CN102432668A - Method capable of rapidly separating and purifying target recombinant protein by using centrifugal method - Google Patents
Method capable of rapidly separating and purifying target recombinant protein by using centrifugal method Download PDFInfo
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Abstract
The invention relates to a method capable of rapidly separating and purifying a target recombinant protein by using a centrifugal method, comprising the following steps: (1) connecting a target protein gene and a fusion tag protein gene through a restrictive restriction site on the fusion tag protein gene, and introducing to a host bacterium; (2) enabling a fusion tag protein to be subjected to phase change aggregation in a special pH value by using different salt ions and buffer solutions with corresponding concentrations through controlling the temperature of a solution to obtain a protein solution; and (3) purifying the protein solution by using the centrifugal method to obtain a target protein. In the invention, phase change aggregation of the fusion tag protein is induced through adding salt with a certain concentration, and the effect on separating a recombinant protein through optimizing an operation condition by using the centrifugal method is remarkable; and the method is simple, rapid, efficient and economic, wide in application range, easy to operate and low in production cost, special instruments are not needed, and therefore, the method is suitable for industrial production.
Description
Technical field
The present invention relates to a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein.
Background technology
Along with the needs to proteinic research and production, recombinant gene is advanced by leaps and bounds in recent years, a lot of gene engineering products occurred, has a deep effect on mankind itself and environment thereof.And more and more show its importance as the separating and purifying technology of the gene recombinant protein in the downstream engineering of genetic engineering technique.According to statistics, the separation and purification cost of gene engineering product accounts for 60%～80% of its overall cost.Therefore increasing biological worker is engaged in the separation and purification work of recombinant protein.
Similar with traditional protein separation mode, the separation and purification of recombinant protein also is a difference of utilizing its physics and chemical property, promptly with bulk of molecule; Shape; Solubleness, iso-electric point, hydrophilic and hydrophobic and set up with the character such as affinity of other molecule.Current purifying recombinant proteins mainly is to rely on chromatography and electrophoretic technique.At present, affinity tag has become back genomics epoch purification of recombinant proteins conventional means.This method need not to understand proteinic biochemical characteristic or physiologically active, recombination fusion protein that just can be through tape label optionally with chromatography substrate on part combine, thereby be able to any protein of purifying.This method is with conventional chromatography method difference, need develops specific part and method to different protein.Adopt the mild conditions of protected protein matter 26S Proteasome Structure and Function integrity, successfully from crude extract, be purified into multiple recombinant protein through a step affinity chromatography, purity reaches more than 90%.Wherein 6 histidine-tagged affinity chromatographys are present representatives.But this method still comes with some shortcomings: as: cost an arm and a leg with the special aglucon of fusion tag bonded, metallic nickel ions comes off easily to flow out and sneaks into protein solution, pillar non-specific adsorption protein sometimes during ni-sepharose purification; Therefore, limited application in extensive separation and purification.Therefore, development is simple, cheap, the recombinant protein purification process is significant for large-scale production of recombinant proteins and the research of high throughput protein group reliably.
Summary of the invention
In order to address the above problem, the object of the present invention is to provide a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein.The present invention brings out fusion tag albumen through adding finite concentration salt and undergoes phase transition gathering, through operation conditions optimization, adopts the centrifuging separating recombinant proteins, and effect is remarkable; This method is simple, quick, efficient, economical, does not need specific apparatus, applied widely, easy handling, low production cost, thereby the suitable suitability for industrialized production of carrying out.
For reaching above-mentioned purpose, the present invention adopts following technical scheme:
A kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein comprises the steps: 1) through the restriction enzyme site on the fusion tag protein gene, the target protein gene is connected with the fusion tag protein gene, and imports in the host bacterium; 2) buffered soln of use different salt ion and corresponding concentration in specific pH value, makes fusion tag albumen undergo phase transition gathering through the control solution temperature and obtains protein soln; 3) protein soln is passed through centrifuging purification of target albumen.
Described a kind of fusion tag albumen and gene thereof that can be responsive to envrionment conditions; Its called after [KV8F] n; Wherein n is repeat number, and its numerical value is between 1-1000, and this sequence has been carried out bacterial classification patent preservation (China Committee for Culture Collection of Microorganisms common micro-organisms center; Preserving number CGMCC NO.4185, on September 17th, 2010) and applied for patent (number of patent application: 201010562100.5) preservation date:.
Restriction enzyme adopts one or more among ApaI, BglII, KpnI, NheI, SphI, XbaI, SacI, Nde I, Sfi I, EcoR I, pflMI, Hind III and the Xho I in the described step 1); Restriction enzyme is connected with the fusion tag protein gene; The sequence that perhaps between target protein and fusion tag protein gene, adds one section random structure; Be inserted in the expression vector then; Be transformed into further that (the two is the commercialization bacterial strain in e. coli bl21 (DE3) or the BLR bacterial strain; Can directly surpass and grind bio tech ltd, the rich inferior bio tech ltd purchase in Shanghai) from Shanghai; Filter out positive transformant, positive transformant is induced through isopropyl-encoding sox (purchasing the company in Sigma) acquisition fusion is efficiently expressed, and carries out cytoclasis then.
The sequence of described random structure is: (AGAGAGPEG)
n, the span of n is at 1-150.
Described efficiently expressing to low temperature induction expressed, and comprises the steps: 1) glycerol stock (purchasing in Shanghai Jierui Biology Engineering Co., Ltd) is linked into by 1: 100 volume ratio contains cultivation (OD in penbritin (Amp) the LB liquid (100mg/L)
600Value is 0.6-1.0, and the time is 10-12h), 4 ℃ of preservations, centrifugal collecting cell; 2) contain (100mg/L) the Luria-Bertani substratum (LB) of 2ml of penbritin (Amp) with what cell was resuspended to 2ml; (its prescription is: peptone (Tryptone) 10g, yeast extract (Yeast extract) 5.0g, NaCl 10g; Zero(ppm) water 1000ml, pH7.0.) in the liquid, cultivate (OD again
600Value is 0.6-1.0, and the time is 10-12 hour), inoculate with this; Be inoculated in the TB substratum by 1: 100 inoculum size, (the TB culture medium prescription of every 900ml volume is: peptone 12g, yeast powder 24g, USP Kosher 8ml; ) in, place 37 ℃ of shaking tables, with the speed of 200r/min, cultivated 4-5 hour; Adding IPTG inductor (final concentration is 1mM) induced 7 hours; Afterwards, under 4 ℃ with the speed of 4000r/min, centrifugal 15 minutes, according to the resuspended thalline of 1L bacterium liquid 40mlPBS damping fluid.
Supersonic method is adopted in described cytoclasis, comprises the steps: 1) take out adding IPTG inductor, induce the Erlenmeyer flask after 7 hours; Ice bath stops growing cell, and bacterium liquid is sub-packed in the 100ml centrifuge tube; Make the centrifuge tube quality that is in symmetric position equate (trim); Centrifugal, centrifugal condition rotating speed 4000r/min, time 15min; 2) after whizzer stops, getting centrifuge tube, abandon supernatant, (PBS buffered soln: bacterium liquid) ratio added PBS buffered soln washing thalline, and fully vibration on vortex mixer until the thalline at the bottom of the centrifuge tube is rinsed well, is collected in 1: 25; 3) carry out cytoclasis with the ultrasonic disruption machine, condition is ultra 2s, stops 2s, 80 circulations, the broken liquid of collecting cell afterwards.
Described step 2) solution temperature in is selected in 4 ℃ of-45 ℃ of scopes according to the characteristic of target protein.
Described step 2) the salt type of salt ion buffered soln adopts sodium-chlor, sodium sulfate, SODIUMNITRATE, SODIUM PHOSPHATE, MONOBASIC, yellow soda ash in; Repone K, vitriolate of tartar, saltpetre, potassium primary phosphate, salt of wormwood; A kind of in an ammonium nitrate, ammonium sulfate and the ammonium chloride; Its concentration range is 0.1mol/L-2.5mol/L, and the pH value scope of buffered soln is: 5.5-8.5; Above-mentioned salt is joined in the cell crude extract behind the broken wall, assemble to bring out the phase transformation of fusion tag albumen.
Described step 3) centrifuging comprises the steps: 1) to add final concentration be the 0.5%W/V polymine, vibration makes its abundant mixing, removes nucleic acid; 2) with step 2) protein soln that obtains is sub-packed in the centrifuge tube of 10ml, ice bath 15min, centrifugal, its condition is 4 ℃, 14000r/min, centrifugation time 15min; 3) take out centrifuge tube, supernatant is moved on to clean centrifuge tube, abandon deposition, press salts solution: the volume ratio adding salts solution of supernatant=1: 2 makes the solution final concentration between 0.1-2.5mol/L.4 ℃ of-45 ℃ of water bath with thermostatic control 15min, centrifugal, centrifugal condition is a temperature: 4 ℃-38 ℃, rotating speed: 12000-13500r/min, the time is 5-15min; 4) take out centrifuge tube, abandon supernatant rapidly, every pipe adds 2mlPBS buffered soln, slowly drips along tube wall, and the protein on the wall is fully dissolved, and is centrifugal behind the ice bath 15min, and centrifugal condition is 4 ℃, 12000-15000r/min, and the time is 5-10min; 5) repeating step 3) and 4) once; Finally abandon deposition, collect supernatant, be kept in-20 ℃ of refrigerators; 6) dialysis postlyophilization; Wherein salts solution adopts sodium-chlor, sodium sulfate, SODIUMNITRATE, SODIUM PHOSPHATE, MONOBASIC, yellow soda ash, Repone K, vitriolate of tartar, saltpetre, potassium primary phosphate, salt of wormwood, any one in an ammonium nitrate, ammonium sulfate and the ammonium chloride.
Described PBS buffered soln is made up of A liquid and B liquid, and its prescription is: A liquid: the 0.2mol/L disodium phosphate soln comprises: Na
2HPO
428.4g, Na
2HPO
4H
2O 31.61g, Na
2HPO
42H
2O 35.6g, Na
2HPO
47H
2O 53.63g, Na
2HPO
412H
2O 71.64g adds distilled water to 1000ml; B liquid: 0.2mol/L sodium dihydrogen phosphate: NaH
2PO
424.0g, NaH
2PO
4H
2O 27.6g, NaH
2PO
42H
2O, 31.21g adds distilled water to 1000ml; A liquid and the mixing of B liquid are obtained the damping fluid of pH between 5.5-8.5.
The invention has the beneficial effects as follows: use a type elastin polypeptide (Elastin-Like Polypeptide; ELP) [KV8F] n cuts with the target protein gene its gene and to be connected through enzyme, after genetic expression; Through using suitable salt and corresponding concentration; The pH of control solution makes it under specified temp, undergo phase transition gathering, uses centrifuging that target protein is carried out purifying afterwards.The required raw-material expense of every purifying 1kg protein is respectively: utilize His tail Qin Hecengxi $838124.73, use IMPACT-CN Chun Huaxitong $989606.23, and adopt ELP system Jin $74509.75, less than the above two 10%.It is simple and easy, with low cost to adopt ELP to carry out non-chromatogram purification operation, is expected to bring revolutionary variation to the separating and purifying technology of recombinant protein.That is to say that the invention provides a cover controls the method for ELP phase transformation fast and effectively, available centrifugal easy with the target protein purifying, but protein concentrate solution has simultaneously been avoided the troublesome operation of chromatographic separation and to the dilution of protein sample.And have following characteristics: 1) can be simple, link with the target protein gene fast, realize efficiently expressing; 2) target protein structure and normal function there are not obvious influence, safe; 3) the protein expression condition is simple, convenient purification, low production cost, thereby the suitable suitability for industrialized production of carrying out.The present invention has broad application prospects in the bioseparation field for the separation of recombinant protein provides a new way.
Description of drawings
Fig. 1 is the design of graphics of the expression plasmid pET-22b-ELP-dhaT in the embodiment of the invention 1;
Fig. 2 is the electrophoretogram that the SDS-PAGE protein electrophoresis of the embodiment of the invention 1 detects 1,3 methyl glycol oxidoreductase;
Fig. 3 is the design of graphics of the expression plasmid pET-22b-SoxB-M2-S-ELP in the embodiment of the invention 2;
Fig. 4 is the electrophoretic analysis collection of illustrative plates of fusion rotein zytase in the embodiment of the invention 2.
Embodiment
Through specific embodiment, the present invention is done further description below.
Present embodiment separates at first construction of fusion protein expression plasmid of 1,3 methyl glycol oxidoreductase with this method, and step is as shown in Figure 1, and Fig. 1 is the design of graphics of the expression plasmid pET-22b-ELP-dhaT in the embodiment of the invention 1; Process is: pUC19-ELP (is the commercialization bacterial strain from bacillus coli DH 5 alpha; Can be directly from Shanghai ultraly grind bio tech ltd, the rich inferior bio tech ltd in Shanghai is bought) extract; With the ELP gene fragment of restriction enzyme Nde I and Hind III double digestion generation 318bp, cut glue and reclaim.Cut pET-22b (+) plasmid with identical double digestion, and be connected up with the ELP gene fragment that Nde I and Hind III will reclaim just now and obtain pET-22b-ELP, be transformed among the E.coli DH5 α and preserve.Extracting pUC57-dhaT plasmid through pcr amplification, is contained the dhaT gene of Xho I and Hind III recognition site in a large number.DhaT mrna length after cutting glue purification is 1211bp, and the pET-22b-ELP that obtains is before handled with Hind III and Xho I double digestion, and being connected with the dhaT gene order then forms the pET-22b-ELP-dhaT plasmid.This construction of recombinant plasmid is identified with Hind III and Xho I double digestion respectively after accomplishing; Nde I and Xho I double digestion is identified; Hind III single endonuclease digestion is identified; Positive colony is delivered to Genscript Co., the Ltd. order-checking.The correct plasmid extraction that will check order at last also is transformed into E.coli BL21 (DE3) respectively and BLR (DE3) bacterial strain (be the commercialization bacterial strain, can directly surpass from Shanghai and grind bio tech ltd, the rich inferior bio tech ltd purchase in Shanghai).
Step is afterwards: 1. bacterial strain activation: the E.coli BL21 (DE3) and the BLR (DE3) 1 that will contain plasmid pET-22b-ELP-dhaT) glycerol stock is linked in the LB liquid that contains penbritin Amp (100mg/L) by 1: 100 volume ratio cultivates, OD wherein
600Value is 0.6-1.0, and the time is 10-12h; 4 ℃ of preservations, centrifugal collecting cell; 2) (its prescription is: peptone (Tryptone) 10g cell to be resuspended to the Luria-Bertani substratum LB that contains penbritin Amp (100mg/L) 2ml of 2ml; Yeast extract (Yeast extract) 5.0g; NaCl 10g, zero(ppm) water 1000ml, pH7.0.) in the liquid, cultivate again, wherein OD
600Value is 0.6-1.0, and the time is 10-12 hour; Inoculate with this; Be inoculated in the TB substratum (prescription: the TB culture medium prescription of every 900ml volume is: peptone 12g, yeast powder 24g, USP Kosher 8ml) by 1: 100 inoculum size, place 37 ℃ of shaking tables,, cultivated 4-5 hour with the speed of 200r/min; Adding final concentration is 1mM isopropyl-IPTG inductor, induces 7 hours; Afterwards, under 4 ℃ with the speed of 4000r/min, centrifugal 15 minutes, according to the resuspended thalline of 1L bacterium liquid 40mlPBS damping fluid (its proportioning sees that table 1pH is 7.0 prescription).Carry out cytoclasis afterwards, the steps include: 1) with the bacterium liquid ice bath after the low temperature induction expression, cell is stopped growing; Bacterium liquid is sub-packed in the 100ml centrifuge tube, the centrifuge tube quality that is in symmetric position is equated, centrifugal; Centrifugal condition rotating speed 4000r/min, time 15min; 2) after whizzer stops, getting centrifuge tube, abandon supernatant; In PBS buffered soln: the ratio of bacterium liquid=1: 25 adds PBS buffered soln washing thalline (its proportioning sees that table 1pH is 7.0 prescription); Fully vibration on vortex mixer until the thalline at the bottom of the centrifuge tube is rinsed well, is collected; 3) carry out cytoclasis with the ultrasonic disruption machine, condition is ultra 2s, stops 2s, 80 circulations, the broken liquid of collecting cell afterwards.
The cell crude extract that obtains is clicked step process, obtain 1,3 methyl glycol oxidoreductase of purifying.1) adding final concentration is the 0.5%W/V polymine, and vibration makes its abundant mixing, removes nucleic acid; 2) with step 2) protein soln that obtains is sub-packed in the centrifuge tube of 10ml, ice bath 15min, centrifugal, its condition is 4 ℃, 14000r/min, centrifugation time 15min; 3) take out centrifuge tube; Supernatant is moved on to clean centrifuge tube, abandon deposition, press salts solution (sodium-chlor, sodium sulfate, SODIUMNITRATE, SODIUM PHOSPHATE, MONOBASIC, yellow soda ash; Repone K, vitriolate of tartar, saltpetre, potassium primary phosphate, salt of wormwood; A kind of in an ammonium nitrate, ammonium sulfate and the ammonium chloride solution): the volume ratio of supernatant=1: 2 adds salts solution, makes its final concentration between 0.1mol/L-2.5mol/L, and pH value scope is: 5.5-8.5; Assemble to bring out the phase transformation of fusion tag albumen.4 ℃ of-45 ℃ of water bath with thermostatic control 15min, centrifugal, centrifugal condition is a temperature: 4 ℃-38 ℃, rotating speed: 12000-13500r/min, the time is 5-15min; 4) take out centrifuge tube, abandon supernatant rapidly, every pipe adds 2mlPBS buffered soln, and (described PBS buffered soln is made up of A liquid and B liquid, and its prescription is: A liquid: the 0.2mol/L disodium phosphate soln comprises: Na
2HPO
428.4g, Na
2HPO
4H
2O 31.61g, Na
2HPO
42H
2O 35.6g, Na
2HPO
47H
2O 53.63g, Na
2HPO
412H
2O 71.64g adds distilled water to 1000ml; B liquid: 0.2mol/L sodium dihydrogen phosphate: NaH
2PO
424.0g, NaH
2PO
4H
2O 27.6g, NaH
2PO
42H
2O, 31.21g adds distilled water to 1000ml; A liquid and B liquid can be obtained the damping fluid of pH between 5.5-8.5 according to the mixing of table 1 proportioning.) slowly drip along tube wall, the protein on the wall is fully dissolved, centrifugal behind the ice bath 15min, centrifugal condition is 4 ℃, 12000-15000r/min, the time is 5-10min; 5) repeating step 3) and 4) once; Finally abandon deposition, collect supernatant, be kept in-20 ℃ of refrigerators; 6) dialysis postlyophilization.
Obtain 1,3 methyl glycol oxidoreductase of purifying at last, electrophorogram is seen Fig. 2; Fig. 2 is the electrophoretogram that the SDS-PAGE protein electrophoresis of the embodiment of the invention 1 detects 1,3 methyl glycol oxidoreductase; Wherein the M swimming lane is albumen Marker; 1 swimming lane is a cell-free extract; 2 swimming lanes are through 2.0M NaCl purifying fusion rotein once; 3 swimming lanes are through 0.5M K
2CO
3The fusion rotein of purifying after once; 4 swimming lanes are through 0.5M Na
2CO
3The fusion rotein of purifying after once.Through calculating, its purity can reach 98%.
Present embodiment separates zytase with this method, construction expression plasmid pET-22 b-SoxB-M2-S-ELP at first, and in this example, goal gene SoxB-M2-S-ELP is that full gene is synthetic, expression vector and host bacterium are respectively pET-22b and E.coli BLR (DE3).The structure flow process of recombinant expression plasmid pET-22b-SoxB-M2-S-ELP is seen Fig. 3.Extract plasmid afterwards, it is transformed into the intestinal bacteria BLR (be the commercialization bacterial strain, can directly surpass from Shanghai and grind bio tech ltd, the rich inferior bio tech ltd purchase in Shanghai).Fig. 3 is the design of graphics of the embodiment of the invention 2 expression plasmid pET-22b-SoxB-M2-S-ELP.
The sequence that pET-22b-ELP designs for us has voluntarily been carried out the preservation of bacterial classification patent (China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number CGMCC NO.4185, preservation date: on September 17th, 2010.); SoxB-M2 gene order wherein derives from document Zhang S; Zhang K; Chen X; Et al.Five mutations in N-terminus confer thermostability on mesophilic xylanase.Biochemical and biophysical research communications, 2010,395 (2): 200-206.; And the sequence of S derives from another piece document Wheeldon IR; Campbell E; Banta S.A chimeric fusion protein engineered withdisparate functionalities-enzymatic activity and self-assembly.Journal of molecular biology; 2009,392 (1): two pieces of documents of 129-142. are all published.
After plasmid imports the reorganization bacterium, yeast culture and collect step and be: 1) glycerol stock is linked in the LB liquid that contains penbritin Amp (100mg/L) by 1: 100 volume ratio cultivates, wherein OD
600Value is 0.6-1.0, and the time is 10-12h; 4 ℃ of preservations, centrifugal collecting cell; 2) (its prescription is: peptone (Tryptone) 10g cell to be resuspended to the Luria-Bertani substratum LB that contains penbritin Amp (100mg/L) 2ml of 2ml; Yeast extract (Yeast extract) 5.0g; NaCl 10g, zero(ppm) water 1000ml, pH7.0.) in the liquid, cultivate again, wherein OD
600Value is 0.6-1.0, and the time is 10-12 hour; Inoculate with this; Be inoculated in the TB substratum (prescription: the TB culture medium prescription of every 900ml volume is: peptone 12g, yeast powder 24g, USP Kosher 8ml) by 1: 100 inoculum size, place 37 ℃ of shaking tables,, cultivated 4-5 hour with the speed of 200r/min; Adding final concentration is 1mM isopropyl-IPTG inductor, induces 7 hours; Afterwards, under 4 ℃ with the speed of 4000r/min, centrifugal 15 minutes, according to the resuspended thalline of 1L bacterium liquid 40mlPBS damping fluid (its proportioning sees that table 1pH is 7.0 prescription).Carry out cytoclasis afterwards, the steps include: 1) with the bacterium liquid ice bath after the low temperature induction expression, cell is stopped growing; Bacterium liquid is sub-packed in the 100ml centrifuge tube, the centrifuge tube quality that is in symmetric position is equated, centrifugal; Centrifugal condition rotating speed 4000r/min, time 15min; 2) after whizzer stops, getting centrifuge tube, abandon supernatant; In PBS buffered soln: the ratio of bacterium liquid=1: 25 adds PBS buffered soln washing thalline (its proportioning sees that table 1pH is 7.0 prescription); Fully vibration on vortex mixer until the thalline at the bottom of the centrifuge tube is rinsed well, is collected; 3) carry out cytoclasis with the ultrasonic disruption machine, condition is ultra 2s, stops 2s, 80 circulations, the broken liquid of collecting cell afterwards.
The cell crude extract that obtains is clicked step process, obtain the zytase of purifying.
The cell crude extract that obtains is clicked step process, obtain 1,3 methyl glycol oxidoreductase of purifying.1) adding final concentration is the 0.5%W/V polymine, and vibration makes its abundant mixing, removes nucleic acid; 2) with step 2) protein soln that obtains is sub-packed in the centrifuge tube of 10ml, ice bath 15min, centrifugal, its condition is 4 ℃, 14000r/min, centrifugation time 15min; 3) take out centrifuge tube; Supernatant is moved on to clean centrifuge tube, abandon deposition, press salts solution (sodium-chlor, sodium sulfate, SODIUMNITRATE, SODIUM PHOSPHATE, MONOBASIC, yellow soda ash; Repone K, vitriolate of tartar, saltpetre, potassium primary phosphate, salt of wormwood; A kind of in an ammonium nitrate, ammonium sulfate and the ammonium chloride solution): the volume ratio of supernatant=1: 2 adds salts solution, makes its final concentration between 0.1mol/L-2.5mol/L, and pH value scope is: 5.5-8.5; Assemble to bring out the phase transformation of fusion tag albumen.4 ℃ of-45 ℃ of water bath with thermostatic control 15min, centrifugal, centrifugal condition is a temperature: 4 ℃-38 ℃, rotating speed: 12000-13500r/min, the time is 5-15min; 4) take out centrifuge tube, abandon supernatant rapidly, every pipe adds 2mlPBS buffered soln, and (described PBS buffered soln is made up of A liquid and B liquid, and its prescription is: A liquid: the 0.2mol/L disodium phosphate soln comprises: Na
2HPO
428.4g, Na
2HPO
4H
2O 31.61g, Na
2HPO
42H
2O 35.6g, Na
2HPO
47H
2O 53.63g, Na
2HPO
412H
2O 71.64g adds distilled water to 1000ml; B liquid: 0.2mol/L sodium dihydrogen phosphate: NaH
2PO
424.0g, NaH
2PO
4H
2O 27.6g, NaH
2PO
42H
2O, 31.21g adds distilled water to 1000ml; A liquid and B liquid can be obtained the damping fluid of pH between 5.5-8.5 according to the mixing of table 1 proportioning.) slowly drip along tube wall, the protein on the wall is fully dissolved, centrifugal behind the ice bath 15min, centrifugal condition is 4 ℃, 12000-15000r/min, the time is 5-10min; 5) repeating step 3) and 4) once; Finally abandon deposition, collect supernatant, be kept in-20 ℃ of refrigerators; 6) dialysis postlyophilization.Obtain the zytase of purifying at last, electrophorogram is seen Fig. 4.Fig. 4 is the electrophoretic analysis collection of illustrative plates of fusion rotein zytase in the embodiment of the invention 2.Through calculating, its purity can reach 64.3%.
Because fusion tag albumen ELP-KV8F can undergo phase transition phenomenon, it does not have obvious influence to the structure of wanting isolating other target protein simultaneously.Therefore; With its encoding sox and after needing isolating target protein to merge, the fusion rotein that gives expression to can be through accurately controlling salt type and concentration and pH of buffer value; Make fusion tag albumen ELP that unique phase transition phenomena take place; Separate through high speed centrifugation, the quick, simple of target protein separated, and this method need be by chromatography commonly used at present thereby reach.
Claims (10)
1. an ability is carried out the method for centrifuging fast separating and purifying to target recombinant protein; It is characterized in that comprising the steps: 1) through the restriction enzyme site on the fusion tag protein gene; The target protein gene is connected with the fusion tag protein gene, and imports in the host bacterium; 2) buffered soln of use different salt ion and corresponding concentration in specific pH value, makes fusion tag albumen undergo phase transition gathering through the control solution temperature and obtains protein soln; 3) protein soln is passed through centrifuging purification of target albumen.
2. a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein as claimed in claim 1; It is characterized in that: described fusion tag albumen and gene thereof, its called after [KV8F] n, wherein n is repeat number; Its numerical value is between 1-1000; This sequence has been carried out the preservation of bacterial classification patent (China Committee for Culture Collection of Microorganisms common micro-organisms center, preserving number CGMCC NO.4185, preservation date: on September 17th, 2010).
3. a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein as claimed in claim 1 is characterized in that: restriction enzyme adopts one or more among ApaI, BglII, KpnI, NheI, SphI, XbaI, SacI, Nde I, Sfi I, EcoRI, pflM I, Hind III and the Xho I in the described step 1); Restriction enzyme is connected with the fusion tag protein gene; The sequence that perhaps between target protein and fusion tag protein gene, adds one section random structure; Be inserted in the expression vector then; Further be transformed in e. coli bl21 (DE3) or the BLR bacterial strain; Filter out positive transformant, positive transformant is induced through isopropyl-IPTG encoding sox acquisition fusion is efficiently expressed, and carries out cytoclasis then.
4. a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein as claimed in claim 3, it is characterized in that: the sequence of described random structure is: (AGAGAGPEG) n, the span of n is at 1-150.
5. a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein as claimed in claim 3; It is characterized in that: described efficiently expressing is the low temperature induction expression; Comprise the steps: 1) glycerol stock is linked in the LB liquid that contains penbritin Amp (100mg/L) by 1: 100 volume ratio cultivates, OD wherein
600Value is 0.6-1.0, and the time is 10-12h; 4 ℃ of preservations, centrifugal collecting cell; 2) cell is resuspended in the Luria-Bertani substratum LB liquid that contains penbritin Amp (100mg/L) 2ml of 2ml, cultivates again, wherein OD
600Value is 0.6-1.0, and the time is 10-12 hour; Inoculate with this; Be inoculated in the TB substratum by 1: 100 inoculum size, place 37 ℃ of shaking tables,, cultivated 4-5 hour with the speed of 200r/min; Adding final concentration is 1mM isopropyl-IPTG inductor, induces 7 hours; Afterwards, under 4 ℃ with the speed of 4000r/min, centrifugal 15 minutes, according to the resuspended thalline of 1L bacterium liquid 40mlPBS damping fluid.
6. a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein as claimed in claim 3 is characterized in that described cytoclasis employing supersonic method, comprises the steps: 1) with the bacterium liquid ice bath after the low temperature induction expression; Cell is stopped growing; Bacterium liquid is sub-packed in the 100ml centrifuge tube, the centrifuge tube quality that is in symmetric position is equated, centrifugal; Centrifugal condition rotating speed 4000r/min, time 15min; 2) after whizzer stops, getting centrifuge tube, abandon supernatant, in PBS buffered soln: the ratio of bacterium liquid=1: 25 adds the fully vibration on vortex mixer of PBS buffered soln washing thalline, until the thalline at the bottom of the centrifuge tube is rinsed well, collects; 3) carry out cytoclasis with the ultrasonic disruption machine, condition is ultra 2s, stops 2s, 80 circulations, the broken liquid of collecting cell afterwards.
7. a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein as claimed in claim 1 is characterized in that: the solution temperature described step 2) is selected in 4 ℃ of-45 ℃ of scopes according to the characteristic of target protein.
8. a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein as claimed in claim 1; It is characterized in that: the salt type of salt ion buffered soln adopts sodium-chlor, sodium sulfate, SODIUMNITRATE, SODIUM PHOSPHATE, MONOBASIC, yellow soda ash described step 2); Repone K, vitriolate of tartar, saltpetre, potassium primary phosphate, salt of wormwood; A kind of in an ammonium nitrate, ammonium sulfate and the ammonium chloride, its concentration range is 0.1mol/L-2.5mol/L, the pH value scope of buffered soln is: 5.5-8.5; Above-mentioned salt is joined in the cell crude extract behind the broken wall, assemble to bring out the phase transformation of fusion tag albumen.
9. a kind of method that can carry out the centrifuging fast separating and purifying to target recombinant protein as claimed in claim 1; It is characterized in that described step 3) centrifuging comprises the steps: 1) to add final concentration be the 0.5%W/V polymine; Vibration makes its abundant mixing, removes nucleic acid; 2) with step 2) protein soln that obtains is sub-packed in the centrifuge tube of 10ml, ice bath 15min, centrifugal, its condition is 4 ℃, 14000r/min, centrifugation time 15min; 3) take out centrifuge tube, supernatant is moved on to clean centrifuge tube, abandon deposition, press salts solution: the volume ratio adding salts solution of supernatant=1: 2 makes the solution final concentration between 0.1-2.5mol/L.4 ℃ of-45 ℃ of water bath with thermostatic control 15min, centrifugal, centrifugal condition is a temperature: 4 ℃-38 ℃, rotating speed: 12000-13500r/min, the time is 5-15min; 4) take out centrifuge tube, abandon supernatant rapidly, every pipe adds 2mlPBS buffered soln, slowly drips along tube wall, and the protein on the wall is fully dissolved, and is centrifugal behind the ice bath 15min, and centrifugal condition is 4 ℃, 12000-15000r/min, and the time is 5-10min; 5) repeating step 3) and 4) once; Finally abandon deposition, collect supernatant, be kept in-20 ℃ of refrigerators; 6) dialysis postlyophilization; Wherein salts solution adopts sodium-chlor, sodium sulfate, SODIUMNITRATE, SODIUM PHOSPHATE, MONOBASIC, yellow soda ash, Repone K, vitriolate of tartar, saltpetre, potassium primary phosphate, salt of wormwood, any one in an ammonium nitrate, ammonium sulfate and the ammonium chloride.
10. like claim 5 or the 6 or 9 described a kind of methods that can carry out the centrifuging fast separating and purifying to target recombinant protein; It is characterized in that: described PBS buffered soln is made up of A liquid and B liquid; Its prescription is: A liquid: the 0.2mol/L disodium phosphate soln comprises: Na
2HPO
428.4g, Na
2HPO
4H
2O 31.61g, Na
2HPO
42H
2O 35.6g, Na
2HPO
47H
2O 53.63g, Na
2HPO
412H
2O 71.64g adds distilled water to 1000ml; B liquid: 0.2mol/L sodium dihydrogen phosphate: NaH
2PO
424.0g, NaH
2PO
4H
2O 27.6g, NaH
2PO
42H
2O, 31.21g adds distilled water to 1000ml; A liquid and the mixing of B liquid are obtained the damping fluid of pH between 5.5-8.5.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972220A (en) * | 2012-12-11 | 2013-03-20 | 湛江师范学院 | Simple method for testing disease resistance of pathogenic bacteria quorum-quenching gene prokaryotic expression product |
| CN106701699A (en) * | 2016-12-23 | 2017-05-24 | 重庆医科大学 | Biocatalyst as well as preparation method and application thereof |
| WO2018030499A1 (en) | 2016-08-10 | 2018-02-15 | Ｓｐｉｂｅｒ株式会社 | Production method for insoluble recombinant protein aggregate |
| CN112279899A (en) * | 2020-10-16 | 2021-01-29 | 成都天邦生物制品有限公司 | Method for purifying prokaryotic expression protein |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418290A (en) * | 2008-12-02 | 2009-04-29 | 中山大学 | High efficiency ELP fusion protease as well as preparation and application thereof |
| CN101955960A (en) * | 2010-08-03 | 2011-01-26 | 中山大学 | Method for purifying recombinant lactase by using elastin like protein tag |
| CN102127156A (en) * | 2010-11-16 | 2011-07-20 | 华侨大学 | Fusion tagging protein for nonchromatographic sepration of target protein and coding gene and preparation method thereof |
-
2011
- 2011-11-25 CN CN2011103805066A patent/CN102432668A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101418290A (en) * | 2008-12-02 | 2009-04-29 | 中山大学 | High efficiency ELP fusion protease as well as preparation and application thereof |
| CN101955960A (en) * | 2010-08-03 | 2011-01-26 | 中山大学 | Method for purifying recombinant lactase by using elastin like protein tag |
| CN102127156A (en) * | 2010-11-16 | 2011-07-20 | 华侨大学 | Fusion tagging protein for nonchromatographic sepration of target protein and coding gene and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 蓝东明: "ELP融合蛋白酶的应用和SARS疫苗研发的研究", 《中山大学博士学位论文》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102972220A (en) * | 2012-12-11 | 2013-03-20 | 湛江师范学院 | Simple method for testing disease resistance of pathogenic bacteria quorum-quenching gene prokaryotic expression product |
| WO2018030499A1 (en) | 2016-08-10 | 2018-02-15 | Ｓｐｉｂｅｒ株式会社 | Production method for insoluble recombinant protein aggregate |
| AU2017310785B2 (en) * | 2016-08-10 | 2022-06-30 | Spiber Inc. | Production method for insoluble recombinant protein aggregate |
| AU2017310785B9 (en) * | 2016-08-10 | 2022-07-21 | Spiber Inc. | Production method for insoluble recombinant protein aggregate |
| CN106701699A (en) * | 2016-12-23 | 2017-05-24 | 重庆医科大学 | Biocatalyst as well as preparation method and application thereof |
| CN106701699B (en) * | 2016-12-23 | 2020-09-18 | 重庆医科大学 | Biocatalyst and preparation method and application thereof |
| CN112279899A (en) * | 2020-10-16 | 2021-01-29 | 成都天邦生物制品有限公司 | Method for purifying prokaryotic expression protein |
| CN112279899B (en) * | 2020-10-16 | 2023-08-29 | 成都史纪生物制药有限公司 | Method for purifying prokaryotic expression protein |
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