CN1336432A - Production process and biological activity of recombinant exarid kinase - Google Patents
Production process and biological activity of recombinant exarid kinase Download PDFInfo
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- CN1336432A CN1336432A CN 00115922 CN00115922A CN1336432A CN 1336432 A CN1336432 A CN 1336432A CN 00115922 CN00115922 CN 00115922 CN 00115922 A CN00115922 A CN 00115922A CN 1336432 A CN1336432 A CN 1336432A
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Abstract
The present invention relates to production method of bacillus subtilis kinase and its bioactivity, on the basis of self screened bacillus subtilis fermentation producing bacillus subtilis kinase, said enzyme gene is cloned, then establishes gene engineering bacteria fermentation, induce expression, inclusion body separation, washing, fragmentation, denature, renaturation and protein purification etc to obtain recombination bacillus subtilis kinse purified product. The recombination bacillus subtilis kinase can be used as new generation gene engineering thrombolysis medicine.
Description
The invention belongs to technical field of bioengineering.Relate to the method for producing recombinant cumic kinase, specifically set up the fermentation of a whole set of engineering bacteria (PBV-NK/JM109), the chromatography purification method of separation and purification excretory cumic kinase from the fermented liquid of cultivating, and after the cultivation thermal induction, collect inclusion body, sex change, renaturation and the method for purifying proteins of dissolving inclusion body and the product of the purifying cumic kinase that obtains.
Existing natural cumic kinase is must be seen in the fermented product of foreign firm isolating Bacillus subtilus (BacillusSubtilis) from natto by Japanese scholar to identify at first.It has very strong fibrinolytic and activates the function that endotheliocyte produces endogenous t-PA.In recent years Japan develops natto food energetically as protective foods, prevention and treatment thrombotic diseases.But because the cumic kinase amount that natural bacteria produces is few, fermentation time is long, is difficult to be applied to scale operation.
The objective of the invention is, by on natural cumic kinase basis, to obtain engineering strain, produce recombinant cumic kinase, solve because natural bacteria generation cumic kinase amount is few, fermentation time is long, is difficult to use in the problem of production.
Realize that concrete technical measures of the present invention are: at first screening and separating goes out the cumic kinase bacterial classification, clone the cumic kinase gene with this understanding, order-checking, structure efficiently expresses the engineering strain of cumic kinase, to obtain high expression engineering strain, and set up the production method of recombinant cumic kinase, through extracting purifying, the active detection determined.Its producing and manufacturing technique comprises: the fermentation of engineering strain, the saturated ammonium sulphate target protein, the dialysis of target protein ammonium sulfate precipitation thing with DEAE-Mierocrystalline cellulose DE-52 column chromatography for separation target protein, is further purified the albumen of purpose with CM-Mierocrystalline cellulose CM-52.Concentrate target protein, steps such as Sephadex G-100 column chromatography purification target protein with PEG-10000.
1. engineering strain and fermentation, reorganization PBV-NK/JM109 is this center construction, from the bacterial classification plate of 4 ℃ of preservations, picking list colony inoculation contains Amp antibiotic 100 μ g/ml in 5mlLB nutrient solution test tube, 30 ℃ of cultivations, 180 rev/mins, shaking culture 18 hours.Be seeded in 200mlLB nutrient solution/1000ml triangular flask by 1: 50, the ammonia benzyl is 100 μ g/ml.30 ℃, 150 rev/mins, cultivate after 24 hours, centrifugal, 6000 rev/mins, 20 minutes, collect supernatant liquor, the content of electrophoresis detection target protein in fermented liquid.
2. saturated ammonium sulphate target protein
With 60% saturated ammonium sulphate cumic kinase.Under 0 ℃ of condition, take by weighing ammonium sulfate, after crushed, join lentamente in the supernatant liquor, the limit edged stirs, after treating all to add, at similarity condition, continue to stir 4 ℃ of refrigerator overnight 2 hours, centrifugal, 6000rpm, 20 minutes, 4 ℃, collecting precipitation, and with pH7.4 in a small amount, 10mM phosphoric acid buffer dissolution precipitation thing.
3. the dialysis of target protein ammonium sulfate precipitation thing
Above-mentioned dissolved throw out is dialysed to distilled water 1200ml at 4 ℃, changes water 1 time in 3-4 hour, dialyse more than 24 hours, and the gained dialyzate, again to 10MmPBS (PH7.4) dialysed overnight, the dialyzate that obtain this moment is for chromatographic separation usefulness.
4. with DEAE-Mierocrystalline cellulose DE-52 column chromatography for separation target protein, because target protein has positive charge when PH7.4, tropina is a negative charge, therefore, through behind the post, ion-exchange takes place and stays on the post in foreign protein and DE-52, and target protein flows out, thereby most of foreign protein and pigment are removed, obtain the cumic kinase of initial gross separation, target protein concentration nearly 50% in the sample.Target protein is retained in rear portion, the 2nd peak and whole the 2nd peak stays in part when the DE-52 column chromatography.As Fig. 2, shown in Figure 4.Get the 20-30mg target protein from the 6000ml nutrient solution.
5. be further purified target protein with CM-Mierocrystalline cellulose CM-52
As shown in Figure 3, Figure 4, use the chromatography curve of CM-52 column chromatography purification target protein.
With DE-52 is isolating have active fraction to merge after, transfer PH to 6.4, under 4 ℃, with 1: 3 ratio, the static exchange adsorption sample was at least more than 4 hours, suction filtration is collected CM-52 then, and washes CM-52 with the 10mMPBS (PH6.4) of 2-5 times of volume, by the CM-52 after washing, be contained in (0.0cm * 100cm), connect the protein detection instrument, 280mM in the chromatography column, continue to use PBS drip washing, after the instrument baseline is walked directly, change gradient elution, with each 300ml of 10mMPBS (PH6.4) of 0M and 1MNaCl.Behind the fraction collection sample, measure activity, and the electrophoresis detection component, merge single purpose albumen fraction, change the SephadexG-100 column purification over to.
6. concentrate target protein with PEG-10000
When CM-52 post gradient elution, cumic kinase is washed when 0.15-0.2MNaCl concentration, after the collection, with about PEG simmer down to 5ml, crosses SephadexG-100 post desalination and is further purified.
7.SephadexG-100 column chromatography purification target protein
SephadexG-100 post (0.8cm100cm) after 10mMPBS (PH7.4) balance, with the spissated CM-52 post of 5ml sample, slowly adds, and after sample enters glue, adds 10mMPBS (PH7.4).The beginning wash-out.Ultraviolet 280m μ detects, and collects the peak fraction, and elution peak is symmetry shape, and the active fraction electrophoresis detection of gained is the cumic kinase of single band.Be the finished product.
The present invention is on natural cumic kinase basis, the engineering strain of acquisition, and the recombinant cumic kinase of generation, its advantage is with the E.coli incubation time short, yield of enzyme is big, is convenient to extraction, purifying, but suitability for industrialized production.
Accompanying drawing provided by the present invention is:
Fig. 1 is technical process of the present invention
Fig. 2, DE-52 and column chromatography purification curve
Fig. 3, CM-52 purifying chromatography purification curve
Fig. 4, DE-52 (A) and CM-52 (B) chromatography sample SDS electrophorogram
Fig. 5, Sephadex G-100 purifying protein electrophorogram
Fig. 6, the SDS-PAGE electrophorogram of recombinant cumic kinase inclusion body
Fig. 7, the sex change electrophorogram of washing inclusion body
Fig. 8, the SDS-PAGE electrophoresis of purifying cumic kinase and electrophoresis scanning
Fig. 9, external fibrinolytic result
Figure 10, the determination of activity result of purifying cumic kinase
Figure 11, the HPLC of purifying cumic kinase
Figure 12, dissolution in vitro clot result
The present invention's 1,2 pair of accompanying drawing in conjunction with the embodiments further specifies.
Embodiment 1:
One, the technical essential of present embodiment
1. fermentation using bacteria is the low density high expression level.
2. the inclusion body denaturing agent is a urea.
3. behind the renaturing inclusion bodies, column chromatography purification, two steps, DE-52 post and CM-52 post.
Two, experimental implementation step (Fig. 1)
1. engineering strain, reorganization PBV-NK/E.coliJM109 is this center construction.Picking list colony inoculation (contains penbritin 100 μ g/ml) 30 ℃ 5mlLB nutrient solution pipe from the engineering bacteria plate of 4 ℃ of preservations, 180 rev/mins, after shaking culture 16-18 hour, be inoculated in to shake in the bottle by 2: 50 and (contain penbritin 100 μ g/ml), 30 ℃, 180 rev/mins, shaking culture 16 hours is as seed liquor.
2. fermentation, by 2: 50 seed liquor is inserted (contain ammonia benzyl perimycin 100 μ g/ml) in 200mlLB/1000ml triangular flask PH7.2,30 ℃, 180 rev/mins, vibrated 4-5 hour, when treating that the nectar degree reaches OD600 and is 0.3-0.4, be warming up to 42 ℃ rapidly, abduction delivering 4-5 hour, centrifugal collection thalline, 6000 rev/mins, 20 minutes, electrophoretic examinations target protein expression amount.Wet thallus is the 5-7g/ liter.See Fig. 6.
3. the cracking of thalline
Centrifugal collection thalline, supernatant discarded claims thalline weight, adds 20mM/L PBS PH7.4 by 1: 10 and washes thalline 1 time.After centrifugal, still thalline is suspended in (buffer A 50mM/L Tris HCL PH8.0 0.5mM/LEDTA, 50mM/L Nacl in the buffer A by 1: 10,5% glycerine, 0.5mM/LDTT), on ultrasonic cell disruption instrument, ultrasonic 30 minutes, test under microscope, percentage of damage is more than 95%.Add Septochol (DOC) and reach final concentration 0.2% in ultrasonic liquid, behind the mixing, room temperature was placed 10 minutes, and 4 ℃, 12000 rev/mins, centrifugal, 15 minutes, supernatant discarded, precipitation is weighed, and is inclusion body.
4. the washing of inclusion body
(1) with 0.5%TritonX-100 (using buffer A) preparation, by 1: 10 resuspension precipitation, (evenly, particle must not be arranged), and stirring at room 20 minutes, 12000 rev/mins, 20 minutes, abandon supernatant, precipitation is weighed.
(2) with 2M/LNacl (buffer A preparation), 1: 10 washing inclusion body, stirring at room 1 hour, 12000 rev/mins, centrifugal 20 minutes, the precipitation inclusion body of collecting and weigh.
(3) washed the inclusion body stirring at room 20 minutes with 3M/L urea (buffer A preparation), 12000 rev/mins, centrifugal 20 minutes, abandon supernatant collecting precipitation inclusion body, weigh.The washing inclusion body is seen Fig. 7.
5. inclusion body sex change
With 8M/L urea dissolving sex change inclusion body.8M/L urea (preparing with buffer A) added urea soln by 1: 5, and 37 ℃ of dissolvings are more than 2 hours, and 12000 rev/mins, centrifugal 20 minutes, supernatant was an inclusion body sex change liquid, should be colourless or pale yellow solution.
6. renaturing inclusion bodies
(1) 2M/L urea renaturation, inclusion body sex change liquid is measured its protein concentration, and renaturation will be carried out during to 100 μ g/ml albumen its concentration is rare.2M/L urea renaturation is spent the night at 4 ℃, and renaturation solution (preparing with buffer A) Ying Chengqing is transparent, no clustering phenomena, and then, urea is repeatedly removed in dialysis to buffer A.
(2) dilution method renaturation.Sex change liquid is dropwise added (buffer A of 100mM/L urea) in the renaturation solution, make protein concn reach 0.1mg/ml, after the abundant renaturation, the centrifugal precipitation of removing, super Shanghai concentrates, and urea is removed in dialysis.
7. the post layer purifying of recombinant protein
(1) with DEAE-Cellulose DE-52 column chromatography for separation, the DE-52 that balance is good pack in the post (4 * 20cm), after 20mMPBS (PH7.4) balance, the renaturation sample is transferred PH7.4, by this post, collects outflow portion, electrophoretic examinations merges (seeing Fig. 2, Fig. 4 A) with active part.
(2) with CM-Cellulose CM-52 column purification.The CM-52 that handles well takes by weighing a certain amount ofly, has used 20mMPBS PH6.5 equilibrated CM-52, joins to transfer the going up in the active fraction of step of PH6.5, and static exchange is more than 16 hours.Take out Shanghai then, keep supernatant, CM-52 pack in the post (0.8 * 60cm), do not exchange or adherent impurity with the level pad flush away, the detector baseline is walked directly, change 0-1.0M/LNaCl (use 20mMPBS, PH6.5 joins) and carry out gradient elution, collect the peak fraction, activity and electrophoresis detection, be associated with activity, single band portion is highly finished product (seeing Fig. 3, Fig. 4 B).
(3) use the SephadexG-100 chromatography purification, the active fraction of CM-52 is merged, PEG is concentrated into below the 10ml, the SephadexG-100 post that overbalance is good (0.8 * 60cm), with same 20mMPBS, PH7.4 wash-out, single symmetrical peak appears, be the finished product, merge freeze-drying.The results are shown in Figure 5 and Fig. 8, the HPLC analytical results of cumic kinase is unimodal simultaneously, and purity is seen Figure 11 more than 99%.
Embodiment 2:
One, the technical essential of present embodiment:
1. recombinant cumic kinase is external to fibrinous lytic activity
2. the external solvency action of recombinant cumic kinase to natural sludged blood
Two, experimental implementation step
1. external solusphere activity to the Agarose-fibrin plate is got Agarose (1%) solution 16.2ml, scleroproein stoste 16.2ml, zymoplasm 1.3ml, under 45 ℃, be poured into behind the mixing in the square box of 9cm * 9cm * 20cm, room temperature was placed after 1 hour, recombinant cumic kinase 10 μ l with different concns, point after 30 minutes, is put into 37 ℃ at planar surface, 16-18 hour, has very big transparent solusphere.Show recombinant cumic kinase, external to fibrinous solvency action.See Fig. 9, Figure 10.
2. a certain amount of rabbit blood is extracted in the effect of dissolution in vitro sludged blood, pours in the plate, after 4 ℃ of blood coagulations, use PBS, PH7.4 is after the washing, take by weighing 500mg respectively, three, be placed in three small beakers, add 1. physiological saline 10ml then, 2. contain the same solution of 10ml of 2mg recombinant cumic kinase, 3. contain the same solution of 10ml of 2mg recombined streptokinase, 37 ℃, vibrate and weighed 1 time in 30 minutes, record clot weight, continuous 3 times promptly 90 minutes, claim the clot weight record, result such as table 1 and Figure 12 finding at every turn.
Table 1 cumic kinase dissolution in vitro clot experimental result
| Time (branch) | Physiological saline | Streptokinase solution (0.2mg/ml) | Cumic kinase solution (0.2mg/ml) | Remarks |
| Heavy (mg) 30 60 90 of clot | ????500 ????440 ????410 ????370 | ??????????500 ??????????131 ??????????65 ??????????46 | ??????????500 ??????????52 ??????????30 ??????????0 |
From the table the result as seen, recombinant cumic kinase has the function of very strong dissolution in vitro clot.
In a word, the invention discloses a kind of production technology method of recombinant cumic kinase, comprise cultivation, the abduction delivering of engineering bacteria, the separation of inclusion body, washing, cracking sex change, the lytic activity and the determination of activity of refolding method and protein purification technology and external clot to fibrin plate.
The product of purifying of the present invention can be used as a kind of new thrombus preparation, can make the lyophilized powder injection, or makes tablet and capsule confession oral administration prevention cardiovascular and cerebrovascular embolism disease.
Claims (7)
1. the production method of a recombinant cumic kinase comprises: the fermentation of engineering strain, the saturated ammonium sulphate target protein, the dialysis of target protein ammonium sulfate precipitation thing with DEAE-Mierocrystalline cellulose DE-52 column chromatography for separation target protein, is further purified the albumen of purpose with CM-Mierocrystalline cellulose CM-52.Concentrate target protein with PEG-10000, steps such as Sephadex G-100 column chromatography purification target protein, it is characterized in that engineering strain and fermentation, from the bacterial classification plate of 4 ℃ of preservations, picking list colony inoculation contains Amp antibiotic 100 μ g/ml, 30 ℃ of cultivations in 5mlLB nutrient solution test tube, 180 rev/mins, shaking culture 18 hours.Be seeded in 200mlLB nutrient solution/1000ml triangular flask by 1: 50, the ammonia benzyl is 100 μ g/ml.30 ℃, 150 rev/mins, cultivate after 24 hours, centrifugal, 6000 rev/mins, 20 minutes, collect supernatant liquor, the content of electrophoresis detection target protein in fermented liquid.
2. the production method of a kind of recombinant cumic kinase according to claim 1 is characterized in that the saturated ammonium sulphate target protein.Promptly with 60% saturated ammonium sulphate cumic kinase.Under 0 ℃ of condition, take by weighing ammonium sulfate, after crushed, join lentamente in the supernatant liquor, the limit edged stirs, after treating all to add, at similarity condition, continue to stir 4 ℃ of refrigerator overnight 2 hours, centrifugal, 6000rpm, 20 minutes, 4 ℃, collecting precipitation, and with pH7.4 in a small amount, 10mM phosphoric acid buffer dissolution precipitation thing.
3. according to the production technique of claim 1,2 described a kind of recombinant cumic kinases, it is characterized in that the dialysis of target protein ammonium sulfate precipitation thing, be with the dissolved throw out, at 4 ℃ distilled water 1200ml is dialysed, changed water 1 time in 3-4 hour, dialysis is more than 24 hours, the gained dialyzate, to 10MmPBS (PH7.4) dialysed overnight, the dialyzate that obtain this moment is used for chromatographic separation again.
4. the production technique of a kind of recombinant cumic kinase according to claim 1, it is characterized in that with DEAE-Mierocrystalline cellulose DE-52 column chromatography for separation target protein, select target protein when PH7.4, to have positive charge, when tropina was negative charge, through behind the post, ion-exchange took place and stays on the post in foreign protein and DE-52, and target protein flows out, thereby most of foreign protein and pigment are removed, obtain the cumic kinase of initial gross separation, this moment, target protein concentration nearly 50%.Target protein is retained in rear portion, the 2nd peak and whole the 2nd peak stays in part when the DE-52 column chromatography.
5. according to the production technique of claim 1,4 described a kind of recombinant cumic kinases, it is characterized in that being further purified target protein with CM-Mierocrystalline cellulose CM-52.After being about to that DE-52 will be isolating and having active fraction to merge, transfer PH to 6.4, under 4 ℃, with 1: 3 ratio, static exchange absorption was at least more than 4 hours, suction filtration is collected CM-52 then, and washes CM-52 with the 10mMPBS (PH6.4) of 2-5 times of volume, by the CM-52 after washing, be contained in (0.0cm * 100cm), connect the protein detection instrument, 280mM in the chromatography column, continue to use PBS drip washing, after the instrument baseline is walked directly, change gradient elution, with each 300ml of 10mMPBS (PH6.4) of 0M and 1MNaCl.The fraction collection target protein is measured activity, and the electrophoresis detection component, merges single purpose albumen fraction, changes the SephadexG-100 column purification over to.
6. according to the production technique of claim 1,5 described a kind of recombinant cumic kinases, it is characterized in that concentrating target protein with PEG-10000.Promptly when CM-52 post gradient elution, cumic kinase is washed when 0.15-0.2MNaCl concentration, after the collection, with about PEG simmer down to 5ml, crosses SephadexG-100 post desalination and is further purified.
7. according to the production technique of claim 1,6 described a kind of recombinant cumic kinases, it is characterized in that SephadexG-100 column chromatography purification target protein.Be SephadexG-100 post (0.8cm100cm), after 10mMPBS (PH7.4) balance,, slowly add, after sample enters glue, add 10mMPBS (PH7.4) the spissated CM-52 post of 5ml sample.The beginning wash-out.Ultraviolet 280m μ detects, and collects the peak fraction, and elution peak is symmetry shape, and the active fraction electrophoresis detection of gained is the cumic kinase of single band.
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| CN101848924A (en) * | 2007-05-09 | 2010-09-29 | 马斯科马公司 | Gene knockout mesophilic and thermophilic organisms, and methods of use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101848924A (en) * | 2007-05-09 | 2010-09-29 | 马斯科马公司 | Gene knockout mesophilic and thermophilic organisms, and methods of use thereof |
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