CN105039471A - Method of increasing expression quantity of methylotrophic yeast expression system - Google Patents
Method of increasing expression quantity of methylotrophic yeast expression system Download PDFInfo
- Publication number
- CN105039471A CN105039471A CN201510175618.6A CN201510175618A CN105039471A CN 105039471 A CN105039471 A CN 105039471A CN 201510175618 A CN201510175618 A CN 201510175618A CN 105039471 A CN105039471 A CN 105039471A
- Authority
- CN
- China
- Prior art keywords
- precursor
- proteinase inhibitor
- expression
- fermentation
- nsc
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a method of increasing expression quantity of methylotrophic yeast expression system, wherein the method includes the steps of adding a protease inhibitor, such as benzamidine hydrochloride, in methanol and flow-adding the solution into a fermentation system during a fermentation inductive step, so that degradation of proteases in bacteria on recombinant proteins, thereby increasing the expression quantity of the recombinant proteins.
Description
Technical field
The present invention relates to field of microbial fermentation, be specifically related to a kind of method that can improve methanotrophic yeast expression system expression expression of recombinant proteins amount.
Background technology
Yeast expression system is one of current most widely used heterologous gene expression system, and methanotrophic yeast nutrition wherein require low, growth is fast, substratum is cheap, be easy to carry out operating and cultivating; The foreign protein of expressing can be secreted into outside born of the same parents, and the intrinsic protein of secretion is few, and foreign protein separation and purification is easy, and the pichia spp especially in methanotrophic yeast has been widely used in production.
But pichia spp is in expression recombinant protein process, for easy degraded proteinoid during the fermentation easily degrade by its spontaneous enzyme system, this expression to recombinant protein accumulation is very unfavorable.Generally take to improve fermenting process nitrogenous source level to reduce the degraded of thalline oneself protease to recombinant protein, this method easily causes the waste of material and then fermentation costs is raised, and the nitrogenous source increased might not have the expression of recombinant protein and promotes effect preferably, the nitrogenous source of fermentation ends remnants may cause certain difficulty to later stage extraction purification simultaneously.Also have and add proteinase inhibitor in the medium to suppress the scheme of the degraded of recombinant protein, but effect is not remarkable, does not have good method significantly to reduce the degraded of recombinant protein at present, so the method finding arrestin degraded seems very important.
Summary of the invention
The object of the invention is for prior art not enough, develop a kind of method improving methanotrophic yeast expression system expression expression of recombinant proteins amount.
For achieving the above object, the invention provides a kind of method improving methanotrophic yeast expression system expression amount: in methanotrophic yeast fermentation induction period, containing proteinase inhibitor in the methyl alcohol that stream adds.
In some embodiments, proteinase inhibitor is NSC 2020 or phenylmethylsulfonyl fluoride.
In other embodiments, proteinase inhibitor is NSC 2020.
In some embodiments, the content of proteinase inhibitor is 0.005mol/L ~ 0.15mol/L.
In some embodiments, methanotrophic yeast is pichia pastoris phaff.
In some embodiments, the recombinant protein of expression is selected from protaminase precursor, insulin precurosor, insulin precursors analogs, short hyperglycemic-glycogenolytic factor class precursor or trypsinase precursor.
The term " precursor " that the present invention uses refers to the single chain polypeptide that can be obtained target protein by one or more chemistry subsequently and/or enzymatic step." insulin precursors analogs " refers to the insulin precursor molecule that A and/or B chain has one or more sudden change relative to human insulin molecule, substitutes, deletes and/or add.In like manner, as utilized the recombinant expressed short hyperglycemic-glycogenolytic factor class of genetically engineered, Lantus, insulin aspart, moral paddy Regular Insulin, Insulin lispro, trypsinase, carboxypeptidase etc., be all express respective single chain polypeptide, namely precursor protein, it is known that these belong to art technology.
Unless explicitly stated otherwise in contrast, otherwise all scopes that the present invention quotes comprise end value.Such as, " content of proteinase inhibitor is 0.005mol/L ~ 0.15mol/L " represents that the content n of proteinase inhibitor is 0.005mol/L≤n≤0.15mol/L.
Advantage of the present invention is: 1) add proteinase inhibitor content less, cost is low, does not also affect later separation and purifies; 2) mode with methanol feeding imports compared with proteinase inhibitor directly adds with fermentation substrate to fermentation system, and cell growth impact is gentleer, is conducive to increasing expression of recombinant proteins amount further.
Embodiment
The following stated be the preferred embodiment of the present invention, what the present invention protected is not limited to following preferred implementation.It should be pointed out that on the basis of conceiving in these innovation and creation for a person skilled in the art, the some distortion made and improvement, all belong to protection scope of the present invention.
Raw material used in embodiment all can be obtained by commercial sources, bacterial classification construction process used belongs to general knowledge as well known to those skilled in the art, the present invention's bacterial classification host cell used and expression vector, with reference to Multi-CopyPichiaExpressionKit and EasySelect of Invitrogen
tMthe test kit of PichiaExpressionKit illustrates, belongs to technology known.Illustrated embodiment fermentation process of the present invention, carry out with reference in the PichiaFermentationProcessGuidelines of Invitrogen company, its concrete operations are:
Fermention medium is by BSM, and 1% yeast extract (YeastExtract) and PTM1 form.PH, after 121 DEG C of high-temperature sterilization 30min, is adjusted to 5.0 with 50% ammoniacal liquor by BSM and yeast extract, then by 4mL/L add through sterile filtration PTM1 and get final product.
Fermentation is divided into three steps:
The first step: fermention medium cultivates about 20h;
Second step: add 50% glycerine containing 12mL/LPTM1 with the data rate stream of 10mL/h/L, the time is 6h.
3rd step: induce with the methyl alcohol that the data rate stream of 6mL/h/L adds containing 12mL/LPTM1.For making the DO value in Induction Process be not less than 25%, need regulate methanol feeding speed, induction 72h, bacterium liquid OD600 reach after more than 500 and terminate to cultivate.Get fermented liquid in fermentation culture process, get supernatant liquor HPLC after centrifugal and detect protein content.
Embodiment 1
The recombinant expressed protaminase precursor of Pichia pastoris GS115
With methanol feeding abduction delivering, do not add any proteinase inhibitor, get 80h and 110h fermented liquid centrifugal, HPLC measures protaminase precursor expression amount, with 80h concentration for standard value 100 in contrast.Ferment in the same way before experimental group fermentation inducement, methanol induction process is respectively at adding 0.05mol/L NSC 2020 and 0.05mol/L concentration phenylmethylsulfonyl fluoride (PMSF) in methyl alcohol, 80h, 110h sampling is simultaneously centrifugal, HPLC measures protaminase precursor expression amount, and itself and control group Experimental comparison results are as table 1.
Table 1 adds the impact of proteinase inhibitor on protaminase precursor expression amount
Embodiment 2
Pichia spp X33 recombinant expression protein enzyme precursor
With methanol feeding abduction delivering, do not add any proteinase inhibitor, get 80h and 110h fermented liquid centrifugal, HPLC measures trypsinase precursor expression amount, with 80h concentration for standard value 100 in contrast.Ferment in the same way before experimental group fermentation inducement, methanol induction process is respectively at adding 0.05mol/L NSC 2020 and 0.05mol/L concentration phenylmethylsulfonyl fluoride (PMSF) in methyl alcohol, 80h, 110h sampling is simultaneously centrifugal measures trypsinase precursor expression amount with HPLC, and itself and control group Experimental comparison results are as table 2.
Table 2 adds the impact of proteinase inhibitor on trypsinase precursor expression amount
Embodiment 3
The recombinant expressed protaminase precursor of Pichia pastoris GS115
With methanol feeding abduction delivering, do not add any proteinase inhibitor, get 80h and 110h fermented liquid centrifugal, HPLC measures protaminase precursor expression amount, with 80h concentration for standard value 100 in contrast.Ferment in the same way before experimental group fermentation inducement, methanol induction process is respectively at adding 0.001mol/L, 0.005mol/L, 0.05mol/L, 0.10mol/L, 0.15mol/L, 0.20mol/L concentration NSC 2020 and 0.005mol/L, 0.05mol/L, 0.10mol/L, 0.15mol/L, 0.20mol/L concentration PMSF in methyl alcohol, 80h, 110h sampling is simultaneously centrifugal measures protaminase precursor expression amount with HPLC, and itself and control group Experimental comparison results are as following table 3 and table 4.
Table 3 adds the impact of NSC 2020 on protaminase precursor expression amount
Table 4 adds the impact of phenylmethylsulfonyl fluoride on protaminase precursor expression amount
Embodiment 4
Proteinase inhibitor addition manner is on the impact of the recombinant expressed protaminase precursor of Pichia pastoris GS115
With methanol feeding abduction delivering, basestocks adds and the NSC 2020 of methanol feeding equivalent or PMSF respectively, does not add NSC 2020 or PMSF in methyl alcohol, gets 80h and 110h fermented liquid centrifugal, HPLC measures protaminase precursor expression amount, contrasts using 80h concentration as standard value 100.Ferment in the same way before experimental group fermentation inducement, basestocks does not add NSC 2020 or PMSF, methanol induction process is respectively at adding 0.05mol/L concentration NSC 2020 and 0.05mol/L concentration PMSF in methyl alcohol, 80h, 110h sampling is simultaneously centrifugal measures protaminase precursor expression amount with HPLC, and itself and control group Experimental comparison results are as following table 5.
Table 5 adds the impact of phenylmethylsulfonyl fluoride on protaminase precursor expression amount
Claims (6)
1. improve a method for methanotrophic yeast expression system expression amount, it is characterized in that, in the fermentation inducement stage, containing proteinase inhibitor in the methyl alcohol that stream adds.
2. method according to claim 1, is characterized in that, described proteinase inhibitor is selected from NSC 2020 or phenylmethylsulfonyl fluoride.
3. method according to claim 1, is characterized in that, described proteinase inhibitor is NSC 2020.
4. method according to claim 1 and 2, is characterized in that, the content of described proteinase inhibitor is 0.005mol/L ~ 0.15mol/L.
5. method according to claim 1, is characterized in that, described methanotrophic yeast is pichia pastoris phaff.
6. method according to claim 1, is characterized in that, described albumen is selected from protaminase precursor, insulin precurosor, insulin precursors analogs, short hyperglycemic-glycogenolytic factor class precursor or trypsinase precursor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510175618.6A CN105039471B (en) | 2014-04-18 | 2015-04-14 | Method for improving expression quantity of methanol nutritional yeast expression system |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410159377 | 2014-04-18 | ||
| CN2014101593771 | 2014-04-18 | ||
| CN201510175618.6A CN105039471B (en) | 2014-04-18 | 2015-04-14 | Method for improving expression quantity of methanol nutritional yeast expression system |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105039471A true CN105039471A (en) | 2015-11-11 |
| CN105039471B CN105039471B (en) | 2022-11-18 |
Family
ID=54446439
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510175618.6A Active CN105039471B (en) | 2014-04-18 | 2015-04-14 | Method for improving expression quantity of methanol nutritional yeast expression system |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105039471B (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314490A (en) * | 2000-03-21 | 2001-09-26 | 李宏 | Method for producing yeast expressed human tissue plasminogen activator and its activity detection |
| CN101270339A (en) * | 2007-03-20 | 2008-09-24 | 广州伯凯生物技术有限公司 | Cultivation method for saccharomycete excreting expression proteolytic enzyme |
| CN101423845A (en) * | 2008-08-29 | 2009-05-06 | 浙江理工大学 | Method for synthesizing protein using in vitro expression of silkworm baculovirus |
| CN102115718A (en) * | 2010-12-16 | 2011-07-06 | 南京工业大学 | Recombinant strain for expressing beta-galactosidase and construction method and application thereof |
| CN102127533A (en) * | 2010-12-31 | 2011-07-20 | 华南农业大学 | Preparation method of recombinant porcine circovirus type 2 Cap antigen |
| CN103804492A (en) * | 2014-02-26 | 2014-05-21 | 辽宁大学 | Glycosylation method of wild-type chicken cystatin inhibitor |
-
2015
- 2015-04-14 CN CN201510175618.6A patent/CN105039471B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1314490A (en) * | 2000-03-21 | 2001-09-26 | 李宏 | Method for producing yeast expressed human tissue plasminogen activator and its activity detection |
| CN101270339A (en) * | 2007-03-20 | 2008-09-24 | 广州伯凯生物技术有限公司 | Cultivation method for saccharomycete excreting expression proteolytic enzyme |
| CN101423845A (en) * | 2008-08-29 | 2009-05-06 | 浙江理工大学 | Method for synthesizing protein using in vitro expression of silkworm baculovirus |
| CN102115718A (en) * | 2010-12-16 | 2011-07-06 | 南京工业大学 | Recombinant strain for expressing beta-galactosidase and construction method and application thereof |
| CN102127533A (en) * | 2010-12-31 | 2011-07-20 | 华南农业大学 | Preparation method of recombinant porcine circovirus type 2 Cap antigen |
| CN103804492A (en) * | 2014-02-26 | 2014-05-21 | 辽宁大学 | Glycosylation method of wild-type chicken cystatin inhibitor |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105039471B (en) | 2022-11-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US11136373B2 (en) | Fermentation process for increasing production level of recombinant human collagen | |
| KR101157437B1 (en) | Growth medium for c. histolyticum without ingredients of mamalian sources | |
| KR101786386B1 (en) | A manufacturing methods of fermented soybean meal using lactic acid bacteria | |
| CN103937830B (en) | A kind of recombinant bacterium of efficient secretory expression Nattokinase | |
| RU2020127568A (en) | METHOD FOR EXTRACTING PROTEIN MATERIAL AND/OR FIBROUS MATERIAL FROM SPENT BEER GRAINS AND ITS APPLICATION; | |
| CN116240121B (en) | Kluyveromyces marxianus, fermentation method, composition and application thereof | |
| Kang et al. | Enhanced production of leech hyaluronidase by optimizing secretion and cultivation in Pichia pastoris | |
| CN106282274A (en) | A kind of Pichia sp. fermentation process in high density of insulin precursor protein | |
| CN105255771A (en) | Staphylococcus succinus for generating collagenase and application thereof | |
| CN102181419A (en) | Trichoderma reesie protease as well as preparation method and application thereof | |
| CN105316261A (en) | Method for improving thermal stability of lactobacillus preparation | |
| CN103436575A (en) | High-density fermentation medium and high-density fermentation method for pichia pastoris recombinant bacteria | |
| CN105039471A (en) | Method of increasing expression quantity of methylotrophic yeast expression system | |
| ES2656049T3 (en) | Use of roasted glucose for the fermentative production of taxtomine | |
| KR20150004088A (en) | A manufacturing methods of fermented soybean meal using lactic acid bacteria | |
| CN103876005A (en) | Compound microbial agent for promoting settlement of sweet potato starch slurry | |
| CN110663813B (en) | Fish meal microbial fermentation and enzymolysis method | |
| WO2021072963A1 (en) | Candida utilis double gene co-expression strain that hydrolyzes protein components in food waste and construction method therefor | |
| CN103865865B (en) | A kind of sea cucumber enteron aisle produces Sumizyme MP bacterial strain and application thereof | |
| JPH03500969A (en) | fermentation method | |
| CN104099261A (en) | Pichia yeast engineering bacteria producing aspergillus-niger glucose oxidase and application thereof | |
| CN102154140A (en) | High-efficiency preparation method of beta-amylase | |
| CN104450599A (en) | Probiotic corn leavening agent capable of increasing content of L-lactic acid and application of probiotic corn leavening agent | |
| CN104745395A (en) | Method for making wine by applying multi-microorganism bacterial solution | |
| CN104762354B (en) | A kind of method for improving recombinant protein inclusion body and being formed |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200927 Address after: 443300, No. 38, Binjiang Road, Yidu, Hubei, Yichang Applicant after: YICHANG HEC CHANGJIANG PHARMACEUTICAL Co.,Ltd. Address before: 523808 Guangdong city of Dongguan province Hubei Songshan Industrial Park Industrial Road No. 1 Applicant before: SUNSHINE LAKE PHARMA Co.,Ltd. Applicant before: YICHANG HEC CHANGJIANG PHARMACEUTICAL Co.,Ltd. |
|
| GR01 | Patent grant | ||
| GR01 | Patent grant |