CN1303100C - Method for preliminary purification of recombinant human parathormone fusion protein by thermo osmosis shock technology - Google Patents
Method for preliminary purification of recombinant human parathormone fusion protein by thermo osmosis shock technology Download PDFInfo
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- CN1303100C CN1303100C CNB2005100255196A CN200510025519A CN1303100C CN 1303100 C CN1303100 C CN 1303100C CN B2005100255196 A CNB2005100255196 A CN B2005100255196A CN 200510025519 A CN200510025519 A CN 200510025519A CN 1303100 C CN1303100 C CN 1303100C
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Abstract
The present invention discloses a method for the preliminary purification of recombinant human parathormone (PTH) fusion protein by thermo osmosis shock technology with the combination of osmosis shock and heat treatment for replacing a metal chelate chromatographic method. The method comprises the operation steps as following: fermentation liquid pretreatment: supernatant fluid is removed after fermentation liquid is centrifugated to obtain thallus; the thallus is suspended in hypertonic buffer solution to be sufficiently stirred to stand; PTH is in thallus cells; the thermo osmosis shock technology: after the hypertonic solution containing the PTH is stirred, the eccentric thallus is suspended in hypotonic buffer solution to be heated; after treatment, supernatant fluid is collected in a centrifugation mode. The present invention has the advantages of process integration, simple manufacturing process, short periodicity, convenient operation, low cost, etc.
Description
Technical field
The present invention relates to a kind of method of thermoosmosis shock preliminary purification recombinant human parathyroid hormone (PTH1-84) fusion rotein, be about to the osmotic shock technology and combine the method for carrying out the separation and purification recombinant human parathyroid hormone with heat treatment phase.
Background technology
Thermoosmosis shock technology is a kind of integrated isolation technique that osmotic shock technology and combined with heat treatment are got up, osmotic shock be utilize to add increase osmotic pressure material for example sucrose etc. improve environment osmotic pressure, reduce environment osmotic pressure then suddenly, the albumen that causes osmotic shock (osmotic pressure sudden change) will be expressed in the cell periplasmic space discharges (sees " TheRelease of Enaymes from Escherichia coli by Osmotic Shock and during theFormation of Spheroplasts ", be published in The Journal of Biological ChemistryVol.240, No.9, September 1965, the author: Harold C.Neu and Leon A.Heppel).In operation, adding EDTA simultaneously can work to destroy epicyte, increase the epicyte permeability.(people's such as Yu-Cheng Chen " A modified osmotic shock for periplasmic release of a recombinamnt creatinasefrom Escherichia coli " is published in Biochemical English Journal 19 (2004)).Heat treatment technics be utilize albumen heat denatured principle with thermostability and thermally labile albumen sepn a kind of measure.And thermoosmosis shock technology is the osmotic shock technology to be combined with heat treatment phase be used for that the protein isolate periplasmic space is expressed and the strong albumen of thermostability.Come to this a kind of fusion rotein of character of recombinant human parathyroid hormone (PTH1-84) fusion rotein.
Thermoosmosis shock technology is a kind of separation integrated technology, the characteristics that it utilizes albumen to have the thermostability of height are carried out osmotic shock method and thermal treatment purifying integrated, received very gratifying effect, not only improved the purity and the yield of target protein, thereby and made the basic inactivation of proteolytic enzyme be the proteic stable preservation of the Target Fusion condition of providing convenience by high temperature.Thermoosmosis shock technology will in biomacromolecule separates, play convenient flexibly with show the effect that becomes more and more important.
PTH1-84 is a kind of hormone by pth secretion, is made up of 84 amino acid and (sees Henry T.Keutmann, wait the people to be published in HUMAN PARATHYROID HORMONE SEQUENCE VOL.17，NO。" CompleteAmino Acid Sequence of Human Parathyroid Hormone " on 26,1978).PTH1-84 can repair and rebuild the sclerotin of patients with osteoporosis, it is not only the loss that stops bone, can also reverse bone loss, good treatment prospect (seeing that people such as Zhang Keqin, Chen Jiawei are published in Jiangsu medical magazine the 26th the 9th phase of volume of September in 2000 " Rat parathyroid hormone 1-34 is prevented and treated osteoporotic progress ") be arranged based on it.The method of reporting at present some separation and purification recombinant human parathyroid hormones is both at home and abroad mostly taked the isolating method of affinity chromatography after the cytoclasis, (see that people such as Li Jian peak are published in Chinese biological engineering magazine 2006,26 " recombinant human parathyroid hormone peptide (1-34) is efficient amalgamation and expression and the purifying in the colibacillus again "), but incident be exactly long, complicated operating process of operational cycle and cost height.
Summary of the invention
The purpose of this invention is to provide a kind of method simply, not affected by environment, the cycle is short, cost is low method with thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone (PTH1-84) fusion rotein.
To achieve these goals, the technical scheme taked of the present invention is as follows:
A kind of method of thermoosmosis shock preliminary purification recombinant human parathyroid hormone (PTH) fusion rotein, step is as follows:
1) fermentation liquor pretreatment
Fermented liquid is centrifugal through 1000～15000rpm, 1～30min, the thalline that contains PTH after centrifugal is suspended in the buffered soln of 1～50mmol/L Tris-HCl (Tutofusin tris), pH5～10.0, containing 1～25mmol/L EDTA (ethylenediamine tetraacetic acid (EDTA)) and mass ratio in the solution simultaneously is 5%～40% sucrose, we are called high osmotic buffer with this solution, add-on adds 10ml～100ml high osmotic buffer by every gram thalline, solution stirring becomes suspendible, leaves standstill 5min～60min then under 0～30 ℃.
2) thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone fusion rotein:
The high osmotic buffer that contains PTH after leaving standstill is lower than under 30 ℃ with 1000～15000rpm, centrifugal 1～30min, get the thalline after centrifugal, the thalline that contains PTH after centrifugal is suspended in the damping fluid of 1～50mmol/L Tris-HCl, pH5～10.0, contain 1～25mmol/L EDTA in this solution, do not contain sucrose, we are called hypotonic buffer liquid with this solution; Hypotonic buffer liquid add-on is identical with the high osmotic buffer amount that adds before this.Treat in the hypotonic buffer liquid behind the thalline thorough mixing, this suspension solution is heated to 40～100 ℃, and keep 5～60min, suspension solution that at last will be after heat treatment is being lower than the supernatant liquor of collecting with 1000～15000rpm, centrifugal 5～60min under 30 ℃ after centrifugal, promptly obtains the product behind recombinant human parathyroid hormone (PTH) the fusion rotein preliminary purification.
Wherein, above-mentioned fermented liquid is a genetic engineering bacterium Escherichia coli fermentation gained, and target protein PTH expresses for the cell periplasmic space, and the host bacterium is intestinal bacteria.
Advantage of the present invention:
1) technology is simple, with short production cycle
The operating process of this production technique is easy, and required time had only about 1 hour.
2) process integration
Production technique is integrated with cell breakage and product preliminary purification, has obtained satisfied effect.
3) equipment is simple, easy to operate
This technology is compared with traditional purification process affinity chromatography and do not use purifier apparatus except that whizzer, the just preparation of different solutions in the operation.
4) occupation of land is little, cost is low
This separation production operation only needs more than ten square metre laboratory to carry out; Cost is compared with affinity chromatography greatly and is reduced.
Embodiment
The invention will be further described below in conjunction with embodiment, and its purpose only is better to understand content of the present invention and unrestricted protection scope of the present invention:
Embodiment 1
The method of thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone (PTH) fusion rotein, its operation steps is as follows:
1) fermentation liquor pretreatment
Fermented liquid is centrifugal through 6000rpm, 30min, the thalline that contains PTH after centrifugal is suspended in the buffered soln of 20mmol/LTris-HCl, pH7.5, contain 2.5mmol/L EDTA and 20% sucrose in the solution simultaneously, we are called high osmotic buffer with this solution, experiment is got the 1g thalline and is added in the 30ml high osmotic buffer, solution stirring becomes the suspendible shape, leaves standstill 10min under 0～4 ℃.
2) thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone fusion rotein:
With the high osmotic buffer 10000rpm that contains PTH after leaving standstill, 4 ℃ of following centrifugal 10min, get the thalline after centrifugal, the thalline that contains PTH1-84 after centrifugal is suspended in the buffered soln of 30ml 20mmol/L Tris-HCl, pH7.5, contain 2.5mmol/L EDTA in the solution simultaneously, we are called hypotonic buffer liquid with this sucrose-free solution.Treat in the hypotonic buffer liquid behind the thalline thorough mixing, this suspension solution is heated to 80 ℃, keep 15min down at 90 ℃ then, to suspension solution 10000rpm after heat treatment, 4 ℃ of following centrifugal 10min at last, collect the supernatant liquor after centrifugal, promptly obtain the product behind recombinant human parathyroid hormone (PTH1-84) the fusion rotein preliminary purification.PTH1-84 fusion rotein purity 75% after purified, yield 66%.
Embodiment 2
The method of thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone (PTH) fusion rotein, its operation steps is as follows:
1) fermentation liquor pretreatment
Fermented liquid is centrifugal through 8000rpm, 30min, the thalline that contains PTH after centrifugal is suspended in the buffered soln of 20mmol/LTris-HCl, pH9.0, contain 5.0mmol/L EDTA and 30% sucrose in the solution simultaneously, we are called high osmotic buffer with this solution, experiment is got the 1.5g thalline and is added in the 45ml high osmotic buffer, solution stirring becomes the suspendible shape, leaves standstill 10min under 0～4 ℃.
2) thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone fusion rotein:
With the high osmotic buffer 10000rpm that contains PTH after leaving standstill, 4 ℃ of following centrifugal 10min, get the thalline after centrifugal, the thalline that contains PTH after centrifugal is suspended in the buffered soln of 45ml 20mmol/L Tris-HCl, pH9.0, contain 2.5mmol/L EDTA in the solution simultaneously, we are called hypotonic buffer liquid with this sucrose-free solution.Treat in the hypotonic buffer liquid behind the thalline thorough mixing, this suspension solution is heated to 80 ℃, keep 10min down at 100 ℃ then, to suspension solution 10000rpm after heat treatment, 4 ℃ of following centrifugal 10min at last, collect the supernatant liquor after centrifugal, promptly obtain the product behind recombinant human parathyroid hormone (PTH) the fusion rotein preliminary purification.PTH fusion rotein purity 80% after purified, yield 60%.
Embodiment 3
The method of thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone (PTH) fusion rotein, its operation steps is as follows:
1) fermentation liquor pretreatment
Fermented liquid is centrifugal through 7000rpm, 30min, the thalline that contains PTH after centrifugal is suspended in the buffered soln of 20mmol/LTris-HCl, pH8.0, contain 2.5mmol/L EDTA and 20% sucrose in the solution simultaneously, we are called high osmotic buffer with this solution, experiment is got the 2g thalline and is added in the 60ml high osmotic buffer, solution stirring becomes the suspendible shape, leaves standstill 10min under 0～4 ℃.
2) thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone fusion rotein:
With the high osmotic buffer 10000rpm that contains PTH after leaving standstill, 4 ℃ of following centrifugal 10min, get the thalline after centrifugal, the thalline that contains PTH after centrifugal is suspended in the buffered soln of 60ml 20mmol/L Tris-HCl, pH8.0, contain 2.5mmol/L EDTA in the solution simultaneously, we are called hypotonic buffer liquid with this sucrose-free solution.Treat in the hypotonic buffer liquid behind the thalline thorough mixing, this suspension solution is heated to 80 ℃, keep 10min down at 80 ℃ then, to suspension solution 10000rpm after heat treatment, 4 ℃ of following centrifugal 10min at last, collect the supernatant liquor after centrifugal, promptly obtain the product behind recombinant human parathyroid hormone (PTH) the fusion rotein preliminary purification.PTH fusion rotein purity 78% after purified, yield 70%.
Claims (2)
- The method of 1 one kinds of thermoosmosis shock preliminary purification recombinant human parathyroid hormone (PTH) fusion roteins is characterized in that, comprises following operation steps:1) fermentation liquor pretreatmentFermented liquid is centrifugal through 1000～15000rpm, 1～30min, the thalline that contains PTH after centrifugal is suspended in the buffered soln of 20mmol/L Tutofusin tris, pH7.5～9.0, containing 2.5～5mmol/L ethylenediamine tetraacetic acid (EDTA) and mass ratio in the solution simultaneously is 20%～30% sucrose; Add 10ml～100ml high osmotic buffer by every gram thalline, solution stirring becomes suspendible, leaves standstill 5min～60min then under 0～30 ℃;2) thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone fusion rotein:The high osmotic buffer that contains PTH after leaving standstill is lower than under 30 ℃ with 1000～15000rpm, centrifugal 1～30min, get the thalline after centrifugal, the thalline that contains PTH after centrifugal is suspended in the hypotonic buffer liquid of 20mmol/L Tris-HCl, pH7.5～9.0, contains 2.5mmol/L EDTA in this solution, do not contain sucrose; Hypotonic buffer liquid add-on is identical with the high osmotic buffer amount that adds before this; Treat in the hypotonic buffer liquid behind the thalline thorough mixing, this suspension solution is heated to 80～100 ℃, and keep 5～60min, suspension solution that at last will be after heat treatment is at the supernatant liquor of collecting with 1000～15000rpm, centrifugal 5～60min under 4 ℃ after centrifugal, promptly obtains the product behind recombinant human parathyroid hormone (PTH) the fusion rotein preliminary purification.
- 2. the method for a kind of thermoosmosis shock preliminary purification recombinant human parathyroid hormone (PTH) fusion rotein according to claim 1 and 2 is characterized in that, comprises following operation steps:1) fermentation liquor pretreatmentFermented liquid is centrifugal through 7000rpm, 30min, the height that the thalline that contains PTH after centrifugal is suspended in 20mmol/LTris-HCl, pH8.0 oozes in the buffered soln, containing 2.5mmol/L EDTA and mass ratio in the solution simultaneously is 20% sucrose, add-on adds the 30ml high osmotic buffer by every gram thalline, solution stirring becomes suspendible, leaves standstill 10min then under 0～4 ℃;2) thermoosmosis shock technology preliminary purification recombinant human parathyroid hormone fusion rotein:With the high osmotic buffer 10000rpm that contains PTH after leaving standstill, 4 ℃ of following centrifugal 10min, get the thalline after centrifugal, the thalline that contains PTH after centrifugal is suspended in the hypotonic buffer solution of 20mmol/L Tris-HCl, pH8.0, contain 2.5mmol/LEDTA in the solution simultaneously, hypotonic buffer liquid add-on is identical with the high osmotic buffer amount that adds before this; Treat in the hypotonic buffer liquid behind the thalline thorough mixing, this suspension solution is heated to 80 ℃, and at 80 ℃ of following 10min that keep, suspension solution 10000rpm that at last will be after heat treatment, 4 ℃ of down centrifugal 10min collect the supernatant liquor after centrifugal, promptly obtain the product behind recombinant human parathyroid hormone (PTH) the fusion rotein preliminary purification.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2505812B2 (en) * | 1987-07-10 | 1996-06-12 | 旭化成工業株式会社 | Freeze-dried composition of h-PTH (1-34) |
| BR0003051A (en) * | 2000-07-10 | 2002-02-19 | Comissao Nac Energia Nuclear | Process for obtaining human growth hormone (somatropin) in the periplasmic space of bacteria, using recombinant DNA techniques and process to perform its purification until obtaining an injectable product in humans |
| CN1408865A (en) * | 2001-09-25 | 2003-04-09 | 中国人民解放军军事医学科学院生物工程研究所 | Oligonucleotide of coding human parathyroid hormone and its high efficiency expression method |
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- 2005-04-28 CN CNB2005100255196A patent/CN1303100C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2505812B2 (en) * | 1987-07-10 | 1996-06-12 | 旭化成工業株式会社 | Freeze-dried composition of h-PTH (1-34) |
| BR0003051A (en) * | 2000-07-10 | 2002-02-19 | Comissao Nac Energia Nuclear | Process for obtaining human growth hormone (somatropin) in the periplasmic space of bacteria, using recombinant DNA techniques and process to perform its purification until obtaining an injectable product in humans |
| CN1408865A (en) * | 2001-09-25 | 2003-04-09 | 中国人民解放军军事医学科学院生物工程研究所 | Oligonucleotide of coding human parathyroid hormone and its high efficiency expression method |
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