CN100372933C - Recombinant human composite alpha interferon preparation method - Google Patents
Recombinant human composite alpha interferon preparation method Download PDFInfo
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- CN100372933C CN100372933C CNB2005100282850A CN200510028285A CN100372933C CN 100372933 C CN100372933 C CN 100372933C CN B2005100282850 A CNB2005100282850 A CN B2005100282850A CN 200510028285 A CN200510028285 A CN 200510028285A CN 100372933 C CN100372933 C CN 100372933C
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Abstract
The present invention relates to a preparation method of recombination human composite alpha interferon (cIFN) by the expression of eucaryon. The method comprises the following main steps: the cIFN is obtained by processing yeast engineering bacteria (Pichia pastoris GS115/pPIC9K-CIFN) through seed cultivation, fermentation tank cultivation, the induced expression of exogenous proteins and the purification of target proteins. The present invention is characterized in that when the induced expression of exogenous proteins is started, Tween-20, Tween-80, SDS, TritonX-100, TritonX-114, NP-40, CHAPS, Zwittergent3-14, LiDS, sodium tauroglycocholate, sodium deoxycholate, octylglycoside or/and Brij35 and 0.01 to 10 g/L of fermentation liquor are added, and the temperature of the induced expression of exogenous proteins is controlled to be 20 to 30 DEG C; when the exogenous proteins are induced and expressed, the PH value of the fermentation liquor is controlled to be 3.0 to 7.0, and the methanol contents of the fermentation liquor are controlled to be 1.0 to 10.0 g/L of the fermentation liquor. The present invention not only realizes the expression of high density and high fermentation of the cIFN, but also maintains the correct monomer molecular state and bioactivity of the cIFN in the fermentation liquor so that the formation of polymers and degraded products of the cIFN are greatly restrained.
Description
Technical field
(consensus interferon, cIFN) preparation method relate in particular to a kind of through eukaryotic expression and can keep the cIFN preparation method of correct configuration and function to the present invention relates to a kind of recombinant human composite alpha interferon.
Background technology
Nineteen eighty-three, Goedell etc. compare research to the sequence of known 13 kinds of human alpha interferons at that time, and the highest amino acid of the frequency of occurrences is assigned on each corresponding position, obtain a new cIFN (US4,414,150) who is made up of 166 amino acid.Clinical application shows that cIFN is having good performance aspect treatment chronic hepatitis B, the hepatitis C.
Up to now, the method for preparing cIFN mainly is divided into two classes.First prokaryotic expression legal system is equipped with cIFN: promptly in intestinal bacteria (E.coli), express produce cIFN (US4,695,623, US4,897,471, US5,541,293, US5,661,009 and ZL 98114663.5).Yet the prokaryotic expression legal system is equipped with cIFN exists the protein post-translational modification difficulty, produce the purifying process complexity and need except that problems such as thermals source, and the cIFN that E.coli produces is that the N-end has the band methionine(Met), is not with methionine(Met) and three kinds of mixtures of methionine(Met) acetylize, all brings adverse influence for biological activity and clinical application; It two is that the eukaryotic expression legal system is equipped with cIFN: promptly express in yeast and produce cIFN (CN1435486A).Though the eukaryotic expression legal system is equipped with some defectives that cIFN has overcome the prokaryotic expression method, there is target protein (cIFN) polymerization and degraded easily take place, cause very difficult separation and purification to obtain correct configuration and highly active target protein cIFN monomer.Therefore, how overcoming the eukaryotic expression legal system is equipped with the defective that polymerization and degraded easily take place target protein among the cIFN (cIFN) and just becomes the technical issues that need to address of the present invention.
Summary of the invention
Existing utilize the yeast eukaryotic expression to prepare the cIFN[fermention medium to be basic salt (synthesize) substratum] method, though can accomplish the fermentation of high-density high expression level, target protein (cIFN) unusual instability in basic salt (synthesizing) substratum.On the one hand, the cIFN that is secreted in the substratum is easy to form polymer, the outer cIFN monomer of nearly all born of the same parents of being secreted into has all aggregated into the polymer that molecular weight differs in size (as shown in Figure 1), cIFN at first is gathered into soluble polymers, and then the bigger polymer of formation molecular weight, until forming precipitation, run off with cell settlement, have a strong impact on the recovery of target protein and activity stabilized; On the other hand, cIFN is easy to again degrade, degraded is by due to the protease hydrolysis, and cIFN degrades in fermented liquid and forms molecular weight is lower than 10,000 all size at a relatively-stationary 4-5 segment and molecular weight more than 10,000 protein micromolecular and polypeptide (as Fig. 2 A, Fig. 2 C).Because the cIFN intramolecularly has two disulfide linkage, the degraded segment is still linked to each other by disulfide linkage in the irreducibility environment, apparent molecular weight and complete cIFN monomer the same (Fig. 2 A and Fig. 2 B), but the integrity of cIFN molecule and activity all are subjected to very big destruction.
The present inventor thinks and causes the reason of above-mentioned phenomenon to be: the tension force effect of liquid-gas interface is one of principal element that inspires polymerization of protein in the solution.When protein soln is agitated, can produce a large amount of bubbles, form liquid-gas interface, the protein receptor that is on the interface is separated folding to surface action, cause the hydrophobic grouping of protein molecule inside to expose, increase the interaction chance between protein and the protein, thereby cause polymerization of protein.In the fermenting process, in order to increase dissolved oxygen and mixing of materials, stir one of the parameter that is absolutely necessary, add the characteristic of fermented liquid itself, producing foam (liquid-gas interface) is inevitably, therefore very easily causes instability problems such as proteinic polymerization.Given this, the inventor proposes following optimization fermentating controling process.
The said recombinant human composite alpha interferon of the present invention (cIFN) preparation method, its key step is: with Yeast engineering bacteria (Pichiapastoris GS115/pPIC9K-cIFN) through seed culture, fermentor cultivation, obtain cIFN behind foreign protein abduction delivering and the target protein purifying, it is characterized in that, when starting, the foreign protein abduction delivering adds Tween-20, Tween-80, SDS, TritonX-100, Triton X-114, NP-40, CHAPS, Zwittergent3-14, LiDS, Sodium cholic acid, Sodium desoxycholate, suffering is crawled glucosides (Octylglucoside) or/and Brij35, and add-on is the 0.01-10g/L fermented liquid; The temperature of foreign protein abduction delivering is controlled at 20～30 ℃ (preferred 20～25 ℃); The pH value of fermented liquid is controlled at 3.0～7.0 (preferred 4.0～6.0 during the foreign protein abduction delivering, the best is 4.5～5.5) and fermented liquid in methanol content be controlled at 10.0g/L fermented liquid following (below the preferred 6.0g/L fermented liquid, the best is below the 3.0g/L fermented liquid).
In the present invention: the seed culture medium of fermentation culture Yeast engineering bacteria (Pichia pastoris GS115/pPIC9K-cIFN) and fermention medium (fermentor tank with) are known substratum, as: YPD flat board, BMGY liquid nutrient medium, basic salt (synthesizing) substratum are (referring to " High Cell-Density Fermentation in Pichia Protocols ", edited by David R.Higgins and James M.Cregg, Vol.103,107-120,1998) and the FM22 substratum; The method of the seed culture of Yeast engineering bacteria (Pichia pastorisGS115/pPIC9K-cIFN), fermentor cultivation, foreign protein abduction delivering, be method of mentioning on the Pichiafermentation process guidelines (Invitrogen Corporation, 2002) or the method for revising on this basis; Target protein (cIFN) purification procedures is referring to (CN 1435486A); Crawl glucosides (Octylglucoside) and Brij35 of said Tween-20, Tween-80, SDS, Triton X-100, Triton X-114, NP-40, CHAPS, Zwittergent3-14, LiDS, Sodium cholic acid, Sodium desoxycholate, suffering is commercially available product.
The present invention has following advantage:
1. target protein (cIFN) polymerization and degraded in fermented liquid have been suppressed, stablized configuration and the biological activity of cIFN, overcome and utilized basic salt (synthesizing) substratum to carry out being difficult to obtain in the high-density high expression level process the monomeric difficult problem of target protein (cIFN) of activated and correct configuration;
2. prolong the monomeric working lipe of cIFN of expressing correct configuration and active function being arranged, thereby improved single batch of total expression amount of cIFN.Compared with prior art, the monomeric induction duration of stably express cIFN prolongs more than at least 1 times, that is: induce still to lose in 100 hours and have tangible polymer to form increase the weight of (Fig. 3 B) of (Fig. 3 A) or degraded, even see from Fig. 3 and to induce that the target protein amount still is increase trend after 100 hours, and reference examples (Fig. 4) is just very obvious degraded in 21 hours, and increase the weight of almost thoroughly degraded after 50 hours gradually.
Description of drawings
Fig. 1 is the natural electrophorogram of the fermented supernatant fluid of complex medium and basic salt (synthesizing) substratum,
Wherein: swimming lane 1 is the complex medium culture supernatant (A), and 2 is the pure product of cIFN, and 3,4 is basic salt (synthesizing) culture medium culturing supernatant liquor, target protein (cIFN) band in the arrow indication sample; (B) figure is the Western Blotting corresponding with swimming lane 3,4 among (A) figure.
The SDS-PAGE electrophorogram of the fermented supernatant fluid of Fig. 2 basis salt (synthesizing) substratum and WesternBlotting figure,
Wherein: A is that the fermented supernatant fluid of basic salt (synthesizing) substratum is through reductibility SDS-PAGE electrophorogram, swimming lane 1 is lower molecular weight Marker, swimming lane 2 is the pure product of cIFN, and swimming lane 3 and 4 is the fermented supernatant fluid of basic salt (synthesizing) substratum, arrow indicating target albumen (cIFN) degraded segment;
B be with the same sample of A through irreducibility SDS-PAGE electrophorogram, swimming lane 1,2 is the fermented supernatant fluid corresponding with swimming lane 3,4 among the A figure, 3 is the pure product of cIFN;
C): swimming lane 1,2 is the reductibility SDS-PAGE electrophorogram of fermented supernatant fluid, and swimming lane 3,4 is the Western Blotting figure corresponding to swimming lane 1,2 samples.
Fig. 3: the electrophorogram in the fermented liquid behind the adding Tween-20, the numeral induction time that marks on each swimming lane of two figure.
Wherein: (A) natural electrophorogram, show that polymer is suppressed, monomer occurs; (B) reductibility electrophorogram shows that the cIFN degraded product obviously reduces.
Fig. 4: in contrast, do not add Tween-20, show cIFN by the phenomenon of severely degrade, band is the target protein degraded product in the square frame.
Embodiment
The said recombinant human composite alpha interferon of the present invention (cIFN) preparation method, it comprises the steps:
(1) seed culture:
With Yeast engineering bacteria (Pichia pastoris GS115/pPIC9K-cIFN), be grown on the YPD flat board, picking mono-clonal inoculation is in the BMGY liquid nutrient medium, and 180-250rpm, shaking culture 16-24 hour, makes thalline OD by 20-30 ℃
600Reach 5-20% (preferred 10-15%);
(2) fermentation culture
Above-mentioned 50ml seed culture fluid transferred in the 5.0L fermented liquid, contain 1.5-2.5L basis salt (synthesizing) substratum (referring to Stratton, J.Chiruvolu V.and Meagher, M. " High Cell-Density Fermentation in Pichia Protocols ", edited by David R.Higgins and James M.Cregg, Vol.103,107-120,1998) or in the FM22 substratum ferment (fermentation divides three phases), fs: glycerine vegetative period, pH is controlled at 4-6 (by the control of 28% ammoniacal liquor), leavening temperature is 25-30 ℃, mixing speed 200-900rpm, air flow quantity 0.5-2.0L/Lmin, dissolved oxygen (DO) is greater than 30%, after the glycerine in the substratum has been consumed (dissolved oxygen rebound suddenly go up to 100% about), begin to flow glycerol adding, enter subordinate phase: glycerine feed supplement/batch culture phase, the feed supplement glycerol concentration is 50% (W/V), every liter of glycerine adds 0-12ml PTM1 (the FM22 substratum is PTM4) simultaneously, glycerine is added the ratio of speed according to dissolved oxygen, is the 30-50% saturation ratio with the control dissolved oxygen generally.The feed supplement time is 2-20 hour, the glycerine feed supplement enters the phase III after finishing: the methanol induction phase, begin to mend methyl alcohol (100%), every liter of methyl alcohol adds 0-12ml PTM1 (the FM22 substratum is PTM4), the methyl alcohol electrode on-line monitoring in addition of the stream of methyl alcohol, keep methanol concentration in jar interior substratum for not being higher than the 10g/L fermented liquid, when starting, methanol induction adds Tween-20, Tween-80, SDS, Triton X-100, Triton X-114, NP-40, CHAPS, Zwittergent3-14, LiDS, Sodium cholic acid, Sodium desoxycholate, suffering is crawled glucosides (Octylglucoside) or/and Brij35, and add-on is 0.001～1.0%0.01～10g/L fermented liquid; Is the temperature of foreign protein abduction delivering is controlled at 20～30 ℃, and (preferred 20～25 ℃, the best 22 ℃? the best it may be noted that?); The pH value of fermented liquid is controlled at 3.0～7.0 (preferred 4.0～6.0 during the foreign protein abduction delivering, the best is 4.5～5.5) and fermented liquid in methanol content be controlled at and be lower than the 10.0g/L fermented liquid (below the preferred 6.0g/L fermented liquid, the best is that the 3.0g/L fermented liquid is following), 48-150 hour methanol induction time.Adopt the separation purification method that CN1435486A reported to obtain target protein (cIFN), the content of cIFN reaches more than the 2g/L fermented liquid.
The present invention is further elaborated below in conjunction with embodiment, should be understood that its purpose only is better to understand content of the present invention and unrestricted protection scope of the present invention:
Detection method:
The detection method of content of total protein adopts the Coomassie brilliant blue staining in the fermented supernatant fluid;
Target protein is qualitative in the fermented supernatant fluid adopts SDS-PAGE electrophoresis, WesternBlotting method with quantitative detecting method, and uses the protein electrophoresis computed in software;
The target protein activity test method adopts the WISH-VSV method in the fermented supernatant fluid;
The target protein polymer forms detection method and adopts Native PAGE electrophoretic method, Western Blotting method in the fermented supernatant fluid.
The preparation of reorganization cIFN:
(1) seed culture
The Yeast engineering bacteria that contains cIFN (Pichiapastoris GS115/pPIC9K-cIFN) with the 1mL freezing, directly insert in the 50mL/BMGY liquid nutrient medium, or be coated on earlier on the YPD flat board, picking mono-clonal bacterium colony one ring is inoculated in the 50mL/BMGY liquid nutrient medium again.220rpm, 30 ℃, shaking culture 18 hours makes thalline OD600 reach 10-15%, in the aseptic access 5L fermentor tank.
(2) fermentation culture
The 50ml seed culture fluid transferred be equipped with in salt (synthesizing) substratum of 2.0L basis in the 5L fermentor tank, used basic salt (synthesizing) substratum of the present invention is with reference to Pichia Protocols, fermention medium is with basic salt (synthesizing) substratum (BSM), the inorganic salt dosage is 1 * BSM, also can between 0.1-2 times of original formulation, change, the PTM1 dosage is: add 4mL/L in the basestocks, add 12mL/L in feed supplement glycerine and the methyl alcohol, also can change between 0-12mL/L.Fermentation control divides three phases: the fs is glycerine vegetative period, pH is controlled at 5.4, control by 28% ammoniacal liquor, leavening temperature is 30 ℃, mixing speed 200-900rpm, air flow quantity 0.5-2.0L/Lmin (wm), keep dissolved oxygen (DO) greater than 30%, after the glycerine in basic salt (synthesizing) substratum had been consumed, dissolved oxygen rebounded suddenly to about 100%, begins to flow glycerol adding, enter subordinate phase---glycerine feed supplement-batch culture phase, the feed supplement glycerol concentration is 50% (W/V), and every liter of glycerine adding 12mlPTM1 glycerine is added the level of speed according to dissolved oxygen simultaneously, is the 30-50% saturation ratio with the control dissolved oxygen generally.The feed supplement time is 6 hours.After the glycerine feed supplement finishes, enter the phase III---the methanol induction phase, begin to mend methyl alcohol (100%), every liter of methyl alcohol adds 12ml PTM1, the stream of regulating methyl alcohol is kept methanol concentration in jar interior substratum for not being higher than 0.8%, and with the on-line monitoring of methyl alcohol electrode, 70 hours methanol induction time.Induce the biomass of later stage pichia spp to reach as high as 170g/L (dry cell weight), target protein in the fermented supernatant fluid (cIFN) content reaches more than the 2g/L.Fermented liquid separates through multiple chromatographic column, does not obtain the cIFN monomer, and the protein of collecting mainly is distributed in the macromole zone of molecular weight greater than 80KD.Confirm to have the cIFN monomer molecule hardly in the fermented supernatant fluid through natural electrophoretic analysis (Fig. 1), and the analyses of reductibility and non-reduced SDS-PAGE electrophoresis (Fig. 2) are found also to have tangible cIFN signs of degradation in the fermented liquid.Measure the antiviral biological activity of fermented supernatant fluid, the high reactivity of result has only 5.39 * 10
7IU/mL fermented liquid (table 1).
Optimize the stabilization of fermentation control strategy to target protein cIFN
Carry out fermentation culture according to the method among the embodiment 1, begin to add protein stabiliser from the methanol induction phase.Stablizer can be one or more mixtures in following several material, and the adding mode is once to add or repeatedly to add, the also mode that can adopt Continuous Flow to add, and add-on is to add one of following additive of 0.1～5.0g in every liter of fermented liquid.
Stablizer: Tween-20, Tween-80, SDS, Triton X-100, Triton X-114, NP-40, CHAPS, Zwittergent3-14, LiDS, Sodium cholic acid, Sodium desoxycholate, suffering crawl glucosides (Octylglucoside) and/or Brij35 etc.
This example is to add the stabilization of protein stabiliser to target protein cIFN in the stablizer explanation fermenting process with Tween-20.The adding mode adopts disposable adding, and add-on is to add 0.01～0.5g Tween-20 in every liter of fermented liquid.
Carry out fermentation culture according to the method among the embodiment 1, begin culture temperature from the methanol induction phase and be adjusted downward to 20-25 ℃.Inductive phase, pH was controlled at 4.0～6.0, and best pH is 4.5～5.5.
With embodiment 1, the methanol induction time is 70 hours.Induce the biomass of later stage pichia spp to reach as high as 165g/L (dry cell weight), target protein in the fermented supernatant fluid (cIFN) content reaches more than the 1.2g/L.Fermented liquid obtains 160mgcIFN monomer/fermented liquid through the chromatrographic separation step identical with embodiment.Confirm that through natural electrophoretic analysis (Fig. 3 A) the cIFN molecule mainly exists with monomeric form in the fermented supernatant fluid, and reductibility SDS-PAGE electrophoresis (Fig. 3 B) analysis finds that the cIFN degraded product in the fermented liquid obviously reduces.Measure the antiviral biological activity of fermented supernatant fluid, the high reactivity of result reaches 9.46 * 10
8Activity in the IU/mL fermented liquid (table 1), every liter of fermented liquid is more than 17 times of embodiment 1, compares than living with the cIFN in two kinds of situation bottom fermentation liquid, improves 29.2 times.
The high biological activity of table 1 different fermentations training method cIFN relatively
| Training method | Unit volume activity (* 10 7IU/mL) |
| Embodiment 1[Abmax (52h)] | 5.39 |
| Embodiment 2[Aimax (66h)] | 94.6 |
| Aimax/Abmax | 17.55 doubly |
In sum, the present invention has not only realized the high fermentation expression of high-density of recombinant human composite alpha interferon (cIFN), and making target protein (cIFN) in fermented liquid, keep correct monomer molecule state and biological activity, separation and purification has obtained high stability, highly purified cIFN.
In addition; though the present invention is that example illustrates content of the present invention with recombination yeast (Pichia pastoris GS115/pPIC9K-cIFN); but protection scope of the present invention is not limited to this, that is: the expression system that optimisation strategy of the present invention is used for other cIFN all should be considered as in protection scope of the present invention.
Claims (6)
1. a recombinant human composite alpha interferon (cIFN) preparation method, its key step is: with Pichia pastorisGS115/pPIC9K-cIFN bacterial strain through seed culture, fermentor cultivation, obtain cIFN behind foreign protein abduction delivering and the target protein purifying, it is characterized in that, when starting, the foreign protein abduction delivering adds Tween-20, Tween-80, SDS, TritonX-100, Triton X-114, NP-40, CHAPS, Zwittergent3-14, LiDS, Sodium cholic acid, Sodium desoxycholate, octyl glucoside (Octylglucoside) is or/and Brij35, and add-on is 0.01～10g/L fermented liquid; The temperature of foreign protein abduction delivering is controlled at 20～30 ℃; During the foreign protein abduction delivering pH value of fermented liquid be controlled at 3.0～7.0 and fermented liquid in methanol content be controlled at below the 10g/L fermented liquid.
2. preparation method as claimed in claim 1 is characterized in that, wherein the temperature of foreign protein abduction delivering is controlled at 20～25 ℃.
3. preparation method as claimed in claim 1 is characterized in that, wherein the pH value of fermented liquid is controlled at 4.0～6.0 during the foreign protein abduction delivering.
4. preparation method as claimed in claim 3 is characterized in that, wherein the pH value of fermented liquid is controlled at 4.5～5.5 during the foreign protein abduction delivering.
5. preparation method as claimed in claim 1 is characterized in that, wherein the methanol content in the fermented liquid is controlled at below the 6.0g/L fermented liquid during foreign protein abduction delivering.
6. preparation method as claimed in claim 5 is characterized in that, wherein the methanol content in the fermented liquid is controlled at below the 3.0g/L fermented liquid during foreign protein abduction delivering.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1062565C (en) * | 1998-06-29 | 2001-02-28 | 深圳九先生物工程有限公司 | Recombination human alpha type composite interferon, prepn. method and use therefor |
| CN1435486A (en) * | 2002-01-28 | 2003-08-13 | 上海贸基生物工程科技有限公司 | Human composite alpha-interferon engineered gene, its expression and production thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1062565C (en) * | 1998-06-29 | 2001-02-28 | 深圳九先生物工程有限公司 | Recombination human alpha type composite interferon, prepn. method and use therefor |
| CN1435486A (en) * | 2002-01-28 | 2003-08-13 | 上海贸基生物工程科技有限公司 | Human composite alpha-interferon engineered gene, its expression and production thereof |
Non-Patent Citations (1)
| Title |
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| 酿酒酵母重组人α2a干扰素补料分批培养. 储炬，胡千德，郭元昕，叶勤，林志伟，徐伦.华东理工大学学报，第27卷第4期. 2001 * |
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