CN105732820A - Renaturation method of restructured human insulin prokaryotic-fusion protein - Google Patents
Renaturation method of restructured human insulin prokaryotic-fusion protein Download PDFInfo
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Abstract
The invention relates to a renaturation method of restructured human insulin prokaryotic-fusion protein and belongs to the field of protein purification. The renaturation method includes steps of 1) smashing escherichia coli expressing restructured human insulin prokaryotic-fusion protein, and collecting inclusion bodies; 2) washing the inclusion bodies, dissolving the inclusion bodies and modifying the fusion protein; 3) renaturating protein. The renaturation method is needless of protein purification in advance, directly performs protein renaturation, and finally obtains high-concentration restructured insulin prokaryotic-fusion protein of natural structure. By the renaturation method, the defect of low protein concentration after protein renaturation and resultantly easy protein accumulation and precipitation is overcome, renaturation efficiency is up to 80-90%, production efficiency is improved, and the renaturation method is applicable to industrial production.
Description
Technical field
The invention belongs to a kind of protein renaturation method, the refolding method of a kind of recombinant insulinum primary fusion protein.
Background technology
Diabetes are the metabolism disorders being characterized with chronic hyperglycemia that Different types of etiopathogenises causes, and are owing to internal insulin is absolute
Or the syndrome that insulin insensitivity is caused by shortage or target tissue relatively.In recent years, along with people's living standard
Improving, the sickness rate of diabetes presents the trend risen year by year, becomes and is only second to the third-largest lethal of cardiovascular and tumor
Disease, the data announced according to IDF (IDF), global diabetics quantity (20 years old to 79 in 2014
Year) reach 3.87 hundred million people, added 5,000,000 people relatively last year, it is contemplated that be up to 5.92 hundred million people in 2035 years.Substantial amounts of
Clinical practice confirms, can obtain preferable effect by recombinant human insulin injection treatment diabetes.
Proinsulin gene, at many system such as escherichia coli, yeast, is obtained for expression in bacillus subtilis and streptococcus,
The expression system of current more employing is escherichia expression system.But when exogenous proteins efficient table in escherichia coli
Being commonly formed soluble when reaching, inactive aggregation, also known as inclusion body, inclusion body must make its shape by renaturation in vitro operation
The higher structure becoming correct just can obtain having the bioactive destination protein of expection.
Recombinant insulinum primary fusion protein refolding method mainly includes following several at present:
(1) dilution refolding: be directly added into water or buffer is diluted, stand overnight afterwards.Dilution method mainly has:
Once dilution, segmentation dilution and three kinds of modes of serial dilution；
(2) dialysis renaturation: do not increase volume, carrys out controlled denaturation agent removal by being gradually lowered extravasation transparent liquid concentration.
(3) on-column refolding: inclusion body after degeneration, renaturation on a column.
There is techniques below defect in existing protein renaturation method:
(1) dilution refolding: increase volume relatively big, denaturant dilution rate is too fast, wayward, and during renaturation, albumen contains
Measuring relatively low, general below 2mg/mL, renaturation yield is about 60%.
(2) dialysis renaturation: be not suitable for large-scale production, it is impossible to be applied to production scale, and be easily formed inactive protein matter
Specifically.
(3) on-column refolding: complex manufacturing, and increase production cost.
Therefore develop a kind of recombinant insulinum primary fusion protein refolding method being suitable to industrialized production, overcome existing work
The defect that after skill renaturation recombinant protein concentration is little, albumen is easily assembled, while simplifying production technology, improves protein renaturation rate,
Improve production efficiency, meet the demand of vast diabetics medication, not only there is important economic implications, and have
Important social meaning.
Summary of the invention
The refolding method of escherichia coli expression recombinant insulinum primary fusion protein, through in-depth study, is carried out by inventor
Improve.Through extensively experiment, improve dissolving, reduction, denaturing conditions, when finally overcoming renaturation protein concentration little,
The defect of the easy aggregate and precipitate of albumen, makes recombinant insulinum primary fusion protein carry out renaturation in higher concentrations, simplifies raw
While production. art, renaturation yield improves to 80%-90%, improves production efficiency, is suitable for industrialized production.
Therefore, the technical problem to be solved is to provide a kind of escherichia coli expression restructuring being suitable to industrialized production
The refolding method of human proinsulin fusion protein, during to overcome renaturation present in prior art, protein concentration is relatively low, produce
The technological deficiency that complex process, renaturation yield are relatively low.
The present invention provides the refolding method of a kind of recombinant insulinum primary fusion protein, comprises the following steps:
1) escherichia coli expressing recombinant insulinum primary fusion protein are crushed, collect inclusion body；
2) using inclusion body cleaning mixture to wash for inclusion body, described inclusion body cleaning mixture is containing final concentration
10-100mM Tris-HCl, 1-10mM EDTA, the solution of mass ratio 1-5%Triton-X100,1-5M carbamide, this bag
The pH containing body cleaning mixture is 8.5-9.5；
3) after removing cleaning mixture, adding denaturant in inclusion body, described inclusion body denaturant is containing final concentration 10-100mM
Tris-HCl, the EDTA of final concentration 1-10mM, the solution of carbamide of final concentration 5-10M, the pH of this inclusion body denaturant is
8-9.5；
4) adding reducing agent, described reducing agent is SH-group reductant, uses final concentration of 5-20mM, it is thus achieved that reduction albumen is molten
Liquid；
5) renaturation buffer prepared is slowly added to 4) in reduction protein solution in, gentle agitation, carry out recombined human
The renaturation of proinsulin fusion protein, each component final concentration of described renaturation buffer be respectively the carbamide of 0.50-1.50M,
The oxidized form of glutathione of 1-10mM, the reduced glutathion of 1-5mM, the EDTA of 1-5mM, final concentration 1-5mM β-
Mercaptoethanol, adjusts pH8-9.5, obtains the recombinant protein solution optimized.
It is an advantage of the current invention that the coli somatic expressing recombinant insulinum primary fusion protein is forgiven through broken collection
Body, wash, dissolve, the most purified, directly carry out refolding strategy and obtain solvable and that there is natural structure recombined human islets of langerhans
The former fusion protein of element.Recombinant insulinum primary fusion protein concentration after degeneration is reduced is 50～100mg/mL.
Present invention also offers a kind of refolding method, the described Mechanical Crushing method that is broken for, preferably ultrasonication, high pressure
Bacterial cell disruption or homogenizer crush.
Present invention also offers a kind of refolding method, described step 2) to be that one is configured to the denseest for described inclusion body cleaning mixture
Degree 50mM Tris-HCl, 2mM EDTA, the solution of mass ratio 1%Triton-X100,3M carbamide, its pH is adjusted to 9.0-9.5.
The washing of the refolding method of the present invention is preferably and repeatedly washs, and more preferably washing times is 2-5 time, most preferably 2 times.
A large amount of non-recombinant human proinsulin fusion protein can be removed in aforementioned manners easily.
Present invention also offers a kind of refolding method, step 3) described in inclusion body denaturant be containing final concentration 50mM's
Tris-HCl, the EDTA of final concentration 2mM, the solution of carbamide of final concentration of 8M, the pH of this solution is 9.0-9.5.With this
Inclusion body is carried out dissolving degenerative treatments by solution, is conducive to reducing agent renaturation subsequently to process.
Present invention also offers a kind of refolding method, wherein, step 4) described in SH-group reductant be dithiothreitol, DTT,
Use final concentration of 10mM.That is, the dithiothreitol, DTT of final concentration of 10mM is used to make recombinant insulinum primary fusion protein wrong
Join disulfide bond to open, it is possible to obtain reduction protein solution.
Present invention also offers a kind of refolding method, wherein, step 4) described in the protein concentration of reduction protein solution be
50-100mg/ml。
Present invention also offers a kind of refolding method, wherein preferred steps 5) in the final concentration of each component of renaturation buffer divide
Wei 0.80M carbamide, the oxidized form of glutathione of 1mM, the reduced glutathion of 1M, the EDTA of 2mM, final concentration 2mM
Beta-mercaptoethanol, adjusting its pH is 9.0-9.5.
In renaturation buffer, oxidized form of glutathione, reduced glutathion, the existence of beta-mercaptoethanol can accelerate weight
The exchange of group human proinsulin fusion protein intrachain disulfide bond, forms the recombinant insulinum primary fusion protein of correct structure；
It is unnecessary that EDTA is possible to prevent enzymatic activity that may be present to cause the correct recombinant insulinum primary fusion protein folded
Enzyme action, and then loss of activity；The existence of non-recombinant human proinsulin fusion protein can reduce recombinant insulinum primary and merge
The probability of mutually collision between albumen, reduces precipitation and produces.
The above recombinant insulinum primary fusion protein is former with natural human insulin identical or different, including complete people's pancreas
The A chain of island element and B chain, its N end adds one section of sequence, is that the Protocols in Molecular Biology that those skilled in the art commonly use designs
Fusion protein, constitutes the recombinant insulinum primary fusion protein of the present invention, when the existence of this section of sequence can increase renaturation, and weight
The solubility of group human proinsulin fusion protein so that it is be difficult to assemble producing precipitation.
The protein concentration of the recombinant protein solution finally optimized is 5-10mg/ml.
Protein renaturation method of the present invention, involved various reagent can be obtained by multiple commercial sources, and place
Reason method is simple.
The recombinant insulinum primary fusion protein refolding method of the present invention has the most useful technique effect: the present invention is directed to
A kind of recombinant insulinum primary fusion protein, it is provided that a kind of simple and effective refolding method, renaturation concentration is by prior art
0.3～2mg/mL raising to 5～10mg/mL, improve renaturation yield to 80%-90%, subsequent technique is through simple purification.
Present invention reagent employed in whole technique can be obtained by multiple commercial sources, and each step operation is simple, soon
Efficiently, all operations process is carried out under room temperature or 4 DEG C of environment speed, is substantially reduced production cost, is suitable for industrialization extensive
The production of recombinant human insulin.
Accompanying drawing explanation
Fig. 1: renaturation process flow chart；
Fig. 2: the recombinant insulinum primary fusion protein HPLC testing result after degeneration is reduced；
Fig. 3: solvable and there is the recombinant insulinum primary fusion protein HPLC testing result of natural structure after renaturation.
Detailed description of the invention
Preferred embodiment below is only in order to illustrate technical scheme and unrestricted, although passing through embodiments discussed below
The present invention is described in detail, it is to be understood by those skilled in the art that can be the most right
It makes various change, without departing from claims of the present invention limited range.
Embodiment 1
The preparation of recombinant insulinum primary fusion protein
Step 1: collect recombinant insulinum primary fusion protein inclusion body
Through Escherichia coli fermentation thalline, washing twice by purified water, fixed weight, every 10g thalline adds 100mL purified water, mixing,
Crush under the conditions of 900-950bar in homogenizer, after thalline is completely broken, be centrifuged 10 with centrifuge 12 000g
Minute, collect precipitation, the precipitation obtained is mainly inclusion body, washes twice by following method, removes major part non-recombinant
Human proinsulin fusion protein: every 10g precipitation uses 100mL lavation buffer solution, containing 50mM Tris-HCl (pH9.0), 2mM
EDTA, 1%Triton-X100 (mass ratio), 3M carbamide.Stirring and evenly mixing under room temperature, stand 30min, 12 000g from
The heart 10 minutes, such twice, collects precipitation.
Step 2: the dissolving of recombinant insulinum primary fusion protein, reduction
Inclusion body after washing, dissolves degeneration with following solution: the Tris-HCl of final concentration of 50mM, final concentration of 2mM
EDTA, the carbamide of final concentration of 8M, adjust pH9.0-9.5.After inclusion body is the most molten, the static 10min of room temperature, add the denseest
Degree is the DTT of 10mM, mixing, and room temperature stands 1 hour.
Step 3: the renaturation of recombinant insulinum primary fusion protein
By the renaturation buffer prepared, slowly and gentle agitation adds in the reducing solution of step 2, makes each concentration of component be: eventually
The carbamide of concentration 0.8M, the oxidized form of glutathione of final concentration 1mM, the reduced glutathion of final concentration 1mM, eventually the denseest
The degree EDTA of final concentration 2mM, the beta-mercaptoethanol of final concentration 2mM, recombinant insulinum primary fusion protein concentration are
5mg/mL, tune pH9.0-9.5.In 4 DEG C of placements, can be overnight.In observable renaturation solution, solution remains transparence,
Produce without precipitation.
Embodiment 2
The preparation of recombinant insulinum primary fusion protein
Recombinant insulinum primary fusion protein after degeneration is reduced, in renaturation buffer, makes protein concentration reach
7.5mg/mL, other each concentration of component are: the carbamide of final concentration 0.8M, the oxidized form of glutathione of final concentration 1mM, end
The reduced glutathion of concentration 1mM, the EDTA of final concentration 2mM, the beta-mercaptoethanol of final concentration 2mM, adjust
PH9.0-9.5, in 4 DEG C of placements.In observable renaturation solution, solution remains transparence, produces without precipitation.Other behaviour
Make with embodiment 1.Be computed recombinant insulinum primary fusion protein renaturation yield is 85.34%.
Embodiment 3
The preparation of recombinant insulinum primary fusion protein
Recombinant insulinum primary fusion protein after degeneration is reduced, in renaturation buffer, makes protein concentration reach
10mg/mL, other each concentration of component are: the carbamide of final concentration 0.8M, the oxidized form of glutathione of final concentration 5mM, eventually the denseest
The degree reduced glutathion of 1mM, the EDTA of final concentration 2mM, the beta-mercaptoethanol of final concentration 3mM, adjust pH9.0-9.5,
In 4 DEG C of placements.In observable renaturation solution, solution remains transparence, produces without precipitation.Other operations are with embodiment 1.
Be computed recombinant insulinum primary fusion protein renaturation yield is 82.59%.
Embodiment 4
The RP-HPLC detection of recombinant insulinum primary fusion protein
The recombinant insulinum primary fusion protein denaturing sample of embodiment 1 and renaturation sample, press Chinese Pharmacopoeia (2015 respectively
Version) method carry out RP-HPLC detection, see Fig. 2, Fig. 3.
Fig. 2 is restructuring human proinsulin fusion protein inclusion body RP-HPLC collection of illustrative plates of sample after degeneration is reduced, and it retains
Time, peak area was 7557461 near 24.25 minutes, and percentage ratio is 42.05%；
Fig. 3 is the RP-HPLC collection of illustrative plates of sample after restructuring human proinsulin fusion protein renaturation, and its retention time is 22.49
Near minute, peak area is 3109782, and percentage ratio is 44.23%
Embodiment 5
The recombinant insulinum primary fusion protein of the embodiment 1 also quantification of 29131g of raw sample total protein, in RP-HPLC
At 24.25 minutes, peak area percent is 42.05%；Recombinant insulinum primary fusion protein renaturation sample total protein is quantification of
In 24890g, RP-HPLC, at 22.49 minutes, peak area percent is 44.23%, be computed recombinant insulinum primary melts
Hop protein renaturation yield is: 24890 × 44.23%/29131 × 42.05%=89.87%.
Use conventional refolding method to carry out renaturation, after collecting inclusion body, use cleaning mixture (100mM Tris, 2M urea, matter
Amount ratio 1% TritonX) wash inclusion body twice after, then with 100mM Tris brine once, be dissolved in containing 20mM
In the 8M urea solution of DTT pH8.0, make recombinant insulinum primary fusion protein concentration in 20-40mg/mL, stirring and dissolving two
After hour centrifugal, supernatant add 10 times of volumes containing 3-5mM cystine, 0.3-0.6mM cysteine, pH10.5
Solution, recombinant insulinum primary fusion protein is 0.3～2mg/ml, carries out renaturation in 4 DEG C, and the renaturation time is little more than 48
Time, renaturation yield about 60%.
It can be seen that use the refolding method of the recombinant insulinum primary fusion protein of the present invention, renaturation yield is significantly higher than often
Rule method renaturation yield.
Claims (9)
1. the refolding method of a recombinant insulinum primary fusion protein, it is characterised in that comprise the following steps:
1) escherichia coli expressing recombinant insulinum primary fusion protein are crushed, collect inclusion body；
2) using inclusion body cleaning mixture to wash for inclusion body, described inclusion body cleaning mixture is containing final concentration
10-100mM Tris-HCl, 1-10mM EDTA, the solution of mass ratio 1-5%Triton-X100,1-5M carbamide, this bag
The pH containing body cleaning mixture is 8.5-9.5；
3) after removing cleaning mixture, adding denaturant in inclusion body, described inclusion body denaturant is containing final concentration
The Tris-HCl of 10-100mM, the EDTA of final concentration 1-10mM, the solution of carbamide of final concentration 5-10M, this inclusion body degeneration
The pH of agent is 8-9.5；
4) adding reducing agent, described reducing agent is SH-group reductant, uses final concentration of 5-20mM, it is thus achieved that reduction albumen
Solution；
5) be that the renaturation buffer prepared is slowly added to 4) in reduction protein solution in, gentle agitation, recombinate
The renaturation of human proinsulin fusion protein, each component final concentration of described renaturation buffer be respectively the carbamide of 0.50-1.50M,
The oxidized form of glutathione of 1-10mM, the reduced glutathion of 1-5mM, the EDTA of 1-5mM, final concentration 1-5mM β-
Mercaptoethanol, adjusts pH8-9.5, obtains the recombinant protein solution optimized.
2. refolding method as claimed in claim 1, it is characterised in that step 1) described in be broken for ultrasonication, height
Pressure bacterial cell disruption or homogenizer crush.
3. refolding method as claimed in claim 1, it is characterised in that step 2) described inclusion body cleaning mixture is for containing the denseest
Degree 50mM Tris-HCl, 2mM EDTA, the solution of mass ratio 1%Triton-X100,3M carbamide, this inclusion body washs
The pH of liquid is 9.0-9.5.
4. refolding method as claimed in claim 1, it is characterised in that step 2) described in washing for repeatedly to wash, preferably
For 2-5 washing.
5. refolding method as claimed in claim 1, it is characterised in that step 3) described in inclusion body denaturant for containing eventually
The Tris-HCl of concentration 50mM, the EDTA of final concentration 2mM, the solution of carbamide of final concentration of 8M, this inclusion body denaturant
PH is 9.0-9.5.
6. refolding method as claimed in claim 1, it is characterised in that step 4) described in SH-group reductant be two sulfur threoses
Alcohol, uses final concentration of 10mM.
7. refolding method as claimed in claim 1, it is characterised in that step 4) described in the albumen of reduction protein solution
Concentration is 50-100mg/ml.
8. refolding method as claimed in claim 1, it is characterised in that step 5) described in renaturation buffer each component eventually
Concentration is respectively 0.80M carbamide, the oxidized form of glutathione of 1mM, the reduced glutathion of 1M, the EDTA of 2mM, eventually the denseest
The beta-mercaptoethanol of degree 2mM, tune pH is 9.0-9.5.
9. refolding method as claimed in claim 1, it is characterised in that step 5) described in the recombinant protein solution of optimization
Protein concentration be 5-10mg/ml.
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| CN106636024A (en) * | 2016-09-30 | 2017-05-10 | 苏州埃德蒙生物技术有限公司 | Purified renaturation method of L-aspartic acid oxidase inclusion bodies |
| CN111172132A (en) * | 2020-02-20 | 2020-05-19 | 北京理工大学 | Preparation method and application of recombinant polyurethane plastic degrading enzyme |
| CN111574583A (en) * | 2020-04-10 | 2020-08-25 | 上海海路生物技术有限公司 | Protein renaturation reagent and its application |
| CN114075295A (en) * | 2020-08-19 | 2022-02-22 | 苏州鲲鹏生物技术有限公司 | Efficient renaturation liquid of Boc-human insulin fusion protein inclusion body and renaturation method thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106636024A (en) * | 2016-09-30 | 2017-05-10 | 苏州埃德蒙生物技术有限公司 | Purified renaturation method of L-aspartic acid oxidase inclusion bodies |
| CN111172132A (en) * | 2020-02-20 | 2020-05-19 | 北京理工大学 | Preparation method and application of recombinant polyurethane plastic degrading enzyme |
| CN111574583A (en) * | 2020-04-10 | 2020-08-25 | 上海海路生物技术有限公司 | Protein renaturation reagent and its application |
| CN114075295A (en) * | 2020-08-19 | 2022-02-22 | 苏州鲲鹏生物技术有限公司 | Efficient renaturation liquid of Boc-human insulin fusion protein inclusion body and renaturation method thereof |
| CN114075295B (en) * | 2020-08-19 | 2024-05-24 | 苏州鲲鹏生物技术有限公司 | Efficient renaturation solution of Boc-human insulin fusion protein inclusion body and renaturation method thereof |
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