CN106008702B - A kind of method that scale isolates and purifies recombination human source collagen - Google Patents
A kind of method that scale isolates and purifies recombination human source collagen Download PDFInfo
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- CN106008702B CN106008702B CN201610585372.4A CN201610585372A CN106008702B CN 106008702 B CN106008702 B CN 106008702B CN 201610585372 A CN201610585372 A CN 201610585372A CN 106008702 B CN106008702 B CN 106008702B
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- 108010035532 Collagen Proteins 0.000 title claims abstract description 37
- 102000008186 Collagen Human genes 0.000 title claims abstract description 36
- 229920001436 collagen Polymers 0.000 title claims abstract description 36
- 238000005215 recombination Methods 0.000 title claims abstract description 34
- 230000006798 recombination Effects 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 21
- 239000012535 impurity Substances 0.000 claims abstract description 21
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 19
- 239000004202 carbamide Substances 0.000 claims abstract description 19
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 18
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 17
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 13
- 238000001641 gel filtration chromatography Methods 0.000 claims abstract description 10
- 238000001471 micro-filtration Methods 0.000 claims abstract description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 27
- 239000007853 buffer solution Substances 0.000 claims description 18
- 239000006228 supernatant Substances 0.000 claims description 16
- 235000018102 proteins Nutrition 0.000 claims description 15
- 241000235058 Komagataella pastoris Species 0.000 claims description 13
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 12
- 229960001484 edetic acid Drugs 0.000 claims description 11
- 239000004475 Arginine Substances 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000003513 alkali Substances 0.000 claims description 10
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 150000003384 small molecules Chemical class 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 238000000855 fermentation Methods 0.000 claims description 8
- 230000004151 fermentation Effects 0.000 claims description 8
- 229920005654 Sephadex Polymers 0.000 claims description 6
- 239000012507 Sephadex™ Substances 0.000 claims description 6
- 239000012141 concentrate Substances 0.000 claims description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 5
- 235000018417 cysteine Nutrition 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 239000012460 protein solution Substances 0.000 claims description 5
- GSEJCLTVZPLZKY-UHFFFAOYSA-N Triethanolamine Chemical compound OCCN(CCO)CCO GSEJCLTVZPLZKY-UHFFFAOYSA-N 0.000 claims description 2
- 239000000945 filler Substances 0.000 claims description 2
- 238000009987 spinning Methods 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims 2
- 239000007789 gas Substances 0.000 claims 1
- 238000011084 recovery Methods 0.000 abstract description 9
- 238000004519 manufacturing process Methods 0.000 abstract description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 4
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 4
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 3
- 238000000926 separation method Methods 0.000 abstract description 3
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 239000007791 liquid phase Substances 0.000 description 4
- 238000005374 membrane filtration Methods 0.000 description 4
- 238000009777 vacuum freeze-drying Methods 0.000 description 4
- 244000005700 microbiome Species 0.000 description 3
- 241000894006 Bacteria Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 244000286779 Hansenula anomala Species 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 1
- 235000014683 Hansenula anomala Nutrition 0.000 description 1
- 108010093096 Immobilized Enzymes Proteins 0.000 description 1
- 108010050808 Procollagen Proteins 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000012876 carrier material Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 235000013373 food additive Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000030788 protein refolding Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Toxicology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods that scale isolates and purifies recombination human source collagen; by the way that linear recombinant protein is carried out just folding and refolding; the space radius of accurate control protein molecular; not only improve the accurate type selecting of micro-filtration and ultrafiltration membrane aperture; the rate of recovery of albumen can be improved again; use in conjunction micro-filtration and ultrafiltration; macromolecular and small molecular weight impurity are removed respectively; protein concentration is realized in ultrafiltration simultaneously; alleviate the load of later period gel filtration chromatography separation; again by the way that urea is added, realizes that recombinant protein is folded and untwist, avoid the residual of nuisance.The recombination human source collagen that the method for the present invention obtains, purity is up to 99%, and the rate of recovery is higher than 65%, and method is easy to operate, significantly reduces the cost of the large-scale production of recombination human source collagen, is particularly suitable for industrialized production.
Description
Technical field
The invention belongs to bioproduct separation technical field of purification, and in particular to a kind of scale isolates and purifies recombination human source
The method of collagen.
Background technique
Collagen is the most abundant class protein family of content in animal body, can be used as food additives, feed adds
Add agent, flocculant, photoemulsion material, surfactant box Carrier Materials of Immobilized Enzyme etc., is applied to each industrial circle.Mesh
The production of procollagen is mainly obtained from animal tissue using the method for chemical extraction.In order to improve the life of collagen
Object safety, purity and activity widen it in the application in the fields such as cosmetics and medical instrument, and technique for gene engineering is used extensively
In by the hosts such as human-like collagen gene cloning to bacterium, yeast, plant, zooblast, source of people glue is prepared to express
Former albumen.
World patent WO 106494 discloses a kind of human-like collagen and its production method, utilizes Bacillus coli expression
Obtain human-like collagen；Chinese patent 201110327865.5 discloses a kind of recombination human source collagen and its preparation side
Method constructs Pichia pastoris recombination human source collagen gene engineering bacterium expression and obtains human-like collagen, both methods
All have been realized in industrialization.But in human-like collagen industrialization process, main production cost and critical technological point are all
It concentrates on isolating and purifying stage, especially Escherichia coli and also needs to carry out microorganism collection and broken wall, finally also need to carry out albumen multiple
Property.During the isolating and purifying of Pichia anomala expression recombination human source collagen, there are many difficult points, wherein using UF membrane and
The link of film decoloration becomes main bottleneck, and reason is the linear structure of recombination human source collagen, is difficult basis first
The true molecular amount of linear structure albumen chooses suitable film core, and secondly linear structure albumen is difficult to pass through film core bore
Gap is easy to build up, result in blockage, so that film device flux declines rapidly, cannot achieve industrialization large-scale production.
Summary of the invention
The purpose of the present invention is to provide the recombination human source collagen isolation and purification method that is industrially easily achieved of one kind,
By the way that linear recombinant protein is carried out just folding and refolding, the accurate space radius for controlling protein molecular not only improves micro-filtration
With the accurate type selecting of ultrafiltration membrane aperture, and the rate of recovery of albumen can be improved, use in conjunction micro-filtration and ultrafiltration remove macromolecular respectively
And small molecular weight impurity, while ultrafiltration realizes protein concentration, alleviates the load of later period gel filtration chromatography separation, then passes through addition
Urea, untwisting after realizing recombinant protein refolding, avoids the residual of nuisance.
Technical scheme is as follows:
A kind of method that scale isolates and purifies recombination human source collagen, process flow are as follows: pichia pastoris yeast
Bacterium Pichia pastoris fermentation liquid is centrifugated laggard line refolding proteins, and it is miscellaneous to be utilized respectively micro-filtration removal macromolecular
Matter and ultrafiltration remove small molecular weight impurity, and urea induction is added after removal of impurities and untwists, freeze-drying is purified after gel chromatography
Recombination human source collagen, specific steps are as follows: Pichia Pastoris Pichia pastoris fermentation liquid is centrifuged, is collected
Supernatant, supernatant first carries out just folding in the first buffer solution of pH >=9, then is being passed through enough air, 9 pH≤11 <
The second buffer solution in carry out sufficient refolding, be passed through after folding nitrogen stablize albumen, later micro-filtration remove protein solution
In big molecular impurity, ultrafiltration removes small molecular weight impurity, while carrying out the concentration of protein, is added 1~10M's in concentrate
Urea makes refolding proteins untwist, and solution spinning liquid is purified through gel filtration chromatography, is freeze-dried, obtains recombination human source collagen.
Pichia pastoris yeast Pichia pastoris of the present invention is preserved in China on June 29th, 2011
Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC No.5021, and in Chinese patent
It is sufficiently disclosed in 201110327865.5.
Big molecular impurity of the present invention refers to that molecular weight is greater than the impurity of recombination human source collagen.
Small molecular weight impurity of the present invention refers to that molecular weight is less than the impurity of recombination human source collagen.
First buffer solution include following components: 1.5M urea, 0.4M arginine, 5~80mM small molecule alkali,
8mM EDTA (ethylenediamine tetra-acetic acid).
Second buffer solution include following components: 1.5M urea, 25mM cysteine, 150mM arginine, 0.3
~1.5mM DTT (dithiothreitol dithio), 2~50mM small molecule alkali, 5mM EDTA.
The small molecule alkali is triethylamine or triethanolamine, preferably triethylamine.
The concentration of small molecule alkali in first buffer solution is preferably 10~25mM, small in the second buffer solution
The concentration of molecule alkali is preferably 15~35mM.
The concentration of the urea is preferably 4~8M.
The filler of the gel filtration chromatography is preferably Sephadex G-100.
Compared with prior art, remarkable result of the invention is as follows: the present invention by during isolating and purifying to linear
It the refolding of albumen and untwists, effectively controls the molecule space radius of albumen, linear recombination human source collagen is by folding
Supination is conciliate, molecular weight does not change, and removal of impurities is avoided while ensure that the stability of recombination human source collagen
The problem of filter membrane caused by protein accumulation blocks in the process, and improve the rate of recovery of albumen.The recombination that the method for the present invention obtains
Human-like collagen, purity is up to 99%, and the rate of recovery is higher than 65%, and this method is easy to operate, significantly reduces recombination human source glue
The cost of the large-scale production of former albumen is particularly suitable for industrialized production.
Specific embodiment
The strain that the present invention uses are as follows: pichia pastoris yeast Pichia pastoris, deposit number:
CGMCCNo.5021 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on June 29th, 2011
The heart, and sufficiently disclosed in Chinese patent 201110327865.5.
Below with reference to embodiment, the invention will be further described.
Embodiment 1
(1) pichia pastoris yeast Pichia pastoris fermentation liquid is centrifuged, collects supernatant.
(2) it is added in supernatant containing 1.5M urea, 0.4M arginine, 10mM triethylamine, 8mM EDTA, pH is
In 9.0 the first buffer solution, 37 DEG C warm bath 30 minutes.
(3) it is added in supernatant and contains 1.5M urea, 25mM cysteine, 150mM arginine, 0.3mM DTT, 15mM
Triethylamine, 5mM EDTA in the second buffer solution that pH is 9.3, are passed through enough air, 37 DEG C warm bath 8 hours, then pass to foot
Amount nitrogen keeps the recombination human source after overlapping collagen-stabilized.
(4) protein solution after overlapping is removed into big molecular impurity through micro-filtrate membrane filtration, then through ultrafiltration membrance filter, removed
Small molecular weight impurity is simultaneously concentrated albumen.
(5) 4M urea, 40 DEG C of unwindase 12 h are added in protein concentrate solution.
(6) column is filled sufficiently after balance using Sephadex G-100 gel to take 800mL to untwist solution, use nucleic acid-protein instrument
Absorption peak at 220nm is detected, destination protein peak is collected.
(7) albumen after gel filtration chromatography is subjected to vacuum freeze drying, obtains recombination human source collagen.
The recombination human source collagen that the present embodiment obtains, by efficient liquid phase chromatographic analysis, measuring its purity is 98%,
The rate of recovery that BCA measures albumen is 67%.
Embodiment 2
(1) pichia pastoris yeast Pichia pastoris fermentation liquid is centrifuged, collects supernatant.
(2) it is added in supernatant and contains 1.5M urea, 0.4M arginine, 18mM triethylamine, 8mM EDTA, pH 9.5
The first buffer solution in, 37 DEG C warm bath 30 minutes.
(3) it is added in supernatant and contains 1.5M urea, 25mM cysteine, 150mM arginine, 0.9mM DTT, 15mM
Triethylamine, 5mM EDTA in the second buffer solution that pH is 10.0, are passed through enough air, 37 DEG C warm bath 8 hours, then pass to
Enough nitrogen keeps the recombination human source after overlapping collagen-stabilized.
(4) protein solution after overlapping is removed into big molecular impurity through micro-filtrate membrane filtration, then through ultrafiltration membrance filter, removed
Small molecular weight impurity is simultaneously concentrated albumen.
(5) 6M urea, 40 DEG C of unwindase 12 h are added in protein concentrate solution.
(6) column is filled sufficiently after balance using Sephadex G-100 gel to take 800mL to untwist solution, use nucleic acid-protein instrument
Absorption peak at 220nm is detected, destination protein peak is collected.
(7) albumen after gel filtration chromatography is subjected to vacuum freeze drying, obtains recombination human source collagen.
The recombination human source collagen that the present embodiment obtains, by efficient liquid phase chromatographic analysis, measuring its purity is 99%,
The rate of recovery that BCA measures albumen is 68%.
Embodiment 3
(1) pichia pastoris yeast Pichia pastoris fermentation liquid is centrifuged, collects supernatant.
(2) it is added in supernatant and contains 1.5M urea, 0.4M arginine, 25mM triethylamine, 8mM EDTA, pH 10.0
The first buffer solution in, 37 DEG C warm bath 30 minutes.
(3) it is added in supernatant and contains 1.5M urea, 25mM cysteine, 150mM arginine, 1.5mM DTT, 35mM
Triethylamine in the second buffer solution that 5mM EDTA, pH are 11.0, is passed through enough air, 37 DEG C warm bath 8 hours, then pass to
Enough nitrogen keeps the recombination human source after overlapping collagen-stabilized.
(4) protein solution after overlapping is removed into big molecular impurity through micro-filtrate membrane filtration, then through ultrafiltration membrance filter, removed
Small molecular weight impurity is simultaneously concentrated albumen.
(5) 8M urea, 40 DEG C of unwindase 12 h are added in protein concentrate solution.
(6) column is filled sufficiently after balance using Sephadex G-100 gel to take 800mL to untwist solution, use nucleic acid-protein instrument
Absorption peak at 220nm is detected, destination protein peak is collected.
(7) albumen after gel filtration chromatography is subjected to vacuum freeze drying, obtains recombination human source collagen.
The recombination human source collagen that the present embodiment obtains, by efficient liquid phase chromatographic analysis, measuring its purity is 96%,
The rate of recovery that BCA measures albumen is 67%.
Comparative example
(1) pichia pastoris yeast Pichia pastoris fermentation liquid is centrifuged, collects supernatant.
(2) supernatant is removed into big molecular impurity through micro-filtrate membrane filtration, then through ultrafiltration membrance filter, removes small molecular weight impurity
And albumen is concentrated.
(3) column is filled sufficiently after balance using Sephadex G-100 gel to take 800mL to untwist solution, use nucleic acid-protein instrument
Absorption peak at 220nm is detected, destination protein peak is collected.
(4) albumen after gel filtration chromatography is subjected to vacuum freeze drying, obtains recombination human source collagen.This implementation
The recombination human source collagen that example obtains, by efficient liquid phase chromatographic analysis, measuring its purity is that 98%, BCA measures albumen
The rate of recovery is lower than 40%.
Claims (3)
1. a kind of scale from supernatant of the Pichia Pastoris Pichia pastoris fermentation liquid through being collected by centrifugation
Change the method for isolating and purifying recombination human source collagen, which is characterized in that specific step is as follows: by Pichia Pastoris
The centrifugation of Pichia pastoris fermentation liquid, collects supernatant, and supernatant first carries out just folding in the first buffer solution of pH >=9
It is folded, then sufficient refolding is carried out in being passed through enough air, 9 pH≤11 < the second buffer solution, nitrogen is passed through after folding
Gas stablizes albumen, and the big molecular impurity in the protein solution of micro-filtration removal later, ultrafiltration removes small molecular weight impurity, while carrying out albumen
The concentration of matter, the urea that 4~8M is added in concentrate make refolding proteins untwist, and solution spinning liquid is purified through gel filtration chromatography, cold
Be lyophilized it is dry after obtain recombination human source collagen,
Wherein, the deposit number of the pichia pastoris yeast Pichia pastoris is CGMCC No.5021；
First buffer solution includes following components: 1.5M urea, 0.4M arginine, 5~80mM small molecule alkali, 8mM second
Ethylenediamine tetraacetic acid (EDTA)；
Second buffer solution include following components: 1.5M urea, 25mM cysteine, 150mM arginine, 0.3~
1.5mM dithiothreitol dithio, 2~50mM small molecule alkali, 5mM ethylenediamine tetra-acetic acid；
The small molecule alkali is triethylamine or triethanolamine；
The filler of the gel filtration chromatography is Sephadex G-100；Moreover,
The purity of the recombination human source collagen reaches 99%.
2. the method according to claim 1, wherein the concentration of the small molecule alkali in first buffer solution
For 10~25mM.
3. according to the method described in claim 2, it is characterized in that, the concentration of the small molecule alkali in second buffer solution
For 15~35mM.
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Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6653098B1 (en) * | 1998-02-23 | 2003-11-25 | G. D. Searle & Co. | Method of producing mouse and human endostatin |
| CN1260247C (en) * | 2003-11-05 | 2006-06-21 | 上海中科伍佰豪生物工程有限公司 | Large-scale cytorrhyctes compound purifying technology for recombining tPA derivative |
| SG162834A1 (en) * | 2006-07-14 | 2010-07-29 | Genentech Inc | Refolding of recombinant proteins |
| CN102443057B (en) * | 2011-10-26 | 2013-10-30 | 南京理工高新技术发展有限公司 | Recombinant humanized collagen and its preparation method |
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