CN104292325A - Method and reagent for preparing soluble interleukin recombinant protein from inclusion body - Google Patents
Method and reagent for preparing soluble interleukin recombinant protein from inclusion body Download PDFInfo
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Abstract
The invention discloses a method and a reagent for preparing soluble interleukin recombinant protein from inclusion body. The reagent comprises a washing liquid, a thalli lysate, an inclusion body dissolving liquid, a protein renaturation liquid I and a protein renaturation liquid II. The employed inclusion body dissolving liquid is capable of substantially improving the yield of soluble interleukin recombinant protein from inclusion body, thereby solving the problem existed in a process of using an escherichia coli expression system to produce interleukin. Compared with the prior art, the reagent does not contain any protein denaturant, thereby facilitating subsequent protein renaturation. The method is simple in operation, is capable of improving the yield of soluble interleukin recombinant protein by 90% or more, has obvious advantage compared with a routine method which employs a protein denaturant and only has the yield of 5-20%, and has value of popularization and application.
Description
Technical field
The present invention relates to a kind of recombinant protein technology, particularly relate to a kind of from inclusion body, prepare soluble interleukin-6 recombinant protein method and reagent.
Background technology
Interleukin-, is called for short: interleukin, (English name: Interleukin is write a Chinese character in simplified form: IL) is the most critical protein molecule that human immune system regulates and controls also known as lymphocyte activating factor (LAF), has had been found that 35 interleukins at present.Interleukin-, at transmission of information, activates and immunity moderation cell, mediates T, B cell activation, reproduction restraint, inflammatory reaction and play very important regulating and controlling effect in tumour generating process.Interleukin-receives much concern in medical research, new drug development etc., its application is also more and more taken seriously, such as, interleukin II (IL-2) is used as antitumour drug, report in 746 tumour medicine development projects of the last year of 185 new drug development mechanisms in the world have 258 drug targets relevant to interleukin-2 bang path (IL-2Signaling Pathway in Oncology Drug Pipeline Update2011) according to Global InformationInc., visible interleukin-application prospect is very good.
Obtainable interleukin-is mainly by recombinant interleukin albumen that genetically engineered is produced in the market.Recombinant protein can be produced by coli expression system, also can be produced by insect viruses expression system or animal cell expression system.Coli expression system has the advantages such as expressing quantity is high, production cost is low, is also most widely used recombinant protein production system the most ripe in above-mentioned several expression system simultaneously.But research shows to there is a large problem when producing interleukin with coli expression system: most of interleukin recombinant protein exists with insoluble inclusion bodies in coli expression system.Under regular situation, albumen inclusion body can dissolve with high density denaturing agent (as 8M urea), then obtains soluble proteins through dialysis renaturation., the inclusion body of interleukin recombinant protein, namely use 8M urea also difficulty dissolve completely, obtain the harsher 6M Guanidinium hydrochloride of use and dissolve, but by the albumen of 6M guanidine hydrochloride dissolution, the more difficult and poor effect of renaturation.
Summary of the invention
Object of the present invention is just to provide a kind of from inclusion body, prepare soluble interleukin-6 recombinant protein method and reagent to solve the problem.
The present invention is achieved through the following technical solutions above-mentioned purpose:
A kind of reagent preparing soluble interleukin-6 recombinant protein from inclusion body of the present invention, comprise washings, cellular lysate liquid, solubilization of inclusion bodies liquid, protein renaturation liquid-I and protein renaturation liquid-II, described solubilization of inclusion bodies liquid is made up of the Tris-NaOH damping fluid of pH value 10-12, NaCl, Glycerol, EDTA, 2 mercapto ethanol and N-sarcosyl.
Particularly, described solubilization of inclusion bodies liquid is made up of the 10-300mM Tris-NaOH damping fluid of pH value 10-12,50-300mM NaCl, 5-30%Glycerol, 2-20mM EDTA, 1-30mM2-mercaptoethanol and 0.05-0.5%N-sarcosyl.
Further, described washings is made up of 50mM Tris-HCl pH8,50mM NaCL, 5%Glycerol, 5mM EDTA, 3mm2-mercaptoethanol and 0.5%Tween-20; Described cellular lysate liquid is made up of 50mM Tris-HCl pH8,50mM NaCL, 5mM EDTA, 3mm2-mercaptoethanol and 0.5%Tween-20; Described protein renaturation liquid-I is made up of 50mM Tris-NaOH pH11,50mM NaCL, 5%Glycerol, 6mM EDTA and 5mM2-mercaptoethanol; Described protein renaturation liquid-II is made up of 50mM Tris-HCl pH8,50mM NaCL, 5%Glycerol, 5mM EDTA, 3mM2-mercaptoethanol.
Improve as one, the 20mM2-mercaptoethanol in described solubilization of inclusion bodies liquid can adopt 5mM DTT to substitute; 0.5%N-sarcosyl in described solubilization of inclusion bodies liquid can adopt 0.05%SDS to substitute; The carbonate buffer solution of the pH value such as the Tris-NaOH damping fluid in described solubilization of inclusion bodies liquid can adopt substitutes or wherein a kind of in phosphate buffered saline buffer substitutes.
A kind of method preparing soluble interleukin-6 recombinant protein from inclusion body of the present invention, is characterized in that, comprise the following steps:
(1) collected by centrifugation is through the intestinal bacteria of abduction delivering interleukin;
(2) washings is added, mixing, resuspended thalline, centrifugal removing supernatant;
(3) add cellular lysate liquid, mixing, resuspended thalline, uses ultrasonic treatment thalline 1-10 second on ice, and interval is carried out 1-5 time (intermittently 1-10 second) at every turn, centrifugal removing supernatant;
(4) washings is added, mixing, resuspended inclusion bodies of protein (using ultrasonic is assisted resuspended), centrifugal removing supernatant (this washing step can repeat 2-3 time);
(5) add solubilization of inclusion bodies liquid, mixing, resuspended inclusion bodies of protein (using ultrasonic is assisted resuspended), collected by centrifugation supernatant, loads dialysis tubing by this supernatant;
(6) dialysis tubing is put into protein renaturation liquid-I to dialyse;
(7) further dialysis tubing is proceeded in protein renaturation liquid-II and continue dialysis;
(8) collect protein liquid in dialysis tubing, just obtain soluble interleukin-6 recombinant protein;
(9) refolded protein content and purity is detected.
Beneficial effect of the present invention is:
The present invention is a kind of method and reagent of preparing soluble interleukin-6 recombinant protein from inclusion body, and compared with prior art, the present invention containing any protein denaturant, is not conducive to follow-up protein renaturation like this; Simple to operate, can promote soluble interleukin-6 albumen yield and reach more than 90%, relatively conventional use protein denaturant method can only obtain 5-20% and have clear superiority, has the value applied.
Accompanying drawing explanation
Fig. 1 is each component S DS-PAGE electrophorogram after urea-denatured dose of process inclusion body;
Fig. 2 is each component S DS-PAGE electrophorogram after guanidine hydrochloride denaturation agent process inclusion body;
Fig. 3 is with each component S DS-PAGE electrophorogram before and after solubilization of inclusion bodies liquid process inclusion body of the present invention;
Fig. 4 is with each component S DS-PAGE electrophorogram before and after interleukin I L9 in the method for the invention and solubilization of inclusion bodies liquid extraction inclusion body.
Embodiment
Below in conjunction with accompanying drawing, the invention will be further described:
A kind of reagent preparing soluble interleukin-6 recombinant protein from inclusion body of the present invention, comprise washings, cellular lysate liquid, solubilization of inclusion bodies liquid, protein renaturation liquid-I and protein renaturation liquid-II, described solubilization of inclusion bodies liquid is by the Tris-NaOH damping fluid of pH value 10-12, 50-300mM NaCl, 5-30%Glycerol, 2-20mM EDTA, 1-30mM2-mercaptoethanol and 0.05-0.5%N-sarcosyl composition, described washings is by 50mM Tris-HCl pH8, 50mM NaCL, 5%Glycerol, 5mM EDTA, 3mm2-mercaptoethanol and 0.5%Tween-20 composition, described cellular lysate liquid is made up of 50mM Tris-HCl pH8,50mM NaCL, 5%Glycerol, 5mM EDTA, 3mm2-mercaptoethanol and 0.5%Tween-20, described protein renaturation liquid-I is made up of 50mM Tris-NaOH pH11,50mM NaCL, 5%Glycerol, 6mM EDTA and 5mM2-mercaptoethanol, described protein renaturation liquid-II is made up of 50mM Tris-HCl pH8,50mM NaCL, 5%Glycerol, 5mM EDTA, 3mM2-mercaptoethanol.
20mM2-mercaptoethanol in described solubilization of inclusion bodies liquid can adopt 5mM DTT to substitute; 0.5%N-sarcosyl in described solubilization of inclusion bodies liquid can adopt 0.05%SDS to substitute; Wherein a kind of in the carbonate buffer solution of the pH value such as the Tris-NaOH damping fluid in described solubilization of inclusion bodies liquid can adopt or phosphate buffered saline buffer substitutes.
A kind of method preparing soluble interleukin-6 recombinant protein from inclusion body of the present invention, is characterized in that, comprise the following steps:
(1) collected by centrifugation is through the intestinal bacteria of abduction delivering interleukin;
(2) washings is added, mixing, resuspended thalline, centrifugal removing supernatant;
(3) add cellular lysate liquid, mixing, resuspended thalline, uses ultrasonic treatment thalline 1-10 second on ice, and interval is carried out 1-5 time (intermittently 1-10 second) at every turn, centrifugal removing supernatant;
(4) washings is added, mixing, resuspended inclusion bodies of protein (using ultrasonic is assisted resuspended), centrifugal removing supernatant (this washing step can repeat 2-3 time);
(5) add solubilization of inclusion bodies liquid, mixing, resuspended inclusion bodies of protein (using ultrasonic is assisted resuspended), collected by centrifugation supernatant, loads dialysis tubing by this supernatant;
(6) dialysis tubing is put into protein renaturation liquid-I to dialyse;
(7) further dialysis tubing is proceeded in protein renaturation liquid-II and continue dialysis;
(8) collect protein liquid in dialysis tubing, just obtain soluble interleukin-6 recombinant protein;
(9) refolded protein content and purity is detected.
Embodiment one: compare interleukin I L10 recombinant protein inclusion body dissolving situation in different solutions:
A. without denaturing agent: 50mM Tris-HCl (pH8);
B. solubilization of inclusion bodies liquid of the present invention: 50mM Tris-NaOH (pH12);
C. urea-denatured dose: 50mM Tris-HCl (pH8), 8M-Urea;
D. guanidine hydrochloride denaturation agent: 50mM Tris-HCl (pH8), 6MGuanidine;
The centrifugal rear equivalent of the IL10 inclusion body of escherichia coli expression washing is divided into 4 parts, then adds above-mentioned solution respectively, fully mix, resuspended inclusion bodies of protein, by ultrasonic wave suspending 10 seconds, collected by centrifugation supernatant, measured the IL10 protein content dissolved in each supernatant liquor.Result is as following table:
Obviously, solubilization of inclusion bodies liquid of the present invention significantly can dissolve the IL10 recombinant protein in inclusion body.
Embodiment two: compare interleukin recombinant protein inclusion body and dissolve and renaturation situation in different denaturation agent:
The centrifugal rear equivalent of the IL10 inclusion body of escherichia coli expression washing is divided into 4 groups, then following A, the described lysate of B, C, D group is added respectively, abundant mixing, resuspended inclusion bodies of protein, with ultrasonic wave suspending 10 seconds, collected by centrifugation supernatant, then by each for gained supernatant dialysis removing denaturing agent, reclaim the solution in each dialysis tubing, collected by centrifugation supernatant (removing insoluble protein) detects the IL10 protein content dissolved in each supernatant liquor.Result is as following table:
A group:
Without denaturing agent lysate: 50mM Tris-HCl (pH8);
Dialyzate: 50mM Tris-HCl (pH8), 50mM NaCL, 5%Glycerol, 5mM EDTA, 3mM2-mercaptoethanol.
B group:
Solubilization of inclusion bodies liquid (modified version) of the present invention: 50mM Tris-NaOH (pH12), 50mM Tris-NaOH, 5mM EDTA, 5%Glycerol, 20mM2-mercaptoethanol, 50mM NaCl, 0.5%N-sarcosyl;
Dialyzate: by 2 subgradient dialysis method, first time dialysis protein renaturation liquid-I:50mM Tris-NaOH (pH11) of the present invention, 50mM NaCL, 5%Glycerol, 5mM EDTA and 5mM2-mercaptoethanol; Second time dialysis protein renaturation liquid-II:50mM Tris-HCl (pH8), 50mM NaCL, 5%Glycerol, 5mM EDTA, 3mM2-mercaptoethanol.
C group:
Urea lysate: 50mM Tris-HCl (pH8), 8M-Urea, 5mM EDTA, 5%Glycerol, 0.1mMDTT, 0.1M NaCl;
Dialyzate: 50mM Tris-HCl (pH8), 50mM NaCL, 5%Glycerol, 5mM EDTA, 3mM2-mercaptoethanol.
D group:
Guanidine hydrochloride dissolution liquid: 50mM Tris-HCl (pH8), 6M Guanidine, 5mM EDTA, 5%Glycerol, 0.1mM DTT, 0.1M NaCl;
Dialyzate: 50mM Tris-HCl (pH8), 50mM NaCL, 5%Glycerol, 5mM EDTA, 3mM2-mercaptoethanol.
Result is as shown above: the solubilization of inclusion bodies liquid using the present invention to tell and secondary dialysis method can significantly improve the yield obtaining soluble interleukin-6 recombinant protein from inclusion body, thus solution coli expression system produces the problem existing for interleukin.
As shown in Figure 1: SDS-PAGE electrophorogram will prove the result that upper table is concluded further: in figure, 1 for adding the precipitation before urea-denatured dose of 8M-, and 2 for adding the supernatant after urea-denatured dose of 8M-, and 3 for adding the precipitation after urea-denatured dose of 8M-.In figure, urea-denatured dose of 8M-almost can not make interleukin I L10 dissolve.SDS-denaturing polyacrylamide gel electrophoresis (SDS-PAGE) experiment proves, mainly be present in (in figure 1) in insoluble inclusion body through the interleukin I L10 of escherichia coli expression, also only can obtain a small amount of IL10 albumen (in figure 2) major part of dissolving still concentrate on (in figure 3) in undissolved precipitation with dissolving this inclusion body containing the denaturing agent of 8MUrea.
As shown in Figure 2: in figure, 1 for adding the supernatant after 6M-guanidine hydrochloride denaturation agent dissolving, add the supernatant of the supernatant after the agent of 6M-guanidine hydrochloride denaturation after renaturation removing Guanidinium hydrochloride, add the precipitation of the supernatant after the agent of 6M-guanidine hydrochloride denaturation after renaturation removing Guanidinium hydrochloride, the interleukin dissolved through the agent of 6M-guanidine hydrochloride denaturation is when removing after Guanidinium hydrochloride, and major part returns to again not dissolved state.And this metaprotein be there will be a large amount of albumen precipitations (in figure 3) after the agent of dialysis renaturation removing guanidine hydrochloride denaturation.
As shown in Figure 3: in figure, 1 is that IL10 expresses the thalline of bacterium, in figure, 2 be the thalline of contrast bacterium (without IL10 expression), and in figure, 3 add the supernatant (solubility) after solubilization of inclusion bodies liquid for IL10 inclusion body.
Embodiment three:
Interleukin I L9 recombinant protein is prepared with soluble interleukin-6 recombinant protein preparation reagent of the present invention.
The IL9 of A incubated overnight expresses bacterial classification, and press 1:10 dilution inoculation with new nutrient solution, 37 DEG C of 200rpm shaking tables cultivate 3 hours, add IPTG (final concentration 1mM) abduction delivering, continue to cultivate 4 hours at 37 DEG C of 200rpm shaking tables.
B receives bacterium: 3000g, and 4 DEG C centrifugal 10 minutes, supernatant discarded.
C adds washings, and fully suspend thalline, 3000g, and 4 DEG C centrifugal 10 minutes, removing supernatant;
D adds cellular lysate liquid, abundant resuspended thalline, uses ultrasonic treatment thalline 5 seconds on ice, interval 10 seconds, and so repeat ultrasonic treatment thalline 5 minutes, 8000g, 4 DEG C centrifugal 20 minutes, removing supernatant;
E adds washings, mix, resuspended inclusion bodies of protein with precipitation, uses Ultrasonographic suspending 5 seconds-interval 10 seconds, repeat 3 times, at 8000g, at 4 DEG C centrifugal 20 minutes, remove supernatant on ice; Repeat this washing step 2 times;
F adds solubilization of inclusion bodies liquid, fully mixes, resuspended inclusion bodies of protein, and with ultrasonic disruption 5 seconds, interval 10 seconds, repeated 3 times, at 8000g, at 4 DEG C centrifugal 20 minutes, collects supernatant, just must solubilization of inclusion bodies liquid;
This solubilization of inclusion bodies liquid loads in the dialysis tubing of Cut-off1K by G, this dialysis tubing is put into protein renaturation liquid-I, dialyses 12 hours;
H takes out dialysis tubing, is proceeded in protein renaturation liquid-II and continues dialysis 12 hours;
I takes out dialysis tubing, collects protein liquid in dialysis tubing, just soluble interleukin-6 IL9 recombinant protein, be stored in-70 DEG C for subsequent use;
J gets soluble interleukin-6 recombinant protein lysate 10-3 microlitre, refolded protein content and purity is detected with SDS-PAGE protein electrophoresis, (take a morsel respectively simultaneously and induce rear whole cell and do not induce bacterium whole cell, carry out SDS-PAGE electrophoresis in contrast together with soluble interleukin-6 recombinant protein with after the cracking of SDS-sample liquid).As shown in Figure 4, in figure, 1 is the cellular lysate liquid of abduction delivering interleukin I L9 to result, and in figure, 2 is the cellular lysate liquid of not inducing, and in figure, 3 is the lysate of interleukin I L9 after purifying,
The result of embodiment three proves to adopt method of the present invention and reagent, need not can prepare soluble interleukin-6 albumen efficiently by any protein denaturant from inclusion body, and its first purification degree is up to about 80%.
More than show and describe ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should understand; the present invention is not restricted to the described embodiments; what describe in above-described embodiment and specification sheets just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications, and these changes and improvements all fall in the claimed scope of the invention.Application claims protection domain is defined by appending claims and equivalent thereof.
Claims (7)
1. from inclusion body, prepare the reagent of soluble interleukin-6 recombinant protein for one kind, it is characterized in that: comprise washings, cellular lysate liquid, solubilization of inclusion bodies liquid, protein renaturation liquid-I and protein renaturation liquid-II, described solubilization of inclusion bodies liquid is made up of the Tris-NaOH damping fluid of pH value 10-12, NaCl, Glycerol, EDTA, 2 mercapto ethanol and N-sarcosyl.
2. a kind of reagent preparing soluble interleukin-6 recombinant protein from inclusion body according to claim 1, is characterized in that: described solubilization of inclusion bodies liquid is made up of the 10-300mM Tris-NaOH damping fluid of pH value 10-12,50-300mM NaCl, 5-30%Glycerol, 2-20mM EDTA, 1-30mM2-mercaptoethanol and 0.05-0.5%N-sarcosyl.
3. a kind of reagent preparing soluble interleukin-6 recombinant protein from inclusion body according to claim 1, is characterized in that: described washings is made up of 50mM Tris-HCl pH8,50mM NaCL, 5%Glycerol, 5mM EDTA, 3mm2-mercaptoethanol and 0.5%Tween-20; Described cellular lysate liquid is made up of 50mM Tris-HCl pH8,50mM NaCL, 5mM EDTA, 3mm2-mercaptoethanol and 0.5%Tween-20; Described protein renaturation liquid-I is made up of 50mM Tris-NaOH pH11,50mM NaCL, 5%Glycerol, 6mM EDTA and 5mM2-mercaptoethanol; Described protein renaturation liquid-II is made up of 50mM Tris-HCl pH8,50mM NaCL, 5%Glycerol, 5mM EDTA, 3mM2-mercaptoethanol.
4. a kind of reagent preparing soluble interleukin-6 recombinant protein from inclusion body according to claim 3, is characterized in that: the 20mM2-mercaptoethanol in described solubilization of inclusion bodies liquid can adopt 5mM DTT to substitute.
5. a kind of reagent preparing soluble interleukin-6 recombinant protein from inclusion body according to claim 3, is characterized in that: the 0.5%N-sarcosyl in described solubilization of inclusion bodies liquid can adopt 0.05%SDS to substitute.
6. a kind of reagent preparing soluble interleukin-6 recombinant protein from inclusion body according to claim 1 and 2, is characterized in that: can to adopt etc. in the carbonate buffer solution of pH value or phosphate buffered saline buffer wherein a kind of substitutes for the Tris-NaOH damping fluid in described solubilization of inclusion bodies liquid.
7. from inclusion body, prepare a method for soluble interleukin-6 recombinant protein, it is characterized in that, comprise the following steps:
(1) collected by centrifugation is through the intestinal bacteria of abduction delivering interleukin;
(2) washings is added, mixing, resuspended thalline, centrifugal removing supernatant;
(3) add cellular lysate liquid, mixing, resuspended thalline, uses ultrasonic treatment thalline 1-10 second on ice, and interval is carried out 1-5 time (intermittently 1-10 second) at every turn, centrifugal removing supernatant;
(4) washings is added, mixing, resuspended inclusion bodies of protein (using ultrasonic is assisted resuspended), centrifugal removing supernatant (this washing step can repeat 2-3 time);
(5) add solubilization of inclusion bodies liquid, mixing, resuspended inclusion bodies of protein (using ultrasonic is assisted resuspended), collected by centrifugation supernatant, loads dialysis tubing by this supernatant;
(6) dialysis tubing is put into protein renaturation liquid-I to dialyse;
(7) further dialysis tubing is proceeded in protein renaturation liquid-II and continue dialysis;
(8) collect protein liquid in dialysis tubing, just obtain soluble interleukin-6 recombinant protein;
(9) refolded protein content and purity is detected.
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| CN111676261A (en) * | 2020-06-24 | 2020-09-18 | 宁波博睿瀚达生物科技有限公司 | Preparation process of high-purity recombinant interleukin-2 |
| CN111777676A (en) * | 2020-06-24 | 2020-10-16 | 宁波博睿瀚达生物科技有限公司 | Method for dissociative adsorption and concentration of recombinant interleukin-2 renaturation solution |
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| CN112679599A (en) * | 2021-02-01 | 2021-04-20 | 大连理工大学 | Preparation method of recombinant human interleukin-6 |
| CN113136407A (en) * | 2021-05-26 | 2021-07-20 | 武汉华美生物工程有限公司 | Renaturation method of inclusion body and kit |
| CN118291598A (en) * | 2024-04-24 | 2024-07-05 | 恒敬合创生物医药（浙江）有限公司 | Method for high-throughput screening of mutant protein expressed in inclusion body form |
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| CN104292325B (en) | 2018-08-03 |
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