CN104479029A - Purification and renaturation method of Notch ligand Delta-like1 fusion protein - Google Patents
Purification and renaturation method of Notch ligand Delta-like1 fusion protein Download PDFInfo
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Abstract
A purification and renaturation method of Notch ligand Delta-like1 fusion protein is characterized by comprising steps as follows: 1), an inclusion body containing HD1R is repeatedly washed and centrifuged by a washing buffer and is dissolved in a strong denaturant buffer to obtain crudely separated HD1R protein; 2), a denatured leaching solution containing human proinsulin is subjected to direct sample introduction to a flowing phase A balanced chromatographic column and is subjected to isocratic elution by a flowing phase B line of PBS (poly butylenes succinate), 1 mol/L of NaCl or Tris and 1 mol/L of NaCl, target peak chromatographic fraction is collected, and renatured and purified HD1R chromatographic fraction is obtained; and 3), the collected target peak chromatographic fraction is subjected to direct sample introduction, is further desalinated by the flowing phase A balanced chromatographic column of 20 mmol/L of PBS and is continuously eluted for 30 min, the elution flow velocity is 0.5-1.5 ml/min, and the renatured and purified chromatographic fraction with higher purity and higher activity is obtained. According to the method, the process is simple, large-scale production can be realized, and the obtained fraction with high yield and high purity can provide a reliable experimental material for intensive study of Notch signal pathways.
Description
Invention field
The invention belongs to metaprotein renaturation technical field in biotechnology, in particular to a kind of purifying of Notch part Delta-like1 fusion rotein and the method for renaturation, utilize ion-exchange chromatography and exclusion chromatography purifying and renaturation Notch part Delta-like1 fusion rotein.
Technical background
Notch finds that in fruit bat fractional mutations can cause breach (Notch) at the edge of fruit bat wing and gain the name because of Morgan in 1916.Notch signal be by local cells between interaction to control a kind of approach of cell fate, evolution camber guard, wide expression is in embryo and adult tissue.This signal pathway comprises part DSL (Delta/Serrate/Lag-2, DSL), acceptor and downstream molecules and Molecular regulator composition.Find 4 kinds of Notch receptor proteins in current Mammals, be respectively Notch1-4; These acceptor molecules have 5 kinds of parts, are respectively Delta-like1,3,4 and Jagged 1,2.These receptor/ligand structurally have high homology, and transcription factor RBP-J (recombination binding protein-J, RBP-J) is the main core internal effect thing of Mammals Notch signal pathway.When after the part and receptors bind of Notch, discharge NICD after triggering three endonuclease reactions and enter nucleus.Enter nuclear NICD by RAM structural domain and transcription factor CSL (CBF1/Suppressor of Hairless/Lag-1, CSL) interact, make to combine with CSL be made up of SMR, SHARP, CIR and HDAC equimolecular transcribe co-suppression complex dissociation, and raise for it and transcribe co-activation mixture by GCN5, P300, SKIP and MAML1 decile is molecular, CSL is turned by Transcription inhibition and switches to transcriptional activation, thus regulate the expression of downstream gene.Wherein people Delta-like 1 (Dll1) is made up of 723 amino acid, is a transmembrane protein, has 8 EGF spline structure territories and 1 DSL motif in its extracellular fragment.It with identical or different Notch receptors bind, can activate Notch signal path, participates in growing of regulation and control Various Tissues, all has expression on surfaces such as hematopoietic cell, thymocyte, bone marrow stromal cells, plays an important role in their differentiation of induction.
Intestinal bacteria (E.coli) produce the most frequently used expression system as engineered protein, have the feature that can obtain high-density recombinant protein while expressing fast and look at by the emperor himself by people very much always.But, the target protein of high expression for want of translate after processing and modification, often can exist with non-activity, insoluble inclusion bodies and limit the application of recombinant protein.The appearance of the external assisted protein renaturation methods such as dilution method, dialysis method, hailstorm suppression of continuing, protein folding liquid phase chromatography (PFLC) method has become a kind of more effective protein renaturation and Simultaneous purification method.Wherein high performance ion exchange chromatography (HPIEC) utilizes the alive part of different proteins strong and weak different with adsorbing between opposite charges stationary phase from surface, by changing the moving phase of different eluting power, carry out absorption-desorption attached-renaturation of adsorbing again, and be purified simultaneously, this method also exists potential advantage [Wang CZ on extensive purifying protein, et al, Appl Biochem Biotechnol2008; 144:181-189], utilize weak anion exchange column DEAE-FF to recombinant human alpha-fetoprotein (rhAFP) renaturation and purifying in 3h, folding productivity ratio dilution method is high 9 times, purity reach 95% [Chen Y, et al, JChromatogr A.2009; 1216:4877-4886].By to the when optimization such as pH of urea concentration, GSH/GSSG concentration in moving phase, macrophage colony stimulating factor of recombinant human granulocyte (rhGM-CSF) [the Bai Q achieving high purity and mass recovery is exchanged with strong anion, et al, Biotechnol Prog, 2007; 23:1138-1142].Although, compared to ion exchange chromatography, high narrow spectrum affinity chromatography can bring higher purity multiple to the purifying of recombinant protein and renaturation, but, except stationary phase cost this shortcoming high, need to add the purification tags such as GST, His when the structure to recombined protein carrier, after renaturation completes, also albumen will be separated [Wilkinson RJ with purification tag by methods such as RPLC, et al, Protein Expres Purifi.2004; 35:334-343], process is loaded down with trivial details, and is not suitable for a large amount of production.Therefore, in the renaturation of recombinant protein and purge process, when especially needing scale operation, how under the condition of short period, low cost, a large amount of highly purified recombinant protein is obtained significant.
Summary of the invention
For overcoming above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of purifying of Notch part Delta-like1 fusion rotein and the method for renaturation, it is the ligandin Dll1 of soluble human Notch acceptor and the product of RGD amalgamation and expression, the present invention's PFLC method, is specially anion-exchange chromatography and exclusion chromatography has carried out renaturation and Simultaneous purification to the inclusion body obtained; The method technique is simple, and can accomplish scale production, the further investigation that the high yield obtained and highly purified cut can be Notch signal pathway provides effective experimental tool.
For achieving the above object, the technical scheme adopted in the present invention is: a kind of purifying of Notch part Delta-like1 fusion rotein and the method for renaturation, comprise the following steps:
1) express in the intestinal bacteria (E.coli) somatic cells after centrifugal, ultrasonic disruption, repeatedly thick purifying, be dissolved in 8.0mol/L urea damping fluid and obtain inclusion body sex change extract.
2) the inclusion body lavation buffer solution containing HD1R is repeatedly washed, centrifugal, be dissolved in strong denaturant damping fluid, obtain the HD1R albumen of roughing out;
3) chromatographic column will balanced to mobile phase A containing people HD1R protein denaturation extract direct injection, use the Mobile phase B line isocratic elution of PBS, 1mol/LNaCl or Tris, 1mol/LNaCl again, collect target peak chromatographic fraction, obtain the chromatographic fraction of the HD1R after renaturation and purifying;
4) the target peak chromatographic fraction direct injection will collected, the further desalination of chromatographic column balance by the moving phase of 20mmol/LPBS, lasting wash-out 30min, elution flow rate is 0.5-1.5ml/min, obtains renaturation and the higher active chromatographic fraction of degree of purification.
Described chromatographic column adopts soluble high-expression post optional HiTrap CaptoQ or HiTrap CaptoDEAE, be GE Healthcare company produce, specification is 0.7 × 2.5cm, 1mL, also can load DEAE filler voluntarily in the Column XK16 of GE Healthcare company production.
In described mobile phase A, PBS concentration is 20-40mmol/L.
In described mobile phase A, the pH of PBS is 7.5-8.0.
In described mobile phase A, the concentration of Tris is 20-40mmol/L.
In described mobile phase A, the pH of Tris is 7.5-8.0.
In described Mobile phase B, PBS concentration is 20-40mmol/L.
In described Mobile phase B, the pH of PBS is 6.0-7.0.
In described Mobile phase B, the concentration of Tris is 20-40mmol/L.
In described Mobile phase B, the pH of Tris is 7.5-8.0.
Described elution flow rate is 0.5-1.5mL/min.
The mode of described lasting wash-out is after mobile phase A balance, and Mobile phase B isocratic elution 5-10min, extends 10min.
Chromatographic column after described further desalination is HiTrap Desalting column.
The invention has the beneficial effects as follows:
The present invention utilizes anion-exchange chromatography and the inclusion body of exclusion chromatography to Notch part Dll1 fusion rotein to carry out renaturation and Simultaneous purification.Overcome affinity chromatography stationary phase cost large, also to carry out the deficiencies such as aftertreatment to the recombinant protein of the tape label be purified into; adopt isocratic elution; in elution process, pH and ionic strength are all constant; do not need gradient mixer; the purge process cycle is short; cost is low, purity and mass recovery high, be particularly suitable for large-scale production.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE analysis chart of HPIEC and HPSEC method purifying and renaturation HD1R, and M is albumen Marker; 1st road is the HD1R do not induced; 2nd road is after HD1R induces 5h; 3rd road is 8.0mol/L urea extract; 4th road is that HiTrap Capto Q is to the purifying of HD1R and renaturation; 5-7 road is HiTrap Capto DEAE to secondarily purified to a step cut desalination of the purifying of HD1R and renaturation; It is secondarily purified that 8th road is that the cut corresponding to the 7th road carries out desalination.
Fig. 2 is that the present invention adopts HiTrap Capto Q chromatographic column to the color atlas of HD1R protein purification and renaturation.
Fig. 3-5 adopts HiTrap Capto DEAE chromatographic column to the color atlas of HD1R purifying and renaturation for the present invention.
Fig. 3-5 three groups of results, in the mobile phase A using chromatographic column to carry out adopting in purifying and renaturation and Mobile phase B composition slightly difference, cause the purity of obtained HD1R albumen to be respectively 97%, 96% to 91%.。
Embodiment
Describe technical scheme of the present invention in detail below in conjunction with embodiment and accompanying drawing, but protection domain is not by this restriction.In embodiment, equipment used or raw material all can obtain from market.
Embodiment one
The purifying of Notch part Delta-like1 fusion rotein and a method for renaturation, comprise the following steps:
1) the inclusion body lavation buffer solution containing HD1R is repeatedly washed, centrifugal, be dissolved in strong denaturant damping fluid, obtain the HD1R albumen of roughing out;
2) will containing people HD1R protein denaturation extract direct injection to mobile phase A (20mmol/LPBS, pH8.0) chromatographic column balanced, again with containing 20mmol/LPBS, 1mol/LNaCl, the Mobile phase B isocratic elution of pH7.5, collect target peak chromatographic fraction, obtain the chromatographic fraction of the HD1R after renaturation and purifying;
3) the target peak chromatographic fraction direct injection will collected, the further desalination of chromatographic column balance by the moving phase of 20mmol/LPBS, lasting wash-out 30min, flow velocity 1.5mL/min, obtain renaturation and the higher active chromatographic fraction of degree of purification.
Embodiment two
The purifying of Notch part Delta-like1 fusion rotein and a method for renaturation, comprise the following steps:
1) the inclusion body lavation buffer solution containing HD1R is repeatedly washed, centrifugal, be dissolved in strong denaturant damping fluid, obtain the HD1R albumen of roughing out;
2) will containing people HD1R sex change extract direct injection to mobile phase A (40mmol/LPBS, or 20-40mmol/LTris, pH7.6) chromatographic column balanced, again with containing 40mmol/LTris, 1mol/LNaCl, the Mobile phase B isocratic elution of pH8.0, collect target peak chromatographic fraction, obtain the chromatographic fraction of the HD1R after renaturation and purifying;
3) the target peak chromatographic fraction direct injection will collected, the further desalination of chromatographic column balance by the moving phase of 20mmol/LPBS, lasting wash-out 30min, flow velocity 1.5mL/min, obtain renaturation and the higher active chromatographic fraction of degree of purification.
Embodiment three
The purifying of Notch part Delta-like1 fusion rotein and a method for renaturation, comprise the following steps:
1) the inclusion body lavation buffer solution containing HD1R is repeatedly washed, centrifugal, be dissolved in strong denaturant damping fluid, obtain the HD1R albumen of roughing out;
2) will containing people HD1R protein denaturation extract direct injection to mobile phase A (30mmol/LPBS, pH7.7) chromatographic column balanced, again with the Mobile phase B line isocratic elution containing 30mmol/LPBS, 1mol/LNaCl pH7.8, collect target peak chromatographic fraction, obtain the chromatographic fraction of the HD1R after renaturation and purifying;
3) the target peak chromatographic fraction direct injection will collected, the further desalination of chromatographic column balance by the moving phase of 20mmol/LPBS, lasting wash-out 30min, flow velocity 0.5mL/min, obtain renaturation and the higher active chromatographic fraction of degree of purification.
Embodiment four
The purifying of Notch part Delta-like1 fusion rotein and a method for renaturation, comprise the following steps:
1) the inclusion body lavation buffer solution containing HD1R is repeatedly washed, centrifugal, be dissolved in strong denaturant damping fluid, obtain the HD1R albumen of roughing out;
2) will containing people HD1R protein denaturation extract direct injection to mobile phase A (35mmol/LTris, pH7.9) chromatographic column balanced, again with containing 38mmol/LTris, 1mol/LNaCl, the Mobile phase B line isocratic elution of pH8.0, collect target peak chromatographic fraction, obtain the chromatographic fraction of the HD1R after renaturation and purifying;
3) the target peak chromatographic fraction direct injection will collected, the further desalination of chromatographic column balance by the moving phase of 20mmol/LPBS, lasting wash-out 30min, flow velocity 1.2mL/min, obtain renaturation and the higher active chromatographic fraction of degree of purification.
Embodiment five
The purifying of Notch part Delta-like1 fusion rotein and a method for renaturation, comprise the following steps:
1) the inclusion body lavation buffer solution containing HD1R is repeatedly washed, centrifugal, be dissolved in strong denaturant damping fluid, obtain the HD1R albumen of roughing out;
2) will containing people HD1R protein denaturation extract direct injection to mobile phase A (25mmol/LTris, pH7.6) chromatographic column balanced, again with containing 25mmol/LPBS, 1mol/LNaCl, the Mobile phase B line isocratic elution of pH7.6, collect target peak chromatographic fraction, obtain the chromatographic fraction of the HD1R after renaturation and purifying;
3) the target peak chromatographic fraction direct injection will collected, the further desalination of chromatographic column balance by the moving phase of 20mmol/LPBS, lasting wash-out 30min, flow velocity 1.4mL/min, obtain renaturation and the higher active chromatographic fraction of degree of purification.
Embodiment six
The purifying of Notch part Delta-like1 fusion rotein and a method for renaturation, comprise the following steps:
1) the inclusion body lavation buffer solution containing HD1R is repeatedly washed, centrifugal, be dissolved in strong denaturant damping fluid, obtain the HD1R albumen of roughing out;
2) will containing people HD1R protein denaturation extract direct injection to mobile phase A (33mmol/LPBS, pH7.5) chromatographic column balanced, again with containing 20-40mmol/LTris, 1mol/LNaCl, the Mobile phase B line isocratic elution of pH7.5-8.0, collect target peak chromatographic fraction, obtain the chromatographic fraction of the HD1R after renaturation and purifying;
3) the target peak chromatographic fraction direct injection will collected, the further desalination of chromatographic column balance by the moving phase of 20mmol/LPBS, lasting wash-out 30min, flow velocity 1.5mL/min, obtain renaturation and the higher active chromatographic fraction of degree of purification.
The preparation of embodiment six: Human Delta-like 1-RGD (HD1R is that Human Delta-like 1-RGD English is write a Chinese character in simplified form) sex change extract;
With E.coli as host cell expression the fusion rotein of notch part Delta-like1 and RGD, again by zymocyte liquid after centrifugal, cytoclasis and the thick purifying that repeatedly washs, the inclusion body obtained is dissolved in 8.0mol/L urea, in the denaturing agent of pH8.0, sex change extract concentration is 3.71mg/mL, rhPI purity is more than 65% (see Fig. 1, lane3).
Embodiment seven: soluble high-expression renaturation and purifying HD1R
First use 100% mobile phase A (20.0mmol/LPBS, pH7.5) HPIEC post (HiTrap CaptoQ, 0.7 × 2.5cm, 1mL is balanced, GE Healthcare), sex change extract direct injection 0.1mL, flow velocity 1.0mL/min, wash away non-adsorbable foreign protein by 100% mobile phase A, use 100% Mobile phase B (20mmol/LPBS again, 1mol/LNaCl, pH7.5) wash-out 10 minutes, extend 5min.HD1R mass recovery is 70.3%, and purity reaches more than 98% (Fig. 1, lane4; Fig. 2).
Embodiment eight: soluble high-expression renaturation and purifying HD1R
First use 100% mobile phase A (20.0mmol/LTris, pH7.8 balances HPIEC post, and (Column XK16 loads Capto DEAE filler 1.6x4.0cm, 9ml, GE Healthcare), sex change extract direct injection 0.6mL, flow velocity 1.5mL/min, wash away non-adsorbable foreign protein by 100% mobile phase A, then use 100% Mobile phase B (20.0mmol/LTris, 1mol/LNaCl, pH7.8) wash-out 10 minutes, extends 10min.HD1R mass recovery is 23.0%, and purity reaches more than 97% (Fig. 1, lane5; Fig. 3).
Embodiment nine: soluble high-expression renaturation and purifying HD1R
First use 100% mobile phase A (40.0mmol/LTris, pH8.0 balances HPIEC post, and (Column XK16 loads Capto DEAE filler 1.6x4.0cm, 9ml, GE Healthcare), sex change extract direct injection 0.7mL, flow velocity 3.0mL/min, wash away non-adsorbable foreign protein by 100% mobile phase A, then use 100% Mobile phase B (40.0mmol/LTris, 1mol/LNaCl, pH8.0) wash-out 10 minutes, extends 10min.HD1R mass recovery is 31.8%, and purity reaches more than 96% (Fig. 1, lane 6; Fig. 4).
Embodiment ten: soluble high-expression renaturation and purifying HD1R
First use 100% mobile phase A (20.0mmol/LTris, pH8.0 balances HPIEC post, and (ColumnXK16 loads Capto DEAE filler 1.6x 4.0cm, 9ml, GE Healthcare), sex change extract direct injection 1.5mL, flow velocity 3.0mL/min, wash away non-adsorbable foreign protein by 100% mobile phase A, then use 100% Mobile phase B (20.0mmol/LTris, 1mol/LNaCl, pH8.0) wash-out 10 minutes, extends 10min.HD1R mass recovery is 72.8%, and purity reaches more than 91% (Fig. 1, lane 7; Fig. 5).
Embodiment 11: with the secondarily purified HD1R of desalination chromatographic column
The desalting column (HiTrap Desaltingcolumn) balanced by the moving phase of 20mmol/LPBS, pH6.0-7.0, by gained target fraction direct injection in embodiment ten, continues wash-out 30min, flow velocity 1.0ml/min.The purity of HD1R reaches more than 99%.(Fig. 1, lane8)
Claims (10)
1. the purifying of Notch part Delta-like1 fusion rotein and a method for renaturation, is characterized in that, comprise the following steps:
1) the inclusion body lavation buffer solution containing HD1R is repeatedly washed, centrifugal, be dissolved in strong denaturant damping fluid, obtain the HD1R albumen of roughing out;
2) chromatographic column will balanced to mobile phase A containing proinsulin human's sex change extract direct injection, use the Mobile phase B line isocratic elution of PBS, 1mol/LNaCl or Tris, 1mol/LNaCl again, collect target peak chromatographic fraction, obtain the chromatographic fraction of the HD1R after renaturation and purifying;
3) the target peak chromatographic fraction direct injection of will collect, the further desalination of chromatographic column balanced by the moving phase of 20mmol/LPBS, continue wash-out 30min, elution flow rate is 0.5-1.5ml/min, obtains renaturation and purifying more high purity and active chromatographic fraction.
2. a kind of purifying of Notch part Delta-like1 fusion rotein according to claim 1 and the method for renaturation, it is characterized in that, described chromatographic column adopts soluble high-expression post optional HiTrapCaptoQ or HiTrap Capto DEAE, be GE Healthcare company produce, specification is 0.7 × 2.5cm, 1mL, also can load DEAE filler voluntarily in the Column XK16 of GE Healthcare company production.
3. a kind of purifying of Notch part Delta-like1 fusion rotein according to claim 1 and the method for renaturation, it is characterized in that, in described mobile phase A, PBS concentration is 20-40mmol/L; In described mobile phase A, the concentration of Tris is 20-40mmol/L.
4. a kind of purifying of Notch part Delta-like1 fusion rotein according to claim 1 and the method for renaturation, it is characterized in that, in described mobile phase A, the pH of PBS is 7.5-8.0; In described mobile phase A, the pH of Tris is 7.5-8.0.
5. a kind of purifying of Notch part Delta-like1 fusion rotein according to claim 1 and the method for renaturation, it is characterized in that, in described Mobile phase B, PBS concentration is 20-40mmol/L; In described Mobile phase B, the concentration of Tris is 20-40mmol/L.
6. a kind of purifying of Notch part Delta-like1 fusion rotein according to claim 1 and the method for renaturation, it is characterized in that, in described Mobile phase B, the pH of PBS is 7.5-8.0; In described Mobile phase B, the pH of Tris is 7.5-8.0.
7. a kind of purifying of Notch part Delta-like1 fusion rotein according to claim 6 and the method for renaturation, it is characterized in that, in described Mobile phase B, the pH of PBS is 6.0-7.0.
8. a kind of purifying of Notch part Delta-like1 fusion rotein according to claim 1 and the method for renaturation, it is characterized in that, described elution flow rate is 0.5-1.5mL/min.
9. a kind of purifying of Notch part Delta-like1 fusion rotein according to claim 1 and the method for renaturation, is characterized in that, the mode of described lasting wash-out is after mobile phase A balance, and Mobile phase B isocratic elution 5-10min, extends 10min.
10. a kind of purifying of Notch part Delta-like1 fusion rotein according to claim 1 and the method for renaturation, it is characterized in that, the chromatographic column after described further desalination is HiTrap Desaltingcolumn.
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| CN108472201A (en) * | 2015-09-26 | 2018-08-31 | 财团法人卫生研究院 | Method for treating hair loss and constituent |
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| CN101891824A (en) * | 2010-07-08 | 2010-11-24 | 中国人民解放军第四军医大学 | Vascular targeting soluble fusion protein TrxHis-hDlll-RGD |
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