CN107988291A - The methanol feeding zymotechnique of Pichia anomala expression recombination human source collagen - Google Patents
The methanol feeding zymotechnique of Pichia anomala expression recombination human source collagen Download PDFInfo
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- CN107988291A CN107988291A CN201810048908.8A CN201810048908A CN107988291A CN 107988291 A CN107988291 A CN 107988291A CN 201810048908 A CN201810048908 A CN 201810048908A CN 107988291 A CN107988291 A CN 107988291A
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 title claims abstract description 246
- 230000014509 gene expression Effects 0.000 title claims abstract description 25
- 102000008186 Collagen Human genes 0.000 title claims abstract description 14
- 108010035532 Collagen Proteins 0.000 title claims abstract description 14
- 229920001436 collagen Polymers 0.000 title claims abstract description 14
- 230000006798 recombination Effects 0.000 title claims abstract description 13
- 238000005215 recombination Methods 0.000 title claims abstract description 13
- 235000014683 Hansenula anomala Nutrition 0.000 title claims abstract description 12
- 241000235063 Wickerhamomyces anomalus Species 0.000 title abstract 2
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 23
- 238000000855 fermentation Methods 0.000 claims abstract description 22
- 230000004151 fermentation Effects 0.000 claims abstract description 22
- 230000006698 induction Effects 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 230000001954 sterilising effect Effects 0.000 claims abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 16
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 14
- 229910052760 oxygen Inorganic materials 0.000 claims description 14
- 239000001301 oxygen Substances 0.000 claims description 14
- 244000286779 Hansenula anomala Species 0.000 claims description 13
- 235000011187 glycerol Nutrition 0.000 claims description 7
- 101150051118 PTM1 gene Proteins 0.000 claims description 3
- 229910052602 gypsum Inorganic materials 0.000 claims description 2
- 239000002054 inoculum Substances 0.000 claims description 2
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 2
- 229910052564 epsomite Inorganic materials 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 11
- 238000005516 engineering process Methods 0.000 abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052799 carbon Inorganic materials 0.000 abstract description 4
- 238000009776 industrial production Methods 0.000 abstract description 3
- 230000002503 metabolic effect Effects 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 description 12
- 108090000623 proteins and genes Proteins 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 10
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 7
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 7
- 238000003872 feeding technique Methods 0.000 description 7
- 238000011534 incubation Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 239000000654 additive Substances 0.000 description 3
- 230000000996 additive effect Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 238000010612 desalination reaction Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000001728 nano-filtration Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 108010025188 Alcohol oxidase Proteins 0.000 description 2
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000000908 ammonium hydroxide Substances 0.000 description 2
- 230000037319 collagen production Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 206010054949 Metaplasia Diseases 0.000 description 1
- 241001597008 Nomeidae Species 0.000 description 1
- 206010000210 abortion Diseases 0.000 description 1
- 231100000176 abortion Toxicity 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000013028 medium composition Substances 0.000 description 1
- 230000015689 metaplastic ossification Effects 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000009822 protein phosphorylation Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
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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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
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Abstract
The invention discloses a kind of methanol feeding zymotechnique of Pichia anomala expression recombination human source collagen.Pichia pastoris strain liquid is inoculated into the fermentation medium of sterilizing by the technique, cultivate to methanol feeding induction period, the preliminary stage of 0~32h of methanol feeding induction, methanol feeding speed is 5.25 ± 0.25L/h, the mid-term stage of 33h~96h, methanol feeding speed is 6.75 ± 0.25L/h, and the later stage of 97h~fermentation ends, methanol feeding speed is 5.25 ± 0.25L/h.Make constant speed stream stage by stage into when the present invention is by methanol induction phase to add, it is ensured that demand of the Pichia pastoris in different metabolic period to carbon source.Present invention process determines, workable, easily forms standardization file, reduces the yield caused by technology mistake and declines, is adapted to the large-scale industrial production of recombination human source collagen.
Description
Technical field
The invention belongs to fermentation engineering field, and in particular to a kind of Pichia anomala expression recombination human source collagen
Methanol feeding zymotechnique.
Background technology
Pichia pastoris yeast (Pichia pastoris) expression system is a kind of exogenous protein expression system, is both had
The advantages such as prokaryotic expression system operation is simple, be easy to culture, the speed of growth is fast, expression quantity is high, cost is low, also with prokaryotes
The features such as modification to foreign protein that expression system does not have is as glycosylated, protein phosphorylation.
Invitrogen companies of the U.S. have made up pichia pastoris yeast expression system, and oneself successfully express it is several
Hundred kinds of foreign proteins, become currently used expression system.Its zymotechnique developed, industry are divided into level-one, secondary seed
Culture, glycerine cultivation stage, glycerol feeding stage and methanol induction phase.The technique is current pichia pastoris yeast expression life
Produce foreign protein, more general zymotechnique (Chinese patent 201010602114.5,201110327865.5).Pichia pastoris
Induced expression stage, methanol are not only derivant, and are the sole carbon source and the energy of cell growth or maintenance metabolism, therefore no matter weigh
How is the Methanol Utilization Phenotype of group Pichia pastoris, and control of the methanol concentration during Pichia anomala expression foreign protein is very
It is crucial.Methanol concentration is too low, can not effectively induce the transcription of alcohol oxidase (AOX), and then influence exogenous protein expression efficiency, first
Determining alcohol is too high, then thalline can be caused to be poisoned, and reduces destination protein expression quantity.
Methanol feeding amount is generally to be reached to adjust methanol additive amount, the work according to integrative feedbacks such as oxygen dissolving value, incubation times
Skill is technical strong, and common laborer is difficult to grasp, and is not easy to form standardization file.Actual Pichia anomala expression recombinant protein
In industrialized production, for main operator based on common laborer, excessively complicated operation is easy to cause higher error rate, sends out
Give birth to technology mistake event and then cause proving an abortion for zymotechnique.Therefore, methanol complicated in existing zymotechnique is simplified
Feeding method, is allowed to form workable standardization file, can be grasped by common direct labor, so as to reduce because of technology
Caused by error yield decline, be more suitable for large-scale industrial production, for recombinant protein industrialized production offer convenience and
Great economic value.
The content of the invention
It is an object of the invention to the methanol feeding technique of the existing Pichia anomala expression recombinant protein of simplification, there is provided a kind of
Technique is simple and convenient to operate the Pichia anomala expression recombination human source that feasible, recombinant protein amount is high, is mass produced suitable for industrialization
The methanol feeding zymotechnique of collagen.
Technical scheme is as follows：
The methanol feeding zymotechnique of Pichia anomala expression recombination human source collagen, comprises the following steps：Red ferment will be finished
Female strain liquid is inoculated into the fermentation medium of sterilizing, is cultivated to methanol feeding induction period, 0~32h of methanol feeding induction
Preliminary stage, methanol feeding speed is 5.25 ± 0.25L/h, the mid-term stage of 33h~96h, and methanol feeding speed is 6.75
± 0.25L/h, the later stage of 97h~fermentation ends, methanol feeding speed are 5.25 ± 0.25L/h.
Pichia pastoris yeast Pichia pastoris of the present invention, are 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
Fully disclosed in 201110327865.5.
Pichia pastoris fermentation medium of the present invention uses existing formula, Ke Yishi：85%H3PO46.6~
26.7mL/L CaSO4·2H2O 0.3~1.175g/L, K2SO44.5~18.2g/L, MgSO4·7H23.7~14.9g/L of O,
1.0~4.13g/L of KOH, 0.435~4.35mL/L of glycerine 10.0~40.0g/L, PTM1 solution.
In the zymotechnique of the present invention, the air velocity of preliminary stage is 30m3/ h, the air velocity of mid-term stage are
40m3/ h, the air velocity of later stage is 45m3/h。
In the zymotechnique of the present invention, the inoculum concentration of Pichia pastoris strain liquid is 8~12%, and fermentation temperature is 28~30
DEG C, dissolved oxygen is not less than 20%, when the methanol induction time is 100~120 small.
Compared with prior art, the present invention has the following advantages：
(1) Pichia pastoris methanol feeding technique of the invention, it is no longer necessary to according to integrative feedbacks such as oxygen dissolving value, incubation times
Reach and adjust methanol additive amount, from technical standpoint, be more suitable for industrialized production；
(2) Pichia pastoris methanol feeding technique of the invention, different phase stream adds the amount of methanol to be relatively fixed, easy to be formed
Standardize file, is conducive to production management；
(3) Pichia pastoris methanol feeding technique of the invention, it is simple and easy to do, reduce error probability, personnel easy to produce
Implement.
Compared with former technique, present invention process is simple, though the rate of recovery is suitable with former technique with purity, destination protein yield
5% or so is improved, and the technique mass produces recombination human source collagen more suitable for industrialization.
Embodiment
The methanol feeding technique of the Pichia anomala expression recombinant protein of the present invention, treats pichia pastoris yeast Pichia
Pastoris was cultivated to the methanol feeding stage, and early period (0~32h) bacterium is dense relatively small, methanol feeding speed for 5.25 ±
0.25L/h, air velocity 30m3/h；Mid-term (33h~96h) cell concentration is big, metabolism is vigorous, methanol feeding speed for 6.75 ±
0.25L/h, air velocity 40m3/h；Later stage (97h~fermentation ends) metabolic capability weakens, methanol feeding speed for 5.25 ±
0.25L/h, air velocity 45m3/h。
The methanol feeding technique of existing Pichia anomala expression recombinant protein, it is comprehensive anti-according to oxygen dissolving value, incubation time etc.
Feedback adjusts methanol additive amount, specifically：Set methanol feeding to link with dissolved oxygen, when dissolved oxygen starts stream plus methanol, dissolved oxygen higher than 20%
Stop stream plus methanol less than 20%, reach plateau after inducing 112h, go out tank after inducing 120h.
After induced expression, zymotic fluid is centrifuged, collect the supernatant after centrifugation concentrated successively, ultrafiltration and nanofiltration
Decoloration desalination, obtains recombination human source collagen.
Unless otherwise specified, the strain and each culture medium used in the embodiment of the present invention and comparative example is substantially as follows：
1st, strain：Pichia pastoris yeast Pichia pastoris, deposit number：CGMCCNo.5021, in 2011
June 29 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and in Chinese patent
Fully disclosed in 201110327865.5.
2nd, culture medium：
(1) primary-seed medium is BMGY culture mediums, forms and is：
(2) secondary seed medium composition is：
(3) fermentation medium includes following components：
Wherein PTM1 is formed：
Methanol feeding pre-fermentation process, step are as follows：
(1) first order seed culture
The strain that glycerine preserves is seeded in the shaking flask containing 200mL seed culture mediums, 30 DEG C, 250rpm cultures 24h
~36h, cultivates to thalline weight in wet base and reaches 20 ± 2g/L；
(2) secondary seed culture
Primary seed solution is all transferred in the 100L fermentation tanks containing 60L fermentation mediums, 30 DEG C of cultures, use ammonium hydroxide
Adjust and control ph is 5.0, dissolved oxygen control is cultivated to thalline weight in wet base and reaches 120 ± 10g/L 20%~30%；
(3) fermentation tank culture
Secondary seed solution is transferred in the 1000L fermentation tanks containing 60L fermentation mediums, adds the glycerine of 60g/L, 30 DEG C
Culture, is adjusted with ammonium hydroxide and control ph is 5.0, throughput 30m3/ h, stir speed (S.S.) is 300~500rpm, when dissolved oxygen is steep
When so rising, hungry 1h makes glycerol depletion；
Embodiment 1
Methanol feeding stage, early period (0~32h), methanol feeding speed are 5L/h, air velocity 30m3/h；Mid-term (33h
~96h), methanol feeding speed is 6.5L/h, air velocity 40m3/h；Later stage (97h~fermentation ends), methanol feeding speed are
5L/h, air velocity 45m3/h.Go out tank after methanol induction, the zymotic fluid after induced expression is centrifuged, collect centrifugation
Supernatant afterwards is concentrated successively, ultrafiltration and nanofiltration decoloration desalination, obtain recombination human source collagen.
Embodiment 2
Methanol feeding early period speed is 5.25L/h to the present embodiment as different from Example 1, and mid-term methanol feeding speed is
6.75L/h, later stage methanol feeding speed are 5.25L/h, other steps are same as Example 1.
Embodiment 3
Methanol feeding early period speed is 5.5L/h to the present embodiment as different from Example 1, and mid-term methanol feeding speed is
7L/h, later stage methanol feeding speed are 5.5L/h, other steps are same as Example 1.
Comparative example 1
Methanol feeding early period speed is 4.75L/h to the present embodiment as different from Example 1, and mid-term methanol feeding speed is
6.25L/h, later stage methanol feeding speed are 4.75L/h, other steps are same as Example 1.
Comparative example 2
Methanol feeding early period speed is 5.75L/h to the present embodiment as different from Example 1, and mid-term methanol feeding speed is
7.25L/h, later stage methanol feeding speed are 5.75L/h, other steps are same as Example 1.
Comparative example 3
Methanol induction, induction period temperature are 30 DEG C, and pH value control is 5.0, and setting methanol feeding links with dissolved oxygen, when molten
Oxygen starts stream plus methanol higher than 20%, and dissolved oxygen stops stream plus methanol less than 20%, reaches plateau after inducing 112h, induces 120h
After go out tank；Zymotic fluid after induced expression is centrifuged, collect the supernatant after centrifugation concentrated successively, ultrafiltration and nanofiltration
Decoloration desalination, obtains recombination human source collagen.
The fermentation results of 1 each embodiment of table and comparative example
| Methanol usage | Collagen production | Protein recovery | Purity of protein | |
| Comparative example 1 | 666 | 19.32g/L | 72.7% | 95.7% |
| Comparative example 2 | 786 | 20.16g/L | 71.8% | 96.2% |
| Comparative example 3 | 642 | 17.42g/L | 72.3% | 94.7% |
| Embodiment 1 | 696 | 20.65g/L | 75.7% | 96.8% |
| Embodiment 2 | 726 | 21.14g/L | 76.3% | 98.1% |
| Embodiment 3 | 756 | 20.87g/L | 76.8% | 97.9% |
As can be seen from the table, carried using methanol feeding technique of the present invention, collagen production more than 20g/L, yield
High by more than 5%, the rate of recovery of recombination human source collagen reaches more than 75%, better than comparative example.Utilize HPLC measure purifying
Collagen concentration and purity, expressing quantity significantly improve afterwards, and protein recovery also has corresponding raising, purity and comparative example phase
When.When the present invention is by methanol induction phase, by the linkage of oxygen dissolving value and methanol pump the stream of methanol will be controlled to add originally, made into
Constant speed stream adds stage by stage.Former technique causes Pichia pastoris to be constantly in the shape of carbon source insufficient supply in the express express target protein stage
State, must have an impact the yield of purpose product and former technology is stronger, need to control methanol feeding amount according to incubation time,
Common laborer is difficult to grasp, and is not easy to form standardization file.It can ensure to finish red ferment after making methanol into constant speed stream adding stage by stage
Female demand in different metabolic period to carbon source.The technique of the present invention determines at the same time, workable, easily forms standardization text
Part, reduces the yield caused by technology mistake and declines, be more suitable for large-scale industrial production, be the industrial metaplasia of recombinant protein
Production offers convenience and great economic value.
Claims (5)
1. the methanol feeding zymotechnique of Pichia anomala expression recombination human source collagen, it is characterised in that comprise the following steps：
Pichia pastoris strain liquid is inoculated into the fermentation medium of sterilizing, is cultivated to methanol feeding induction period, methanol feeding induction
0~32h preliminary stage, methanol feeding speed is 5.25 ± 0.25L/h, the mid-term stage of 33h~96h, methanol feeding speed
It is 5.25 ± 0.25L/h to spend for 6.75 ± 0.25L/h, the later stage of 97h~fermentation ends, methanol feeding speed.
2. methanol feeding zymotechnique according to claim 1, it is characterised in that comprise the following steps：By Pichia pastoris
Strain liquid is inoculated into the fermentation training zymotechnique of sterilizing, it is characterised in that the Pichia pastoris is pichia pastoris yeast
Pichia pastoris, deposit number are CGMCC No.5021.
3. methanol feeding zymotechnique according to claim 1, it is characterised in that the Pichia pastoris fermentation medium
Formula be 85%H3PO46.6~26.7mL/L, CaSO4·2H2O 0.3~1.175g/L, K2SO44.5~18.2g/L,
MgSO4·7H2O 1.0~4.13g/L of 3.7~14.9g/L, KOH, glycerine 10.0~40.0g/L, PTM1 solution 0.435~
4.35mL/L。
4. methanol feeding zymotechnique according to claim 1, it is characterised in that the air velocity of the preliminary stage
For 30m3/ h, the air velocity of mid-term stage is 40m3/ h, the air velocity of later stage is 45m3/h。
5. methanol feeding zymotechnique according to claim 1, it is characterised in that the inoculum concentration of Pichia pastoris strain liquid is
8~12%, fermentation temperature is 28~30 DEG C, and dissolved oxygen is not less than 20%, when the methanol induction time is 100~120 small.
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Cited By (8)
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| CN109402039A (en) * | 2018-10-16 | 2019-03-01 | 江南大学 | A kind of reinforcing MutSThe method of type Pichia anomala expression heterologous protein |
| CN109680025A (en) * | 2018-12-25 | 2019-04-26 | 江苏江山聚源生物技术有限公司 | Fermentation process for improving production level of recombinant human collagen and reducing protein degradation speed |
| CN111088304A (en) * | 2019-12-27 | 2020-05-01 | 深圳康泰生物制品股份有限公司 | Fermentation induction process for expressing recombinant enterovirus 71 type virus-like particles, enterovirus 71 type vaccine and preparation method thereof |
| CN111733200A (en) * | 2020-06-30 | 2020-10-02 | 浙江诸暨聚源生物技术有限公司 | Fermentation process for improving production level of recombinant collagen by adjusting addition mode of PTM1 |
| CN113564062A (en) * | 2021-09-10 | 2021-10-29 | 汉肽生物医药集团有限公司 | Fermentation process for shortening culture time and improving collagen content |
| CN113667709A (en) * | 2021-08-28 | 2021-11-19 | 西安德诺海思医疗科技有限公司 | Fermentation method of recombinant humanized collagen |
| CN114045321A (en) * | 2021-12-24 | 2022-02-15 | 江苏江山聚源生物技术有限公司 | Application of glutathione in fermentation production of recombinant human collagen |
| CN117534746A (en) * | 2023-11-10 | 2024-02-09 | 江苏创健医疗科技股份有限公司 | Methanol-induced fermentation process for expressing recombinant collagen |
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