rpms, centrifugal 90 minutes, cleer and peaceful precipitation in SDS-PAGE analysis, result display SUMO-FGF19 was present in precipitation, illustrates that SUMO-FGF19 expresses with inclusion bodies.
Embodiment 1 nickel ion metal chelate affinity chromatography post Solid phase protein renaturation
(1) SUMO-FGF19 inclusion body is precipitated through cracking and collected after centrifugation, i.e. SUMO-FGF19;
(2) by denaturation buffer (25mM tri-(methylol) aminomethane-hydrochloride buffer (Tris-HCl), pH=8.0,150mM NaCl, 8M urea, 50mM beta-mercaptoethanol) the above-mentioned precipitation of suspendible, and at room temperature stir 1 hour.In 4 degrees Celsius, 18200 rpms, centrifugal 30 minutes, get supernatant liquor, Coomassie brilliant blue G250 method detects protein concentration;
(3) with above-mentioned denaturation buffer, pre-balance is carried out to nickel metal ion-chelant affinity chromatographic column.The sex change SUMO-FGF19 solution of 20 milliliters of 4mg/mL is loaded on nickel metal ion-chelant affinity chromatographic column with the flow velocity of 1.5 milliliters of per minutes; After completion of the sample, rinse to remove the albumen be not combined with pillar, until signal-balanced with denaturation buffer; In 8 hours, with the flow velocity of 0.1 milliliter of per minute, moving phase is replaced into renaturation buffer (25mM Tris-HCl from denaturation buffer, pH=8.0,150mM NaCl, 0.5M urea, 1mM GSSG(Sleep-promoting factor B) and 5mMGSH (reduced glutathion)); After completing renaturation, with store buffer liquid A(25mM Tris-HCl, pH=8.0) chromatographic column 5 column volumes are rinsed with 1 milliliter of per minute flow velocity, with store buffer liquid B(25mM Tris-HCl, pH=8.0,0.5M imidazoles) with 1 milliliter of per minute flow velocity elution chromatography post, collect elution peak, and use SDS-PAGE assay products.
Embodiment 2 nickel ion metal chelate affinity chromatography post Solid phase protein renaturation
(1) SUMO-FGF19 inclusion body is through cracking and collected after centrifugation precipitation, i.e. SUMO-FGF19;
(2) by denaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 8M urea, 50mM beta-mercaptoethanol) the above-mentioned precipitation of suspendible, and at room temperature stir 1 hour.In 4 degrees Celsius, 18200 rpms, centrifugal 30 minutes, get supernatant liquor, Coomassie brilliant blue G250 method detects protein concentration;
(3) with above-mentioned denaturation buffer, pre-balance is carried out to nickel metal ion-chelant affinity chromatographic column.The sex change SUMO-FGF19 solution of 20 milliliters of 4mg/mL is loaded on nickel metal ion-chelant affinity chromatographic column with the flow velocity of 1.5 milliliters of per minutes; After completion of the sample, rinse to remove the albumen be not combined with pillar, until signal-balanced with denaturation buffer; In 12 hours, with the flow velocity of 0.1 milliliter of per minute, moving phase is replaced into renaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 0.5M urea, 1mM GSSG and 5mMGSH) from denaturation buffer; After completing renaturation, with store buffer liquid A(25mM Tris-HCl, pH=8.0) chromatographic column 10 column volumes are rinsed with 1 milliliter of per minute flow velocity, with store buffer liquid B(25mM Tris-HCl, pH=8.0,0.5M imidazoles) with 1 milliliter of per minute flow velocity elution chromatography post, collect elution peak, and use SDS-PAGE assay products.
Immunoblotting (Western blotting) identifies solid-phase refolding gained SUMO-FGF19
Renaturation gained SUMO-FGF19 is prepared 10,5,2.5 and 1.25 micrograms per millilitre.SDS-PAGE electrophoretic separation albumen; After electrophoresis terminates, with BIO-RAD system by the protein delivery on gel on pvdf membrane; After transferring film is complete, film is placed in 5% skimmed milk, room temperature concussion closes 2 hours; TBST washes film 3 times, each 7 minutes.FGF19 polyclonal antibody incubated at room, after 2 hours, goes to 4 C overnight and hatches; TBST washes film 3 times, each 7 minutes; Two anti-incubated at room 1 hour, TBST washes film 3 times, each 7 minutes; ECL luminescence analysis, to identify that whether renaturation products therefrom is for SUMO-FGF19.Result shows the monoclonal antibody of product and the FGF19 obtained can specific binding, and along with FGF19 content higher, object band gray scale higher (Fig. 3).
Solid-phase refolding gained SUMO-FGF19 external activity detects
The HepG2 cell of taking the logarithm vegetative period, digesting centrifugal rear complete culture solution (RPMI-1640+10% foetal calf serum+1% penicillin/streptomycin) and be made into single cell suspension, is 5 × 10 by every porocyte concentration
5individual every milliliter is inoculated in 6 well culture plates, and every pore volume is 1 milliliter, at 37 degrees Celsius, 5% CO
2, saturated humidity condition incubator in cultivate 24 hours.Discard the nutrient solution in 6 orifice plates, then clean 1 time with PBS, add 1 milliliter of 1640 hungry nutrient solution containing 0.4% inactivated fetal bovine serum, Nature enemy 24 hours.Discard the nutrient solution in 6 orifice plates, adding respectively containing concentration is the hungry nutrient solution of 20,10,5,2.5 and 0 micrograms per millilitre SUMO-FGF19, at 37 degrees Celsius, 5% CO
2, saturated humidity condition incubator in effect 30 minutes.Discard nutrient solution in hole, clean 3 times with PBS, in every hole, add 60 microlitres of cells lysates, 4 degrees Celsius of cracking 30 minutes, collect the cell pyrolysis liquid in each hole, adopt Western blotting method to analyze ERK1/2 tyrosine phosphorylation level in each group of HepG2 cell.Result display SUMO-FGF19 makes HepG2 intracellular ERK1/2 tyrosine phosphorylation level significantly improve, and increase along with the increase of SUMO-FGF19 dosage, metal ion-chelant affinity chromatographic column Solid phase protein renaturation gained SUMO-FGF19 active good (Fig. 4) is described.
Activity determination in solid-phase refolding gained SUMO-FGF19 proteoplast
Male C57BL/ 6J mouse (10 week age) 12, adaptability is divided into 3 groups after feeding 1 week at random, often organizes each 4.Tail vein injection SUMO-FGF19, each group injected dose is respectively 0,10 and 100 ng/kg, and administration was put to death after 2 hours, and cardiac perfusion, gets liver.Total serum IgE in Trizol extraction process extracting liver, be cDNA by MMLV reversed transcriptive enzyme by RNA reverse transcription again, then the real-time quantitative fluorescence (Quantitative Real-time PCR, QR-PCR) mediated by SYBR-Green detects Cyp7a1mRNA expression level in each group of liver.Result shows, and SUMO-FGF19 suppresses Cyp7a1 mRNA level in-site in mouse liver, and rejection ability strengthens along with the increase of SUMO-FGF19 dosage, and SUMO-FGF19 in animal aspect active good (Fig. 5) is described.
Β-actin the primer sequence of table 1 mouse Cyp7a1 primer sequence and internal reference
Embodiment 3 cobalt ion metal chelate affinity chromatography post Solid phase protein renaturation
(1) SUMO-FGF19 inclusion body is through cracking and collected after centrifugation precipitation, i.e. SUMO-FGF19;
(2) by denaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 6M Guanidinium hydrochloride, 50mM beta-mercaptoethanol) the above-mentioned precipitation of suspendible, and at room temperature stir 1 hour.In 4 degrees Celsius, 18200 rpms, centrifugal 30 minutes, get supernatant liquor, Coomassie brilliant blue G250 method detects protein concentration;
(3) with above-mentioned denaturation buffer, pre-balance is carried out to cobalt ion metal chelate affinity chromatography post.The sex change SUMO-FGF19 solution of 20 milliliters of 4mg/mL is loaded on cobalt ion metal chelate affinity chromatography post with the flow velocity of 1.5 milliliters of per minutes; After completion of the sample, rinse to remove the albumen be not combined with pillar, until signal-balanced with denaturation buffer; In 8 hours, with the flow velocity of 0.1 milliliter of per minute, moving phase is replaced into renaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 0.5M Guanidinium hydrochloride, 1mM GSSG and 5mMGSH) from denaturation buffer; After completing renaturation, with store buffer liquid A(25mM Tris-HCl, pH=8.0) chromatographic column 10 column volumes are rinsed with 1 milliliter of per minute flow velocity, with store buffer liquid B(25mM Tris-HCl, pH=8.0,0.5M imidazoles) with 1 milliliter of per minute flow velocity elution chromatography post, collect elution peak, and use SDS-PAGE assay products.
Embodiment 4 nickel ion metal chelate affinity chromatography post Solid phase protein renaturation
(1) His-FGF19 inclusion body is through cracking and collected after centrifugation precipitation, i.e. His-FGF19
(2) by denaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 8M urea, 50mM beta-mercaptoethanol) the above-mentioned precipitation of suspendible, and at room temperature stir 1 hour.In 4 degrees Celsius, 18200 rpms, centrifugal 30 minutes, get supernatant liquor, Coomassie brilliant blue G250 method detects protein concentration;
(3) with above-mentioned denaturation buffer, pre-balance is carried out to nickel metal ion-chelant affinity chromatographic column.The sex change His-FGF19 solution of 20 milliliters of 4mg/mL is loaded on nickel ion metal chelate affinity chromatography post with the flow velocity of 1.5 milliliters of per minutes; After completion of the sample, rinse to remove the albumen be not combined with pillar, until signal-balanced with denaturation buffer; In 8 hours, with the flow velocity of 0.1 milliliter of per minute, moving phase is replaced into renaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 0.5M urea, 1mM GSSG and 5mMGSH) from denaturation buffer; After completing renaturation, with store buffer liquid A(25mM PB, pH=8.0,150mM NaCl) rinse nickel ion metal chelate affinity chromatography post 5-10 column volume with 1 milliliter of per minute flow velocity, with store buffer liquid B(25mM PB, pH=8.0,150mM NaCl and 0.5M imidazoles) with 1 milliliter of per minute flow velocity eluting nickel metal ion-chelant affinity chromatographic column, collect elution peak, and use SDS-PAGE assay products.
Embodiment 5 nickel ion metal chelate affinity chromatography post Solid phase protein renaturation
(1) His-FGF19 inclusion body is through cracking and collected after centrifugation precipitation, i.e. His-FGF19;
(2) by denaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 6M Guanidinium hydrochloride, 50mM beta-mercaptoethanol) the above-mentioned precipitation of suspendible, and at room temperature stir 1 hour.In 4 degrees Celsius, 18200 rpms, centrifugal 30 minutes, get supernatant liquor, Coomassie brilliant blue G250 method detects protein concentration;
(3) with above-mentioned denaturation buffer, pre-balance is carried out to nickel ion metal chelate affinity chromatography post.The sex change His-FGF19 solution of 20 milliliters of 4mg/mL is loaded on nickel ion metal chelate affinity chromatography post with the flow velocity of 1.5 milliliters of per minutes; After completion of the sample, rinse to remove the albumen be not combined with pillar, until signal-balanced with denaturation buffer; In 12 hours, with the flow velocity of 0.1 milliliter of per minute, moving phase is replaced into renaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 0.5M Guanidinium hydrochloride, 1mM GSSG and 5mMGSH) from denaturation buffer; After completing renaturation, with store buffer liquid A(25mM PB, pH=8.0,150mM NaCl) rinse chromatographic column 5-10 column volume with 1 milliliter of per minute flow velocity, with store buffer liquid B(25mM PB, pH=8.0,150mM NaCl and 0.5M imidazoles) with 1 milliliter of per minute flow velocity elution chromatography post, collect elution peak, and use SDS-PAGE assay products.
Embodiment 6 cupric ion metal chelate affinity chromatography post Solid phase protein renaturation
(1) His-FGF19 inclusion body is through cracking and collected after centrifugation precipitation, i.e. His-FGF19.
(2) by denaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 6M Guanidinium hydrochloride, 50mM beta-mercaptoethanol) the above-mentioned precipitation of suspendible, and at room temperature stir 1 hour.In 4 degrees Celsius, 18200 rpms, centrifugal 30 minutes, get supernatant liquor, Coomassie brilliant blue G250 method detects protein concentration;
(3) with above-mentioned denaturation buffer, pre-balance is carried out to cupric ion metal chelate affinity chromatography post.The sex change His-FGF19 solution of 20 milliliters of 4mg/mL is loaded on cupric ion metal chelate affinity chromatography post with the flow velocity of 1.5 milliliters of per minutes; After completion of the sample, rinse to remove the albumen be not combined with pillar, until signal-balanced with denaturation buffer; In 8 hours, with the flow velocity of 0.1 milliliter of per minute, moving phase is replaced into renaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 0.5M Guanidinium hydrochloride, 1mM GSSG and 5mMGSH) from denaturation buffer; After completing renaturation, with store buffer liquid A(25mM Tris-HCl, pH=8.0) chromatographic column 10 column volumes are rinsed with 1 milliliter of per minute flow velocity, with store buffer liquid B(25mM Tris-HCl, pH=8.0,0.5M imidazoles) with 1 milliliter of per minute flow velocity elution chromatography post, collect elution peak, and use SDS-PAGE assay products.
Embodiment 7 nickel ion metal chelate affinity chromatography post Solid phase protein renaturation
(1) His-FGF20 inclusion body is through cracking and collected after centrifugation precipitation, i.e. His-FGF20;
(2) by denaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 8M urea, 50mM beta-mercaptoethanol) the above-mentioned precipitation of suspendible, and at room temperature stir 1 hour.In 4 degrees Celsius, 18200 rpms, centrifugal 30 minutes, get supernatant liquor, Coomassie brilliant blue G250 method detects protein concentration;
(3) with above-mentioned denaturation buffer, pre-balance is carried out to nickel metal ion-chelant affinity chromatographic column.The sex change His-FGF20 solution of 20 milliliters of 4mg/mL is loaded on nickel ion metal chelate affinity chromatography post with the flow velocity of 1.5 milliliters of per minutes; After completion of the sample, rinse to remove the albumen be not combined with pillar, until signal-balanced with denaturation buffer; In 12 hours, with the flow velocity of 0.1 milliliter of per minute, moving phase is replaced into renaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 0.5M urea, 1mM GSSG and 5mMGSH) from denaturation buffer; After completing renaturation, with store buffer liquid A(25mM Tris-HCl, pH=8.0l) chromatographic column 10 column volumes are rinsed with 1 milliliter of per minute flow velocity, with store buffer liquid B(25mM Tris-HCl, pH=8.0,0.5M imidazoles) with 1 milliliter of per minute flow velocity elution chromatography post, collect elution peak, and use SDS-PAGE assay products.
Embodiment 8 nickel ion metal chelate affinity chromatography post Solid phase protein renaturation
(1) His-FGF21 inclusion body is through cracking and collected after centrifugation precipitation, i.e. His-FGF21;
(2) by denaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 8M urea, 50mM beta-mercaptoethanol) the above-mentioned precipitation of suspendible, and at room temperature stir 1 hour.In 4 degrees Celsius, 18200 rpms, centrifugal 30 minutes, get supernatant liquor, Coomassie brilliant blue G250 method detects protein concentration;
(3) with above-mentioned denaturation buffer, pre-balance is carried out to nickel ion metal chelate affinity chromatography post.The sex change His-FGF21 solution of 20 milliliters of 4mg/mL is loaded on nickel ion metal chelate affinity chromatography post with the flow velocity of 1.5 milliliters of per minutes; After completion of the sample, rinse to remove the albumen be not combined with pillar, until signal-balanced with denaturation buffer; In 12 hours, with the flow velocity of 0.1 milliliter of per minute, moving phase is replaced into renaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 0.5M urea, 1mM GSSG and 5mMGSH) from denaturation buffer; After completing renaturation, with store buffer liquid A(25mM Tris-HCl, pH=8.0) nickel metal ion-chelant affinity chromatographic column 10 column volumes are rinsed with 1 milliliter of per minute flow velocity, with store buffer liquid B(25mM Tris-HCl, pH=8.0,0.5M imidazoles) with 1 milliliter of per minute flow velocity elution chromatography post, collect elution peak, and use SDS-PAGE assay products.
Embodiment 9 zine ion metal chelate affinity chromatography post Solid phase protein renaturation
(1) His-FGF23 inclusion body is through cracking and collected after centrifugation precipitation, i.e. His-FGF23;
(2) by denaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 8M urea, 50mM beta-mercaptoethanol) the above-mentioned precipitation of suspendible, and at room temperature stir 1 hour.In 4 degrees Celsius, 18200 rpms, centrifugal 30 minutes, get supernatant liquor, Coomassie brilliant blue G250 method detects protein concentration;
(3) with above-mentioned denaturation buffer, pre-balance is carried out to zine ion metal chelate affinity chromatography post.The sex change His-FGF23 solution of 20 milliliters of 4mg/mL is loaded on zine ion metal chelate affinity chromatography post with the flow velocity of 1.5 milliliters of per minutes; After completion of the sample, rinse to remove the albumen be not combined with pillar, until signal-balanced with denaturation buffer; In 12 hours, with the flow velocity of 0.1 milliliter of per minute, moving phase is replaced into renaturation buffer (25mM Tris-HCl, pH=8.0,150mM NaCl, 0.5M urea, 1mM GSSG and 5mMGSH) from denaturation buffer; After completing renaturation, with store buffer liquid A(25mM Tris-HCl, pH=8.0) chromatographic column 10 column volumes are rinsed with 1 milliliter of per minute flow velocity, with store buffer liquid B(25mM Tris-HCl, pH=8.0,0.5M imidazoles) with 1 milliliter of per minute flow velocity elution chromatography post, collect elution peak, and use SDS-PAGE assay products.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can to do medical science amendment or improve, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (7)
1. the protein renaturation method that is solid phase carrier with metal ion-chelant affinity chromatographic column, it is characterized in that comprising the following steps: (1) will His or SUMO label be carried and with inclusion bodies express reassemble into fibroblast growth factor albumen, namely His-rFGF or SUMO-rFGF is dissolved in denaturation buffer, makes it to be in the state of reducing denaturation; 2) the above-mentioned protein adsorption of the state of reducing denaturation will be in metal ion-chelant affinity chromatographic column; (3) with renaturation buffer, with the flow velocity of 0.1 milliliter of per minute, described denaturation buffer is replaced; (4) rinse chromatographic column with store buffer liquid A and displace renaturation buffer wherein, and with store buffer liquid B, renaturation His-rFGF or SUMO-rFGF is eluted from chromatographic column.
2., according to the protein renaturation method being solid phase carrier with metal ion-chelant affinity chromatographic column according to claim 1, it is characterized in that: wherein metal ion-chelant affinity chromatographic column refers to chelated nickel ion Ni
2+, zine ion Zn
2+, cupric ion Cu
2+or cobalt ion Co
2+in the IMAC Sepharose HP/6FF of any one metal ion or Chelating Sepharose HP/FF.
3. according to the protein renaturation method being solid phase carrier with metal ion-chelant affinity chromatographic column according to claim 1, it is characterized in that: wherein denaturation buffer is three (methylol) aminomethane-hydrochloride buffer containing 50-150mM NaCl, 6-8M urea and 50-200mM beta-mercaptoethanol.
4. according to the protein renaturation method being solid phase carrier with metal ion-chelant affinity chromatographic column according to claim 1, it is characterized in that: wherein denaturation buffer is three (methylol) aminomethane-hydrochloride buffer containing 50-150mM NaCl, 4-6M Guanidinium hydrochloride and 50-200mM beta-mercaptoethanol.
5. according to the protein renaturation method being solid phase carrier with metal ion-chelant affinity chromatographic column according to claim 1, it is characterized in that: wherein renaturation buffer contains 50-150mM NaCl, 0-0.5M urea, 1mM Sleep-promoting factor B and 5mM reduced glutathion.
6. according to the protein renaturation method being solid phase carrier with metal ion-chelant affinity chromatographic column according to claim 1, it is characterized in that: wherein renaturation buffer contains 50-150mM NaCl, 0-0.2M Guanidinium hydrochloride, 1mM GSSG and 5mM GSH.
7. according to the protein renaturation method being solid phase carrier with metal ion-chelant affinity chromatographic column according to claim 1, it is characterized in that: described store buffer liquid A is three (methylol) aminomethane-hydrochloride buffer or phosphate buffered saline buffer (PB); Described store buffer liquid B is the store buffer liquid A containing 0.1-1M imidazoles.
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| CN105085606A (en) * | 2015-09-30 | 2015-11-25 | 河南科技大学 | Method for separating copper proteome by using copper-chelated magnetic beads |
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| CN107866205B (en) * | 2017-10-31 | 2020-07-31 | 苏州博进生物技术有限公司 | Affinity chromatography medium using glutathione as ligand |
| CN111777659A (en) * | 2020-07-09 | 2020-10-16 | 向开军 | Universal electric field driven protein solid phase renaturation method |
| CN111777659B (en) * | 2020-07-09 | 2023-11-24 | 向开军 | Universal electric field driven protein solid phase renaturation method |
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