CN103665097A - Denatured protein renaturation device and renaturation method - Google Patents
Denatured protein renaturation device and renaturation method Download PDFInfo
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- CN103665097A CN103665097A CN201210328445.3A CN201210328445A CN103665097A CN 103665097 A CN103665097 A CN 103665097A CN 201210328445 A CN201210328445 A CN 201210328445A CN 103665097 A CN103665097 A CN 103665097A
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Abstract
The invention provides a denatured protein renaturation device and renaturation method. Particularly, the denatured protein renaturation device provided by the invention comprises a denatured protein renaturation kettle, an ultra-filtration module, a column renaturation module, a numerical control pump, an electromagnetic valve, an online detector and a processor, wherein the numerical control pump is used for driving liquid to flow; the electromagnetic valve is used for controlling the flow direction of the liquid; the online detector is used for detecting the state of the solution; the processor is connected with the numerical control pump, the electromagnetic valve and the online detector and used for controlling the numerical control pump, the electromagnetic valve and the online detector. By adopting the renaturation device provided by the invention, diluting renaturation, dialysis renaturation, column renaturation and the like and renaturation post-operations (including concentration, purification and the like) are integrated; the denatured protein renaturation device has the advantages of being automatic in operation, time-saving and labor-saving and the like, and is very applicable to development and improvement of a denatured protein renaturation technology.
Description
Technical field
The present invention designs biological technical field, relates to particularly a kind of full-automatic sex change protein renaturation device, and the purposes of this device.Also relate to described device recombinant protein inclusion body is being carried out to denature and renature, thereby obtain the application of activated target protein aspect.
Background technology
Intestinal bacteria are as the heterologous gene expression system being most widely used, and the Main Bottleneck problem of existence is that the foreign protein of high expression level mainly exists with the inclusion body form of non-activity, need to could obtain biological activity by renaturation.From inclusion body, obtain activated target protein, mainly through two steps: the one, with denaturing agent, dissolve inclusion body; The 2nd, remove denaturing agent and make target protein renaturation.
This area wishes that the gratifying metaprotein renaturation technology of exploitation should have following characteristics: the component yield after metaprotein renaturation with activated protein is high, after metaprotein renaturation, can obtain the proteinaceous product that concentration is higher, renaturation technology is easy to realize, easily realize automatization, renaturation process is consuming time less, quick etc.
With regard to the metaprotein refolding method of having developed at present, the refolding method being most widely used is dilution refolding (50%), dialysis renaturation (30%) and post renaturation (11%).In order to realize the best annealing efficiency of metaprotein, people have developed various renaturation technique, such as optimizing metaprotein renaturation solution component or redox system environment, adding corresponding protein renaturation molecular chaperones etc.Usually, these renaturation techniques are all based upon in the methods such as dilution refolding, dialysis renaturation or post renaturation.Yet this area still lacks gratifying, efficient, automatic metaprotein renaturation device up to now.Owing to being limited by the restriction of metaprotein renaturation device, various refolding methods can not be used in combination, and cause operating continuity not good, thereby have limited the extension of metaprotein renaturation technique.
In sum, efficient in the urgent need to developing, the automatic metaprotein renaturation device in this area, thereby renaturation convenient, that realize efficiently metaprotein.
Summary of the invention
The object of the invention is to overcome the defect of prior art, thereby it is strong to develop a kind of operation continuity, the novel metaprotein renaturation device that level of automation is higher.
A first aspect of the present invention, provide a kind of for making metaprotein renaturation form the device of activated target protein, it is characterized in that, this device comprises: metaprotein renaturation still, ultrafiltration module and post renaturation module, and connect the pipeline of described metaprotein renaturation still, ultrafiltration module and post renaturation module and be positioned at one or more pipelines for driving the pump of liquid-flow, wherein said metaprotein renaturation still optionally as dilution module;
And described device also comprises for controlling the electromagnetic valve of flow direction, and described device controls flow direction by described electromagnetic valve, thereby switches the different working modes of renaturation device.
Preferably, each renaturation module running separately in described device, Collaboration or jointly running, thus form the different working modes that carries out metaprotein renaturation, and the switching between described different working modes is that regulation and control by described valve realize.
In another preference, the operating mode of described renaturation device comprises following operating mode:
(a) ultrafiltration module running, and post renaturation module does not operate;
(b) ultrafiltration module does not operate, and the running of post renaturation module;
(c) ultrafiltration module does not operate, and post renaturation module does not operate; With
(d) ultrafiltration module running, and the running of post renaturation module.
In another preference, described renaturation device can, the in the situation that of the running of metaprotein renaturation still act or omission dilution module, switch between above-mentioned (a), (b), (c), (d) four kinds of operating mode.
In another preference, described electromagnetic valve comprises:
For controlling the five-way electromagnetic valve door V6 of dilution module, ultrafiltration module and the combination of post renaturation module, wherein said five-way electromagnetic valve door V6 is connecting numerical control pump, renaturation still, five-way electromagnetic valve door V7 and six three-way electromagnetic valve door V8;
For controlling the five-way electromagnetic valve door V7 of dilution module, ultrafiltration module and the combination of post renaturation module, wherein said five-way electromagnetic valve door V7 is connecting numerical control pump, five-way electromagnetic valve door V6 and chromatography column; With
For being controlled at six three-way electromagnetic valve door V8 of the switching state of thread detector, wherein said six three-way electromagnetic valve door V8 are connecting three-way solenoid valve door V3, three-way solenoid valve door V5 and five-way electromagnetic valve door V6.
In another preference, described five-way electromagnetic valve door V6 and V7 are a kind of adjacent two hole connections, medium pore and the five-way electromagnetic valve door being communicated with being left in two holes; And six described three-way electromagnetic valve door V8 are the six three-way electromagnetic valve doors that are communicated with between a kind of adjacent two holes.
In another preference, the metaprotein renaturation module in described device includes, but is not limited to:
As the metaprotein renaturation still of dilution module, wherein metaprotein carries out renaturation reaction in described renaturation still;
Ultrafiltration module, described ultrafiltration module is communicated with renaturation still and forms a liquid communication loop, and wherein said ultrafiltration module is for making metaprotein renaturation by dialysis renaturation and holding back the albumen of renaturation by ultrafiltration, and/or
Post renaturation module, described post renaturation module is communicated with formation one liquid communication loop with renaturation still, and wherein said post renaturation module is for making metaprotein renaturation by post renaturation; And described modules can be separately or jointly for metaprotein renaturation.
In another preference, metaprotein renaturation still comprises renaturation bottle and whipping appts, and renaturation bottle bottom arranges 3 import and export, and one of them import and export is connected with numerical control pump P2, and remaining two are connected with five-way electromagnetic valve door V6.
In another preference, described ultrafiltration module has opening for feed, holds back mouth and infiltration mouthful, and wherein opening for feed is connected with three-way solenoid valve door V2, holds back mouth and is connected with three-way solenoid valve door V3, and its infiltration mouth is connected with a waste liquid discharge line.
In another preference, the infiltration mouth place of described ultrafiltration module configuration under meter.
In another preference, described ultrafiltration module and/or post renaturation module are freely to dismantle.
In another preference, described post renaturation module is chromatography column; Preferably described chromatography column comprises: affinity column, ion exchange column (comprising negatively charged ion and anion-exchange column) exchange column, hydrophobic chromatography post and molecular sieve column.
In another preference, the opening for feed of described chromatography column is connected with five-way electromagnetic valve door V7, and chromatography column outlet is communicated with three-way solenoid valve door V5.
In another preference, described device also comprise for detection of solution state at thread detector.
In another preference, described comprises under meter, pH, specific conductivity, visible ray-UV-detector at thread detector.
In another preference, the described test section at thread detector (or probe) is positioned at passage, and the import of described passage and outlet be connected with the two-way of six three-way electromagnetic valve door V8, formation one independent loop.
In another preference, described electromagnetic valve also comprises:
The quaternary gradient electromagnetic valve V1 that flows into renaturation still for controlling renaturation solution, wherein said quaternary gradient electromagnetic valve V1 is connected with reservoir (as liquid storage bottle) and the renaturation still of renaturation solution or splendid attire renaturation solution;
The three-way solenoid valve door V2 that flows out albumen hyperfiltration membrane assembly for controlling renaturation still liquid, wherein said three-way solenoid valve door V2 is communicated with ultrafiltration module and renaturation still;
For controlling the three-way solenoid valve door V3 of the liquid flowing out from ultrafiltration module, wherein said the 3rd electromagnetic valve V3 is connected with ultrafiltration module and six three-way electromagnetic valve door V8;
The quaternary gradient electromagnetic valve V4 that flows into chromatography column for controlling solution, wherein said quaternary gradient electromagnetic valve V4 is connected with reservoir (as liquid storage bottle) and the five-way electromagnetic valve door V7 of renaturation solution or splendid attire renaturation solution;
For controlling the three-way solenoid valve door V5 of the liquid flowing out from post renaturation module, wherein said three-way solenoid valve door V5 is connected with post renaturation module and six three-way electromagnetic valve door V8.
In another preference, described quaternary gradient electromagnetic valve V1 is for controlling the quaternary gradient electromagnetic valve of four road flow directions and allocation proportion.
In another preference, described renaturation solution comprises (but being not limited to): elutriant, washing are with damping fluid etc.
In another preference, described three-way solenoid valve door V2 and V3 are pressure resistant type three-way solenoid valve, and the two-way of appointing that can control in three tunnels is connected;
Described quaternary gradient electromagnetic valve V4 is for controlling the quaternary gradient electromagnetic valve of four road flow directions and allocation proportion; And/or;
Described three-way solenoid valve door V5 is into two removing from mould three-way solenoid valves.
In another preference, described device also has the one or more features that are selected from lower group:
-described pump is numerical control pump;
-described device also comprises: with described numerical control pump, electromagnetic valve with thread detector be connected and for controlling described numerical control pump, electromagnetic valve and at the treater of thread detector;
-described device also comprises: with the liquid storage bottle that recombinant protein renaturation still is communicated with, wherein said liquid storage bottle is for splendid attire renaturation solution.
In another preference, described numerical control pump P1 numerical control pump P2 and/or numerical control pump P3 are peristaltic pump or ram pump.
In another preference, described treater comprises (but being not limited to): micro-chip, panel computer etc.
In another preference, described numerical control pump comprises:
For driving renaturation solution to flow into the first numerical control pump P1 of renaturation still;
Be used for the second numerical control pump P2 that drives renaturation still liquid to flow out renaturation still and flow to ultrafiltration module; With
For driving the 3rd numerical control pump P3 of flow direction post renaturation module.
In another preference, at three-way solenoid valve door V5 and five-way electromagnetic valve door V7, by a pipeline, be directly communicated with, thereby for washing shared pipeline, numerical control pump etc.
In another preference, liquid storage is communicated with quaternary gradient electromagnetic valve V1 or V4 opening for feed by corresponding pipeline;
In another preference, the container that described albumen collector and liquid waste collector comprise various routines, such as test tube, bottle, cup etc.
In another preference, described albumen collector is automatic collector.
In another preference, described liquid waste collector is waste liquid bottle.
In another preference, for making metaprotein renaturation, thereby form activated target protein.
A second aspect of the present invention, a kind of method that makes metaprotein renaturation form activated target protein is provided, it is characterized in that, comprise step: with the metaprotein renaturation device described in first aspect present invention, carry out protein renaturation, thereby form or obtain the activated target protein after renaturation.
In another preference, described step comprises: the metaprotein for the treatment of renaturation is entered to renaturation still or chromatography column; Then the renaturation device starting described in first aspect present invention carries out renaturation, thereby forms or obtain the activated target protein after renaturation.
In another preference, described renaturation is included in carries out renaturation under different working modes.Preferably, dilute each renaturation module running separately, Collaboration or the running jointly of module, ultrafiltration module and post renaturation module comprising in described device, thereby form the different working modes that carries out metaprotein renaturation.
In should be understood that within the scope of the present invention, above-mentioned each technical characterictic of the present invention and in below (eg embodiment) specifically described each technical characterictic can combine mutually, thereby form new or preferred technical scheme.As space is limited, at this, tire out and state no longer one by one.
Accompanying drawing explanation
Fig. 1 has shown the structure iron of the metaprotein renaturation device in example of the present invention.
Fig. 2 has shown the schematic diagram of the dialysis part in example of the present invention.
Fig. 3 A, 3B, 3C and 3D have shown respectively the four kinds of operating mode schematic diagram of the five-way electromagnetic valve door V6 in example of the present invention.
Fig. 4 A, 4B and 4C have shown respectively the three kinds of working state schematic representations of the five-way electromagnetic valve door V7 in example of the present invention.
Fig. 5 A and Fig. 5 B have shown respectively the two kinds of working state schematic representations of six three-way electromagnetic valve door V8 in example of the present invention.
Fig. 6 A has shown molecular sieve (Superdex-7510/300) analysis of SDF/CXCL12 in example of the present invention, solid line is the elution curve of the SDF/CXCL12 of 8mg/ml, dotted line is the elution curve of the SDF/CXCL12 of 1.2mg/ml, after showing renaturation, SDF/CXCL12 albumen has self-assembly characteristic, consistent with the characteristic of natural SDF/CXCL12.
Fig. 6 B has shown the circular dichroism spectrum analysis of SDF/CXCL12 in example of the present invention, after renaturation, the circular dichroism spectrum shape of SDF/CXCL12 and characteristic peak and natural SDF/CXCL12's is consistent, after showing renaturation, SDF/CXCL12 albumen has self-assembly characteristic, consistent with the characteristic of natural SDF/CXCL12.
Fig. 7 A has shown the schematic diagram with distilled water rinsing renaturation still timer in example of the present invention.
Fig. 7 B has shown the schematic diagram with distilled water rinsing renaturation still timer in example of the present invention.
Fig. 7 C has shown the schematic diagram with sex change liquid and renaturation solution rinsing renaturation still timer in example of the present invention.
Fig. 7 D has shown the schematic diagram with sex change liquid and renaturation solution rinsing renaturation still timer in example of the present invention.
Fig. 7 E has shown that the metaprotein in example of the present invention adds the schematic diagram of renaturation still timer.
Fig. 7 F has shown that the renaturation solution in example of the present invention adds the schematic diagram of renaturation still timer.
Fig. 7 G has shown the schematic diagram with balance liquid rinsing ultra-filtration membrane timer in example of the present invention.
Fig. 7 H has shown the schematic diagram with balance liquid dialysis renaturation albumen timer in example of the present invention.
Fig. 7 I has shown the schematic diagram with balance liquid and elutriant rinsing purification system timer in example of the present invention.
Fig. 7 J has shown the schematic diagram with balance liquid balance chromatography column timer in example of the present invention.
Fig. 7 K has shown the schematic diagram to chromatography column loading timer from renaturation still in example of the present invention.
Fig. 7 L has shown and residual sample in renaturation still and hyperfiltration membrane assembly has been pushed to the schematic diagram of chromatography column timer in example of the present invention.
Fig. 7 M has shown the schematic diagram of the sample timer in the elution chromatography post in example of the present invention.
Working state schematic representation when Fig. 7 N has shown the washing unit in example of the present invention.
Working state schematic representation when Fig. 7 O has shown the washing unit in example of the present invention.
Working state schematic representation when Fig. 7 P has shown the washing unit in example of the present invention.
Working state schematic representation when Fig. 7 Q has shown the washing unit in example of the present invention.
Working state schematic representation when Fig. 7 R has shown the washing unit in example of the present invention.
In accompanying drawing respectively to identify implication as follows:
A1, A2, A3, A4 and B1, B2, B3, B4 represent respectively the pipeline being connected with a certain liquid storage bottle, are mainly used in renaturation solution to introduce renaturation still or chromatography column.Certainly, these pipelines also can be introduced renaturation still or chromatography column by the metaprotein solution for the treatment of renaturation.
V1, V2, V3, V4, V5, V6, V7 and V8 represent respectively quaternary gradient electromagnetic valve V1, three-way solenoid valve door V2, three-way solenoid valve door V3, quaternary gradient electromagnetic valve V4, three-way solenoid valve door V5, five-way electromagnetic valve door V6, five-way electromagnetic valve door V7 and six three-way electromagnetic valve door V8.
P1, P2 and P3 represent respectively first and second and the 3rd numerical control pump.
Out[1], Out[2], Out[3] and Out[4] solution discharge line in indication device respectively.
L16, Lr6a, Lr6b, Lr2, L38, L47, L58, L57, L67, L68 philosophy represent pipeline, and especially, L57 represents not by the pipeline of chromatography column.
Embodiment
The inventor, through extensive and deep research, has developed a kind of full-automatic sex change protein renaturation device.This installs not only applicable to multiple refolding methods such as dilution refolding, dialysis renaturation and post renaturation, and can between different working modes, switch neatly.This device adds the modes such as multiple solution, coutroi velocity, control strength of solution rate of change or pattern, the multiple refolding method of combination by adopting stream, the practicality of this device is significantly provided, go for the renaturation of various different denaturation albumen, contribute to realize efficiently metaprotein renaturation, and for technician, develop new renaturation technique and provide a great convenience and design space.
Term
As used herein, term " renaturation " is exactly the renaturation process by appropriate design, thereby makes non-activity or active lower metaprotein form activated or active higher target protein.
As used herein, term " apparatus of the present invention " and " metaprotein renaturation device of the present invention " are used interchangeably.
Renaturation device
Apparatus of the present invention comprise: metaprotein renaturation still, ultrafiltration module and post renaturation module, and connect the pipeline of described metaprotein renaturation still, ultrafiltration module and post renaturation module and be positioned at one or more pipelines for driving the pump of liquid-flow; And described device also comprises for controlling the electromagnetic valve of flow direction, and described device controls flow direction by described electromagnetic valve, thereby switches the different working modes of renaturation device.In addition, apparatus of the present invention should comprise for detection of solution state at thread detector.
In the present invention, the parts such as renaturation still, ultrafiltration module, post renaturation module, pipeline, pump and detector, can adopt conventional or commercially available parts.
As shown in Figure 1, in device, a plurality of liquid storage bottle (not shown) are communicated with quaternary gradient electromagnetic valve V1 by A1, A2, A3, A4 pipeline respectively in the present invention; A plurality of liquid storage bottles are communicated with quaternary gradient electromagnetic valve V4 by B1, B2, B3 pipeline respectively.
Renaturation still comprises renaturation bottle (Reservoir) and whipping appts, and wherein the volume of renaturation bottle is not particularly limited, and generally can be 50～5000 milliliters, is preferably 50～1000 milliliters.
In the present invention, renaturation still can be used as dilution refolding module, also can be not as dilution refolding module (only as liquid storage bottle).
The material of renaturation bottle is not particularly limited, and generally can use glass, plastics or stainless steel.
One end of renaturation bottle is provided with lid (as screw cap), which is provided with 3 ducts.
Whipping appts comprises magnetic stirring apparatus (not marking) and stirrer, and wherein stirrer is positioned at renaturation bottle bottom.
Five-way electromagnetic valve door V6 and five-way electromagnetic valve door V7 are a kind of adjacent two hole connections, medium pore and the five-way electromagnetic valve door being communicated with being left in two holes.
Pipeline between quaternary gradient electromagnetic valve V1 and five-way electromagnetic valve door V6 is L16.
The pipeline of two ducts on renaturation still respectively and between five-way electromagnetic valve door V6 is Lr6a, Lr6b.
A remaining duct on renaturation still and the pipeline between three-way solenoid valve door V2 are Lr2.
Between three-way solenoid valve door V2 and three-way solenoid valve door V3, there is albumen hyperfiltration membrane assembly.
Pipeline between three-way solenoid valve door V3 and six three-way electromagnetic valve door V8 is L38.
As shown in Figure 2, the part of the dialysis in the present invention (or module) comprises three-way solenoid valve door V2, three-way solenoid valve door V3 and albumen hyperfiltration membrane assembly and under meter.Three-way solenoid valve door V2 and three-way solenoid valve door V3 are composed in series by two three-way solenoid valve doors (enters two removing from moulds).Three-way solenoid valve door (one enters two removing from moulds) has three holes 1,2 and 3, and wherein No. 1 hole is common aperture.Three-way solenoid valve door (one enters two removing from moulds) can only be communicated with No. 2 holes in No. 1 hole, or No. 1 hole is communicated with No. 3 holes.Two three-way solenoid valve doors (one enters two removing from moulds) are composed in series three-way solenoid valve door V2 or three-way solenoid valve door V3 by common aperture, thereby can realize in two three-way solenoid valve doors (enters two removing from moulds) being communicated with between No. 2 holes and No. 3 holes.
Under meter Flow meter detects outlet Out[2] flow velocity of solution.
The molecular weight cut-off of albumen hyperfiltration membrane assembly (ultrafiltration membrane) is generally 8～30kDa.
Six three-way electromagnetic valve door V8 are the six three-way electromagnetic valve doors that are communicated with between a kind of adjacent two holes.
Passage between five-way electromagnetic valve door V6 and six three-way electromagnetic valve door V8 is L68.
Pipeline between five-way electromagnetic valve door V6 and six three-way electromagnetic valve door V7 is L67.
It is upper that on-line filtration device (online filters) is positioned at pipeline L67, for removing the suspended particle of solution.
On-line filtration device is comprised of wide aperture filter membrane and small-bore filter membrane respectively, and its large pore filter membrane aperture is generally 100～10 μ m, and filter membrane aperture, small-bore is generally 10～1 μ m.
Pipeline between quaternary gradient electromagnetic valve V4 and six three-way electromagnetic valve door V7 is L47.
Between five-way electromagnetic valve door V7 and three-way solenoid valve door V5, there is pipeline L57 and chromatography column.
Pipeline between three-way solenoid valve door V5 and six three-way electromagnetic valve door V8 is L58.
Three-way solenoid valve door V5 only has a three-way solenoid valve door (enters two removing from moulds) to form.Between the common aperture of three-way solenoid valve door (enters two removing from moulds) and six three-way electromagnetic valve door V8, by pipeline L58, be connected.
In numerical control pump P1 control pipeline L16, solution stream is to the flow velocity of renaturation bottle, and flow rates is 0～50ml/min.
In numerical control pump P2 control pipeline Lr2, solution stream is to the flow velocity of albumen hyperfiltration membrane assembly, and flow rates is 0～200ml/min.
Numerical control pump P3 controls the flow velocity of solution in pipeline L47, and flow rates is 0～50ml/min.
Four kinds of working ordeies of five-way electromagnetic valve door V6
As shown in Figure 3, the five-way electromagnetic valve door V6 in the present invention is connecting numerical control pump P1, renaturation still, five-way electromagnetic valve door V7 and six three-way electromagnetic valve door V8.Five-way electromagnetic valve door V6 can switch four kinds of working ordeies, respectively A, B, C, the D in corresponding Fig. 3.
As shown in Figure 3A, five-way electromagnetic valve door V6 is communicated with numerical control pump P1 and renaturation still; Five-way electromagnetic valve door V6 is communicated with renaturation still and six three-way electromagnetic valve door V8 simultaneously.
As shown in Figure 3 B, five-way electromagnetic valve door V6 is communicated with numerical control pump P1 and five-way electromagnetic valve door V7.
As shown in Figure 3 C, five-way electromagnetic valve door V6 is communicated with numerical control pump P1 and six three-way electromagnetic valve door V8; Five-way electromagnetic valve door V6 is communicated with renaturation still and five-way electromagnetic valve door V7 simultaneously.
As shown in Figure 3 D, five-way electromagnetic valve door V6 is communicated with numerical control pump P1 and renaturation still; Five-way electromagnetic valve door V6 is communicated with five-way electromagnetic valve door V7 and six three-way electromagnetic valve door V8 simultaneously.
Three kinds of working ordeies of five-way electromagnetic valve door V7
As shown in Figure 4, the five-way electromagnetic valve door V7 in the present invention is connecting five-way electromagnetic valve door V6, numerical control pump P3, pipeline L57 and chromatography column.Five-way electromagnetic valve door V7 can switch three kinds of working ordeies, respectively A, B, the C in corresponding Fig. 4.
As shown in Figure 4 A, five-way electromagnetic valve door V7 is communicated with five-way electromagnetic valve door V6 and chromatography column, is communicated with numerical control pump P3 and pipeline L57 simultaneously.
As shown in Figure 4 B, five-way electromagnetic valve door V7 is communicated with five-way electromagnetic valve door V6 and pipeline L57.
As shown in Figure 4 C, five-way electromagnetic valve door V7 is communicated with numerical control pump P3 and chromatography column.
Two kinds of working ordeies of six three-way electromagnetic valve door V8
As shown in Figure 5, six three-way electromagnetic valve doors in the present invention connecting three-way solenoid valve door V5, at thread detector, five-way electromagnetic valve door V6 and outlet Out[4].Wherein, described detector comprises (but being not limited to): pH, specific conductivity and ultraviolet-visible(light)detector or device.Six three-way electromagnetic valve door V8 can switch two kinds of working ordeies, respectively A and the B in corresponding Fig. 5.
As shown in Figure 5A, on-line monitoring device connecting pipe L68 and pipeline L38, thereby make thread detector be positioned at dialysis module downstream.
As shown in Figure 5A, on-line monitoring device connecting pipe L58 and outlet Out[4], thereby make to be positioned at thread detector the downstream of chromatography column.
Metaprotein renaturation device of the present invention, the operating process under different operating state, algorithm or operating mode is as shown in Fig. 7 A～7R, comprising (but being not limited to): with distilled water rinsing renaturation still; With sex change liquid and renaturation solution rinsing renaturation still; Metaprotein is added to renaturation still; Renaturation solution is added to renaturation still; With balance liquid rinsing ultra-filtration membrane; With balance liquid dialysis renaturation albumen; With balance liquid and elutriant rinsing purification system; With balance liquid balance chromatography column; From renaturation still to chromatography column loading; Residual sample in renaturation still and hyperfiltration membrane assembly is pushed to chromatography column; Sample in elution chromatography post; Washing unit.Should be understood that above-mentioned different operating state, algorithm or operating mode can be by various different order, combination mutually, thus form different working ordeies or operating mode.
Major advantage of the present invention comprises:
(a) apparatus of the present invention are applicable to multiple conventional refolding method at present.
(b) integrated multiple refolding method and renaturation subsequent operations, in integrated, can be carried out renaturation neatly under different operating mode, therefore can be widely used in different types of protein renaturation.
(c) metaprotein renaturation technique has a significant improvement, and can save a large amount of manpowers, and reduces mistake.
(d) monitoring of metaprotein renaturation process multiparameter can realize automatization.
(e) the mode automatization chronologically of independent operation unit completes.
(f) contribute to exploitation for high efficiency method and the condition of different protein renaturations.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally according to normal condition, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Embodiment 1: the renaturation of recombinant human SDF/CXCL12 albumen
Adopt the device shown in Fig. 1 to carry out the renaturation of metaprotein, renaturation technique is as follows:
Solubilization of inclusion bodies liquid (50mM TrisHCl, 6M GdnHCl, 10mM β-mercaptoethanol, pH8.0) dissolve after inclusion body, lentamente by metaprotein solution dilution in renaturation solution (containing L-arginine), renaturation is after for some time, by dialysis, operates displacement damping fluid, remove L-arginine, final online enrichment and purifying target protein.
The renaturation process of metaprotein on this device can be divided into: preparatory stage, working stage and ending stage.Each concrete operations flow process can be referring to Fig. 7 A-7R.
In the preparatory stage, five-way electromagnetic valve door V6 is communicated with numerical control pump P1 and renaturation solution (as shown in Figure 3A).A1, A2, A3, A4 pipeline that ultrapure water cleans quaternary gradient electromagnetic valve V1 by numerical control pump P1 be to renaturation still, flow velocity 40ml/min, time 1min.Ultrapure water in renaturation still by numerical control pump P2 through outlet Out[1] discharge.A1, A2 pipeline that solubilization of inclusion bodies liquid and renaturation solution clean quaternary gradient electromagnetic valve V1 by numerical control pump P1 be to renaturation still, flow velocity 40ml/min, time 1min.Solution in renaturation still again by numerical control pump P2 through outlet Out[1] discharge.
At working stage, the metaprotein of 30ml (protein concentration is 1.0mg/ml) is introduced in renaturation still by numerical control pump P1, flow velocity 2ml/min.Then, the renaturation solution of 300ml is introduced in renaturation still lentamente by numerical control pump P1, and flow velocity is 0.23ml/min, and the time is about 21 hours.Five-way electromagnetic valve door V6 is communicated with numerical control pump P1 and six three-way electromagnetic valve door V8 (as shown in Figure 3 C).
A3, on-line monitoring device and albumen hyperfiltration membrane assembly that balance liquid cleans quaternary gradient electromagnetic valve V1 by numerical control pump P1 are to exporting Out[1] discharge flow velocity 10ml/min, time 5min.Five-way electromagnetic valve door V6 is communicated with numerical control pump P1 and renaturation solution, and renaturation still and six three-way electromagnetic valve door V8 are communicated with (as shown in Figure 3A) simultaneously.
Balance liquid is introduced in renaturation still continuously by numerical control pump P1, flow velocity 2ml/min.Meanwhile, the solution in renaturation still is introduced albumen hyperfiltration membrane assembly continuously by numerical control pump P2, flow velocity 50ml/min.Albumen filtered liquid is by outlet Out[2] to discharge, setting flow velocity is 2ml/min; Albumen trapped fluid is again by being back in renaturation still at thread detector.
Dialysis operation through 12 hours, five-way electromagnetic valve door is communicated with numerical control pump P1 and five-way electromagnetic valve door V7 (as Fig. 3 B), five-way electromagnetic valve door V7 is communicated with five-way electromagnetic valve door and pipeline L67 (as Fig. 4 B), rotates the downstream (as Fig. 5 B) that six three-way electromagnetic valve door V8 make to be positioned at thread detector chromatography column.
Balance liquid and elutriant are by numerical control pump P1 cleaning layer analysis system, flow velocity 20ml/min, time 1min.Then, again rotating five-way electromagnetic valve door V7 makes five-way electromagnetic valve door and chromatography column (CM-sepharose FF, 5ml) be communicated with (as shown in Figure 4 A).
Balance liquid cleans 5 column volumes of chromatography column, flow velocity 2ml/min by numerical control pump P1.Regulate five-way electromagnetic valve door V6 to be communicated with numerical control pump P1 and the connection of renaturation still, six three-way electromagnetic valve door V8 and five-way electromagnetic valve door V7 are communicated with (as Fig. 3 D) simultaneously.
Albumen in renaturation still is introduced chromatography column, flow velocity 5ml/min by numerical control pump P2.In albumen hyperfiltration membrane assemblies etc., residual protein solution, by numerical control pump P1 introducing portion balance liquid to renaturation still, and then pushes residual protein solution in chromatography column by numerical control pump P2.Regulate five-way electromagnetic valve door V6 to be communicated with numerical control pump P1 and five-way electromagnetic valve door V7 (as shown in Figure 3 B).
Balance liquid is by 5 column volumes of numerical control pump P1 rinsing chromatography column.Finally, balance liquid and elutriant are by quaternary gradient electromagnetic valve V1 with 0～100% linear gradient of 20 column volumes by protein from chromatography column wash-out out.
In the ending stage, ultrapure water cleans whole system.Five-way electromagnetic valve door V7 is communicated with five-way electromagnetic valve door V6 and pipeline L57 (as shown in Figure 4 B).Ultrapure water by numerical control pump P1 through A1, A2 cleaning layer analysis system, flow velocity 20ml/min, time 1min.Then five-way electromagnetic valve door V7 is communicated with five-way electromagnetic valve door V6 and chromatography column (as shown in Figure 3 B) again.
Ultrapure water cleans chromatography column by numerical control pump through A1, A2.Five-way electromagnetic valve door V6 is communicated with numerical control pump P1 and renaturation still (as shown in Fig. 3 A and 3D).
Ultrapure water cleans renaturation still, flow velocity 40ml/min, time 5min by numerical control pump P1 through A1, A2, A3, A4.Ultrapure water in renaturation still by numerical control pump P2 through outlet Out[1] discharge.Five-way electromagnetic valve door V6 is communicated with numerical control pump P1 and six three-way electromagnetic valve door V8 (as shown in Figure 3 C).
Ultrapure water cleans albumen hyperfiltration membrane assembly to exporting Out[1 by numerical control pump P1 through A1, A2, A3, A4] discharge flow velocity 40ml/min, time 5min.
Result:
In the present embodiment 1, dilute the enforcement that combines of module, dialysis module and post renaturation module.In the present embodiment, by micro computer, process, by program arrange, control and executable operations cell command (as the selection of pipeline cleaning, refolding method, pipeline connection etc.), therefore, operate mode automatization chronologically to complete, improved working efficiency.
As shown in chart 1, initial metaprotein quality 30mg, purity of protein approximately 60%; Refolded protein quality 8.7mg, purity of protein approximately 96%.Therefore, the preparation yield of recombinant human SDF/CXCL12 albumen is about 46%.
As shown in Figure 6, the qualitative analysis of SDF/CXCL12 albumen after renaturation.The molecular sieve (Superdex-7510/300) that is as shown in Figure 6A SDF/CXCL12 is analyzed, the elution curve of the SDF/CXCL12 that solid line is 8mg/ml, the elution curve of the SDF/CXCL12 that dotted line is 1.2mg/ml.After Fig. 6 A shows renaturation, SDF/CXCL12 albumen has self-assembly characteristic, consistent with the characteristic of natural SDF/CXCL12.Be the circular dichroism spectrum analysis of SDF/CXCL12 as shown in Figure 6B.After renaturation, the circular dichroism spectrum shape of SDF/CXCL12 and characteristic peak and natural SDF/CXCL12's is consistent.Therefore, bio-physical method is analyzed the characteristic that SDF/CXCL12 after renaturation has natural SDF/CXCL12.
Table 1. recombinant human SDF/CXCL12 protein purification table
A protein concentration is measured by Bradford method.
B purity of protein is measured by SDS-PAGE method.
* be illustrated in the step that instrument drilling has been done.
NA represents to measure.
The renaturation of embodiment 2 bovine serum albumins (BSA)
During the running of renaturation device, adopt gradient dialysis process to reduce gradually denaturing agent concentration and make target protein carry out renaturation.Concrete implementation step is as described below:
Preparatory stage is with described in embodiment 1.
At working stage, the metaprotein solution that is 0.1mg/ml by 110ml protein concentration imports in renaturation still with 10ml/min flow velocity by numerical control pump P1.Then, by 480ml renaturation solution (50mM TrisHCl, 1mM EDTA, 79mM urea, 2.2mM reductive glutathione, 1.1mM GSSG, pH8.5) by numerical control pump P1, with 0.2ml/min speed, import in renaturation still, meanwhile, in renaturation still, solution imports in albumen hyperfiltration membrane assembly with 10ml/min flow velocity by numerical control pump P2, the flow velocity of setting filtrate is 0.2ml/min, and trapped fluid is back in renaturation still by magnetic valve V8 and V6.In renaturation still, urea concentration is reduced to about 0.1mM to dialysis end.The on-line preconcentration that BSA is follow-up and purification step are with described in embodiment 1.
The ending stage is with described in embodiment 1.
In result: embodiment 2, initial metaprotein quality is 11mg, and the soluble protein quality that renaturation finishes rear acquisition is 8.8mg.By RP-HPLC, identify, the quality yield of correct unfolded protein is greater than 90%.
The renaturation of recombinant human SDF/CXCL12 albumen
SDF/CXCL12 protein mass 8.0mg after renaturation, purity of protein approximately 90%.
Above-described embodiment shows, apparatus of the present invention are applicable to multiple conventional refolding method at present, effectively multiple refolding method and renaturation subsequent operations are integrated in one, can under different operating mode, carry out neatly renaturation, therefore can be widely used in different types of protein renaturation, can save a large amount of manpowers, and reduce mistake.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document, are quoted as a reference separately.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. one kind for making metaprotein renaturation form the device of activated target protein, it is characterized in that, this device comprises: metaprotein renaturation still, ultrafiltration module and post renaturation module, and connect the pipeline of described metaprotein renaturation still, ultrafiltration module and post renaturation module and be positioned at one or more pipelines for driving the pump of liquid-flow, wherein said metaprotein renaturation still optionally as dilution module;
And described device also comprises for controlling the electromagnetic valve of flow direction, and described device controls flow direction by described electromagnetic valve, thereby switches the different working modes of renaturation device;
Preferably, each renaturation module running separately in described device, Collaboration or jointly running, thus form the different working modes that carries out metaprotein renaturation, and the switching between described different working modes is that regulation and control by described valve realize.
2. metaprotein renaturation device as claimed in claim 1, is characterized in that, the operating mode of described renaturation device comprises following operating mode:
(a) ultrafiltration module running, and post renaturation module does not operate;
(b) ultrafiltration module does not operate, and the running of post renaturation module;
(c) ultrafiltration module does not operate, and post renaturation module does not operate; With
(d) ultrafiltration module running, and the running of post renaturation module.
3. metaprotein renaturation device as claimed in claim 1 or 2, described electromagnetic valve comprises:
For controlling the five-way electromagnetic valve door V6 of dilution module, ultrafiltration module and the combination of post renaturation module, wherein said five-way electromagnetic valve door V6 is connecting numerical control pump, renaturation still, five-way electromagnetic valve door V7 and six three-way electromagnetic valve door V8;
For controlling the five-way electromagnetic valve door V7 of dilution module, ultrafiltration module and the combination of post renaturation module, wherein said five-way electromagnetic valve door V7 is connecting numerical control pump, five-way electromagnetic valve door V6 and chromatography column; With
For being controlled at six three-way electromagnetic valve door V8 of the switching state of thread detector, wherein said six three-way electromagnetic valve door V8 are connecting three-way solenoid valve door V3, three-way solenoid valve door V5 and five-way electromagnetic valve door V6.
4. metaprotein renaturation device as claimed in claim 3, is characterized in that, described five-way electromagnetic valve door V6 and V7 are a kind of adjacent two hole connections, medium pore and the five-way electromagnetic valve door being communicated with being left in two holes; And six described three-way electromagnetic valve door V8 are the six three-way electromagnetic valve doors that are communicated with between a kind of adjacent two holes.
5. metaprotein renaturation device as claimed in claim 1, is characterized in that, the metaprotein renaturation module in described device comprises:
As the metaprotein renaturation still of dilution module, wherein metaprotein carries out renaturation reaction in described renaturation still;
Ultrafiltration module, described ultrafiltration module is communicated with renaturation still and forms a liquid communication loop, and wherein said ultrafiltration module is for making metaprotein renaturation by dialysis renaturation and holding back the albumen of renaturation by ultrafiltration, and/or
Post renaturation module, described post renaturation module is communicated with formation one liquid communication loop with renaturation still, and wherein said post renaturation module is for making metaprotein renaturation by post renaturation; And described modules can be separately or jointly for metaprotein renaturation.
6. metaprotein renaturation device as claimed in claim 1, is characterized in that, described device also comprise for detection of solution state at thread detector.
7. metaprotein renaturation device as claimed in claim 1, is characterized in that, described electromagnetic valve also comprises:
The quaternary gradient electromagnetic valve V1 that flows into renaturation still for controlling renaturation solution, wherein said quaternary gradient electromagnetic valve V1 is connected with reservoir (as liquid storage bottle) and the renaturation still of renaturation solution or splendid attire renaturation solution;
The three-way solenoid valve door V2 that flows out albumen hyperfiltration membrane assembly for controlling renaturation still liquid, wherein said three-way solenoid valve door V2 is communicated with ultrafiltration module and renaturation still;
For controlling the three-way solenoid valve door V3 of the liquid flowing out from ultrafiltration module, wherein said the 3rd electromagnetic valve V3 is connected with ultrafiltration module and six three-way electromagnetic valve door V8;
The quaternary gradient electromagnetic valve V4 that flows into chromatography column for controlling solution, wherein said quaternary gradient electromagnetic valve V4 is connected with reservoir and the five-way electromagnetic valve door V7 of renaturation solution or splendid attire renaturation solution;
For controlling the three-way solenoid valve door V5 of the liquid flowing out from post renaturation module, wherein said three-way solenoid valve door V5 is connected with post renaturation module and six three-way electromagnetic valve door V8.
8. the metaprotein renaturation device as described in any one in claim 1～4, is characterized in that, described device also has the one or more features that are selected from lower group:
-described pump is numerical control pump;
-described device also comprises: with described numerical control pump, electromagnetic valve with thread detector be connected and for controlling described numerical control pump, electromagnetic valve and at the treater of thread detector;
-described device also comprises: with the liquid storage bottle that recombinant protein renaturation still is communicated with, wherein said liquid storage bottle is for splendid attire renaturation solution.
9. the purposes of metaprotein renaturation device as claimed in claim 1, is characterized in that, for making metaprotein renaturation, thereby forms activated target protein.
10. make metaprotein renaturation form a method for activated target protein, it is characterized in that, comprise step: with metaprotein renaturation device claimed in claim 1, carry out protein renaturation, thereby form or obtain the activated target protein after renaturation.
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| CN110922449A (en) * | 2018-09-20 | 2020-03-27 | 江苏健安生物科技有限公司 | Automatic insoluble recombinant protein resuscitation device |
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