CN103694324B - A kind of method that restructuring rTPA38 inclusion bodies of protein being applicable to industrialized production is consummate - Google Patents
A kind of method that restructuring rTPA38 inclusion bodies of protein being applicable to industrialized production is consummate Download PDFInfo
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Abstract
The invention belongs to biological engineering downstream protein technical field of purification, being specifically related to the method that restructuring rTPA38 inclusion bodies of protein is consummate, concrete is finally obtained consummate rTPA38 albumen by steps such as inclusion body cracking, rTPA38DEAE column purification, rTPA38 albumen dilution refolding, rTPA3830Q column purifications.This method is suitable for industrialized great production, purification renaturation integration, and batch can disposable purification 50g to 200g inclusion body.RTPA38 destination protein purity is high, up to more than 95%, then after turning salt, complies fully with use requirement.
Description
Technical field
The invention belongs to biological engineering downstream protein technical field of purification, be specifically related to the method that restructuring rTPA38 inclusion bodies of protein is consummate.
Background technology
Tuberculosis is to be infected the multi organ infection disease caused, predominantly pulmonary tuberculosis by mycobacterium tuberculosis complex.Wherein China newly sends out patient and is up to 1,500,000.The EPDML key character of tubercule bacillus is the latent infection of mycobacterium tuberculosis.Mycobacterium tuberculosis latent infection is a kind of sub-clinical state, and without radioscopy sign, antibacterial is resting state, and the mycobacterium tuberculosis latent infection crowd that there are about 10% can develop into tuberculosis patient in life.
Tubercule bacillus latent infection crowd there is no diagnosis goldstandard at present, the test of PPD skin test is the common method of diagnosing tubercle bacillus latent infection crowd, but this method has certain defective, because test PPD antigen used is that mycobacterium tuberculosis, nonpathogenic mycobacteria and bacillus calmette-guerin vaccine (i.e. BCG) are common, having cross reaction, specificity is poor.China is the country of universal bcg vaccination, and reaction phenomenologically very difficult differentiation PPD tested due to BCG (Bacille Calmette-Guerin) vaccination person and tubercule bacillus natural infection person, even if using above-mentioned PPD skin test test strong positive as criterion, the infected still suffering from a part of BCG (Bacille Calmette-Guerin) vaccination person and nonpathogenic mycobacteria is included in tubercule bacillus latent infection crowd, therefore causes PPD test practical function in tubercule bacillus latent infection crowd diagnoses extremely limited.A kind of preparation that new specificity is high, side effect is little need to be found replace.Restructuring Mycobacterium tuberculosis albumen 38K D a (r T P A38) it is a kind of lipoprotein and the major antigen of Mycobacterium tuberculosis, effect of its induction Cavia porcellus delayed hypersensitivity D T H is better than other single antigen.Higher for the Sensitivity and Specificity of tuberculosis serological detection.Therefore, r T P A38 albumen is the albumen at present with preferable application prospect.
Escherichia expression system because of genetic background understand, genetic manipulation is simple, expression is high, fermentation costs is low and is prone to the advantages such as commercial scale amplification and becomes medicinal restructuring TPA38KDa(recombinant TPA38KD, rTPA38KDa) preferable host system.Because of MTB and escherichia coli nearly source on evolving, so the TPA38KDa coded sequence of the simple transformation of natural or warp in MTB source is readily available high efficient expression under the control of strong promoter (such as T7 promoter), expression typically can reach more than 30%.With other at the destination protein of E. coli system height efficient expression as, the efficient expression product of TPA38KDa is also a kind of inclusion body protein inactive, water-insoluble, it is necessary to take suitable renaturation and purification process could obtain the product of pharmaceutical grade.But in the preliminary purification of inclusion body, yield is low in prior art, cost is high, it is difficult to be applied to large-scale production.
But in prior art, the response rate of renaturing inclusion bodies is about 15-25%, it occupies escherichia coli and produces most expenses of recombiant protein.Therefore, a bionic challenge the biggest is exactly by this albumen not having active and insoluble albumen to be more effectively converted into solvable and correct folding, if it is possible to find a kind of method of dissolving to make solubilization of inclusion bodies but do not make its secondary structure lose.This is possible not only to make protein renaturation success rate high, and can greatly reduce its expense producing activated protein.
Therefore, for deficiency of the prior art, need badly provide a kind of can industrialization be amplified, is easy to Quality Control, efficiency is high, save the consummate method of the restructuring inclusion body protein of the pharmaceutical grade of production cost.
Summary of the invention
It is an object of the invention to provide: a kind of method that rTPA38 inclusion bodies of protein of recombinating is consummate, collect a large amount of inclusion bodys that amplification obtains, cracking, cross column purification, renaturation, cross column purification step, finally give consummate after there is bioactive r T P A38 albumen.
Technical scheme: a kind of method that rTPA38 inclusion bodies of protein of recombinating is consummate, comprises the steps:
One, inclusion body cracking: take recombinant expressed rTPA38 inclusion body, by lysis buffer volume (mL): the consumption of the ratio-dependent rTPA38 inclusion body lysis buffer of inclusion body weight (g) about 100:1, dissolving is stirred at room temperature, substantially clarify to solution, exist without obvious bulky grain precipitation, lysate is placed in 4 DEG C of refrigerator overnight.Filter: upper prop filters to 20L PP bucket with the filter of 0.45 μm before purification.
Two, rTPA38DEAE column purification: chromatographic column: for Pyrex material, specification is GAG200/550(internal diameter 200mm, post height 550mm), stigma mesh size standard 10 μm, filler is DEAESepharose, post bed height about 16-17cm, bed volume about 5-6L.(1) chromatographic column balance: with rTPA38DEAE column chromatography buffer (8M carbamide, pH8.020mM Tris-HCl) the balance chromatographic column of about 4 bed volumes, equilibrium velocity about 500mL/min.(2) loading: the rTPA38 lysate after filtering pumps into chromatographic column, flow velocity about 500mL/min.(3) wash flat: after end of the sample, wash flat chromatographic column with rTPA38DEAE column chromatography buffer (8M carbamide, pH8.020mM Tris-HCl), balance 3-5 column volume.(4) eluting 1: with rTPA38DEAE column chromatography buffer (8M carbamide, pH8.020mM Tris-HCl, 20mM NaCl) eluting, elution flow rate about 500mL/min for the first time, about rinsing 12-15L, the protein peak eluted is not collected.(5) eluting 2: with rTPA38DEAE column chromatography buffer (8M carbamide, pH8.020mM Tris-HCl, 60mM NaCl) eluting, elution flow rate about 500mL/min for the second time.(6) collect: the main peak (the DEAE column chromatography liquid of the rTPA38 containing destination protein) that collection eluting 2 occurs is in 10L PP bucket, and chromatographic column online treatment, first step eluting completes.
Three, rTPA38 albumen dilution refolding: (1) adds rTPA38DEAE column chromatography liquid: press dilution refolding buffer (1.8M carbamide in 150L dilution refolding tank, 0.5mM GSS, 5mM GSH, 10% glycerol, pH8.020mM Tris-HCl) with the volume ratio 49:1 of the DEAE column chromatography liquid of rTPA38 containing destination protein, be initially charged the DEAE column chromatography liquid of rTPA38 containing destination protein, and open the chilled water in 150L dilution refolding tank chuck and carry out being cooled to keep less than 12 DEG C.(2) dropping dilution refolding buffer (1.8M carbamide, 0.5mM GSS, 5mM GSH, 10% glycerol, pH8.020mM Tris-HCl): with silica gel tube, the inlet of 150L buffer storage tank liquid outlet with 150L dilution refolding tank will be coupled together, centre peristaltic pump clamps silica gel tube, provides power for the dropping of dilution refolding buffer.First the dilution refolding buffer in 150L buffer storage tank is cooled to less than 15 DEG C, starts dropping, dilution refolding buffer (1.8M carbamide, 0.5mM GSS, 5mM GSH, 10% glycerol, pH8.020mM Tris-HCl) it is added dropwise in 150L dilution refolding tank with the flow velocity of 300ml/min, and simultaneously open 150L dilution refolding tank stirring, be stirred with 25HZ, from dilution refolding buffer dropping start calculate, dilution refolding about 16 hours, dilution refolding completes.
Four, rTPA3830Q column purification: chromatographic column: Pyrex material, specification is GAG200/550(internal diameter 200mm, post height 550mm), stigma mesh size standard 10 μm, filler is Source30Q, post bed height about 160-200mm, bed volume about 5-6L.(1) 30Q column equilibration: with rTPA3830Q column chromatography buffer (10% glycerol, 2M carbamide, pH8.020mM Tris-HCl) the balance chromatographic column of about 2-3 bed volume, equilibrium velocity about 500mL/min.(2) loading: the rTPA38 dilution refolding liquid pump after filtering enters chromatographic column, flow velocity about 600mL/min.(3) wash flat: after end of the sample, wash flat chromatographic column with the rTPA3830Q column chromatography buffer (10% glycerol, 2M carbamide, pH8.020mM Tris-HCl) of about 3-5 bed volume.(4) linear gradient elution: by rTPA3830Q column chromatography buffer (10% glycerol, 2M carbamide, pH8.020mM Tris-HCl) the A pump of Access Layer analysis system, rTPA3830Q column chromatography buffer (200mM NaCl, 10% glycerol, 2M carbamide, pH8.020mM Tris-HCl) the B pump of Access Layer analysis system, set type of elution as the B pump washing 100% from the A pump of 100%, elution flow rate about 500mL/min, elution time is 120min, and total elution volume is about 10 column volumes；(5) collect: during eluting, when peak for the purpose of second main peak occurs, start when its 280 light absorption value is more than 254 light absorption value to collect (the rTPA3830Q column chromatography liquid containing target protein) in 10L PP bucket, chromatographic column online treatment, 30Q column purification completes.
The effect rTPA38DEAE column purification rTPA38 destination protein that the present invention reaches: purity (electrophoresis) is more than more than 75%, final rTPA38 destination protein: purity (electrophoresis) is more than more than 95%, protein content is more than 0.3mg/ml.
The advantage that this rTPA38 is consummate: this method is suitable for industrialized great production, purification renaturation integration, batch can disposable purification 50g to 200g inclusion body.RTPA38 destination protein purity is high, up to more than 95%, then after turning salt, complies fully with use requirement.
Accompanying drawing explanation
Fig. 1: rTPA38 destination protein electrophoretogram (1 swimming lane: DEAE post penetrates peak；2 swimming lanes: inclusion body lysate；3 swimming lanes: the destination protein of DEAE column purification；The destination protein of swimming lane 4:30Q column purification)
Fig. 2: rTPA38 consummate method process chart
Detailed description of the invention
Embodiment 1
One, inclusion body cracking: take recombinant expressed rTPA38 inclusion body, by lysis buffer volume (mL): the consumption of the ratio-dependent rTPA38 inclusion body lysis buffer of inclusion body weight (g) about 100:1, dissolving is stirred at room temperature, substantially clarify to solution, exist without obvious bulky grain precipitation, lysate is placed in 4 DEG C of refrigerator overnight.Filter: upper prop filters to 20L PP bucket with the filter of 0.45 μm before purification.
Two, rTPA38DEAE column purification: chromatographic column: for Pyrex material, specification is GAG200/550(internal diameter 200mm, post height 550mm), stigma mesh size standard 10 μm, filler is DEAESepharose, post bed height about 16-17cm, bed volume about 5-6L.(1) chromatographic column balance: with rTPA38DEAE column chromatography buffer (8M carbamide, pH8.020mM Tris-HCl) the balance chromatographic column of about 4 bed volumes, equilibrium velocity about 500mL/min.(2) loading: the rTPA38 lysate after filtering pumps into chromatographic column, flow velocity about 500mL/min.(3) wash flat: after end of the sample, wash flat chromatographic column with rTPA38DEAE column chromatography buffer (8M carbamide, pH8.020mM Tris-HCl), balance 3-5 column volume.(4) eluting 1: with rTPA38DEAE column chromatography buffer (8M carbamide, pH8.020mM Tris-HCl, 20mM NaCl) eluting, elution flow rate about 500mL/min for the first time, about rinsing 12-15L, the protein peak eluted is not collected.(5) eluting 2: with rTPA38DEAE column chromatography buffer (8M carbamide, pH8.020mM Tris-HCl, 60mM NaCl) eluting, elution flow rate about 500mL/min for the second time.(6) collect: the main peak (the DEAE column chromatography liquid of the rTPA38 containing destination protein) that collection eluting 2 occurs is in 10L PP bucket, and chromatographic column online treatment, first step eluting completes.
Three, rTPA38 albumen dilution refolding: (1) adds rTPA38DEAE column chromatography liquid: press dilution refolding buffer (1.8M carbamide in 150L dilution refolding tank, 0.5mM GSS, 5mM GSH, 10% glycerol, pH8.020mM Tris-HCl) with the volume ratio 49:1 of the DEAE column chromatography liquid of rTPA38 containing destination protein, be initially charged the DEAE column chromatography liquid of rTPA38 containing destination protein, and open the chilled water in 150L dilution refolding tank chuck and carry out being cooled to keep less than 12 DEG C.(2) dropping dilution refolding buffer (1.8M carbamide, 0.5mM GSS, 5mMGSH, 10% glycerol, pH8.020mM Tris-HCl): with silica gel tube, the inlet of 150L buffer storage tank liquid outlet with 150L dilution refolding tank will be coupled together, centre peristaltic pump clamps silica gel tube, provides power for the dropping of dilution refolding buffer.First the dilution refolding buffer in 150L buffer storage tank is cooled to less than 15 DEG C, starts dropping, dilution refolding buffer (1.8M carbamide, 0.5mM GSS, 5mM GSH, 10% glycerol, pH8.020mM Tris-HCl) it is added dropwise in 150L dilution refolding tank with the flow velocity of 300ml/min, and simultaneously open 150L dilution refolding tank stirring, be stirred with 25HZ, from dilution refolding buffer dropping start calculate, dilution refolding about 16 hours, dilution refolding completes.
Four, rTPA3830Q column purification: chromatographic column: Pyrex material, specification is GAG200/550(internal diameter 200mm, post height 550mm), stigma mesh size standard 10 μm, filler is Source30Q, post bed height about 160-200mm, bed volume about 5-6L.(1) 30Q column equilibration: with rTPA3830Q column chromatography buffer (10% glycerol, 2M carbamide, pH8.020mM Tris-HCl) the balance chromatographic column of about 2-3 bed volume, equilibrium velocity about 500mL/min.(2) loading: the rTPA38 dilution refolding liquid pump after filtering enters chromatographic column, flow velocity about 600mL/min.(3) wash flat: after end of the sample, wash flat chromatographic column with the rTPA3830Q column chromatography buffer (10% glycerol, 2M carbamide, pH8.020mM Tris-HCl) of about 3-5 bed volume.(4) linear gradient elution: by rTPA3830Q column chromatography buffer (10% glycerol, 2M carbamide, pH8.020mM Tris-HCl) the A pump of Access Layer analysis system, rTPA3830Q column chromatography buffer (200mM NaCl, 10% glycerol, 2M carbamide, pH8.020mM Tris-HCl) the B pump of Access Layer analysis system, set type of elution as the B pump washing 100% from the A pump of 100%, elution flow rate about 500mL/min, elution time is 120min, and total elution volume is about 10 column volumes；(5) collect: during eluting, when peak for the purpose of second main peak occurs, start when its 280 light absorption value is more than 254 light absorption value to collect (the rTPA3830Q column chromatography liquid containing target protein) in 10L PP bucket, chromatographic column online treatment, 30Q column purification completes.From description Fig. 1 swimming lane 4 it can be seen that destination protein purity is more than more than 95%, protein concentration is more than 0.3mg/mL.
The method of the protein renaturation that embodiment 2 is common
Method according to embodiment 1, it is thus achieved that inclusion body solution, loads inclusion body solution in bag filter and carries out renaturation, denaturing conditions change should be the most inviolent, in order to avoid forming cotton-shaped albumen precipitation, generally changing a renaturation solution every 6h, renaturation solution formula is: 2mmol/L reduced glutathion；0.2mmol/L oxidized form of glutathione, renaturation 48h, the protein solution after collection renaturation is after ultraviolet absorption method measures concentration, and-20 DEG C frozen.
Embodiment 3 Activity determination
(1) recombinant protein obtained by the method for embodiment 1 and 2 respectively carries out SDS-PAGE electrophoresis, then enter electrophoretic blotting and albumen is transferred to nitrocellulose filter, closing with defatted milk powder, reacting with tuberculosis patient serum, HRP labelling goat anti-human igg respectively, nitrite ion develops the color.The albumen that result display uses the method for embodiment 1 to obtain can be combined with tuberculosis patient serological specificity, and uses the method for embodiment 2 to obtain albumen, and its intensity being combined with tuberculosis patient serological specificity only has the former 1/2nd.Its activity of albumen that this explanation embodiment 1 method obtains is higher than the albumen that embodiment 2 obtains.
(2) generation of rTPA38 antibody is seen after rTPA38 immune mouse
The protein immunization mice obtained by embodiment 1 and embodiment 2 respectively, observes the generation of anti-rTPA38 antibody.ElISA detection immune mouse antibody is that dilution factor result shows, the albumen of embodiment 1 has preferable immunogenicity, and mice can be stimulated to produce higher anti-rTPA antibody.Data are as follows: the different dilution factor immune mouse of table 1ELISA detection sees rTPA38 antibody horizontal
Claims (1)
1. being applicable to the method that the restructuring rTPA38 inclusion bodies of protein of industrialized production is consummate, it comprises the following steps:
Inclusion body cracks: takes recombinant expressed rTPA38 inclusion body, is that the ratio of 100:1 is true in lysis buffer volume mL: inclusion body weight g
Determine the consumption of rTPA38 inclusion body lysis buffer, dissolving is stirred at room temperature, clarify to solution, exist without obvious bulky grain precipitation, use 0.45 μ
The filter of m filters to 20L PP bucket；
RTPA38 DEAE column purification: (1) chromatographic column balances: balance chromatographic column with the rTPA38 DEAE column chromatography buffer of 4 bed volumes is flat
Weighing apparatus flow velocity 500mL/min, column chromatography buffer formulation is 8M carbamide, pH8.0 20mM Tris-HCl buffer；(2) loading: after filtering
RTPA38 lysate pump into chromatographic column, flow velocity 500mL/min；(3) wash flat: after end of the sample, with rTPA38 DEAE column chromatography buffer,
Wash flat chromatographic column, balance 3-5 column volume；(4) eluting 1: carry out with the rTPA38 DEAE column chromatography buffer adding 20mM NaCl
Eluting for the first time, elution flow rate 500mL/min, rush 12-15L, the protein peak eluted is not collected；(5) eluting 2: with adding 60mM NaCl
RTPA38 DEAE column chromatography buffer, eluting for the second time, elution flow rate 500mL/min；(6) collect: collect the main peak that eluting 2 occurs,
The DEAE column chromatography liquid of the rTPA38 containing destination protein is in 10L PP bucket, and chromatographic column online treatment, first step eluting completes；
RTPA38 albumen dilution refolding: (1) add rTPA38DEAE column chromatography liquid: in 150L dilution refolding tank by dilution refolding buffer with contain
The volume ratio 49:1 of the DEAE column chromatography liquid of the rTPA38 of destination protein, is initially charged the DEAE column chromatography of rTPA38 containing destination protein
Liquid, and open the chilled water in 150L dilution refolding tank chuck and carry out being cooled to keep less than 12 DEG C；(2) dropping dilution refolding buffer, will use
Silica gel tube couples together the inlet of 150L buffer storage tank liquid outlet with 150L dilution refolding tank, and centre peristaltic pump clamps silica gel tube,
Power is provided for the dropping of dilution refolding buffer；First the dilution refolding buffer in 150L buffer storage tank is cooled to less than 15 DEG C, starts to drip
Adding, dilution refolding buffer is added dropwise in 150L dilution refolding tank with the flow velocity of 300ml/min, and opens stirring of 150L dilution refolding tank simultaneously
Mixing, be stirred with 25HZ, start to calculate from the dropping of dilution refolding buffer, dilution refolding 16 hours, dilution refolding completes；
RTPA3830Q column purification: (1) 30Q column equilibration: with the rTPA3830Q column chromatography buffer of 2-3 bed volume, balance chromatographic column is flat
Weighing apparatus flow velocity 500mL/min；RTPA38 30Q column chromatography buffer formulation is on 10% glycerol, 2M carbamide, pH8.0 20mM Tris-HCl (2)
Sample: the rTPA38 dilution refolding liquid pump after filtering enters chromatographic column, flow velocity 600mL/min；(3) wash flat: after end of the sample, with 3-5 post
Flat chromatographic column washed by the rTPA38 30Q column chromatography buffer of bed volume；(4) linear gradient elution: by rTPA38 30Q column chromatography buffer Access Layer
The A pump of analysis system, with formula be 200mM NaCl, the rTPA38 30Q post of 10% glycerol, 2M carbamide, pH8.0 20mM Tris-HCl
Chromatography buffer, the B pump of Access Layer analysis system, set type of elution as the B pump washing 100% from the A pump of 100%, elution flow rate
500mL/min, elution time is 120min, and total elution volume is 10 column volumes；(5) collect: during eluting, when occurring that second main peak is
Purpose peak, starts to be collected in 10L PP bucket when its 280 light absorption value is more than 254 light absorption value, and chromatographic column online treatment, 30Q column purification is complete
Become；Wherein the formula of renaturation buffer is: 1.8M carbamide, 0.5mM GSS, 5mM GSH, 10% glycerol, pH8.0 20mM Tris-HCl；
Wherein DEAE post is Pyrex material, and specification is GAG200/550, internal diameter 200mm, post height 550mm, stigma mesh size standard
10 μm, filler is DEAESepharose, post bed height 16-17cm, bed volume 5-6L；30Q column chromatography post is: Pyrex material,
Specification is GAG200/550, internal diameter 200mm, post height 550mm, stigma mesh size standard 10 μm, and filler is Source30Q, post bed
Highly 160-200mm, bed volume 5-6L.
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| CN101139388A (en) * | 2007-09-26 | 2008-03-12 | 华东理工大学 | Purification renaturation method for antineoplastic medicine TAT(m)-Survivin(T34A) |
| CN102366628A (en) * | 2010-12-31 | 2012-03-07 | 华东理工大学 | Quality stabilization technique of antitumor protein medicines |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101139388A (en) * | 2007-09-26 | 2008-03-12 | 华东理工大学 | Purification renaturation method for antineoplastic medicine TAT(m)-Survivin(T34A) |
| CN102366628A (en) * | 2010-12-31 | 2012-03-07 | 华东理工大学 | Quality stabilization technique of antitumor protein medicines |
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