CN101153269A - High-efficiency representation method of regrouped human growth hormone - Google Patents
High-efficiency representation method of regrouped human growth hormone Download PDFInfo
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- CN101153269A CN101153269A CNA2007101486623A CN200710148662A CN101153269A CN 101153269 A CN101153269 A CN 101153269A CN A2007101486623 A CNA2007101486623 A CN A2007101486623A CN 200710148662 A CN200710148662 A CN 200710148662A CN 101153269 A CN101153269 A CN 101153269A
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Abstract
The invention provides an optimized production process for recombinant human growth hormone as well as related expression carriers. The recombinant human growth hormone has steady secretory expression, improves the expression level by optimizing the fermentation process, and has the advantages of good stability, high expression level and simple production process. Compared with the traditional and classical HGH production method, the invention can effectively, simply and inexpensively obtain the recombinant human growth hormone.
Description
Technical field
The present invention relates to the genetically engineered field, more specifically, the invention provides a kind of high-density, recombinant human somatropin's expression process efficiently.
Background technology
Human growth hormone (hGH) is human endogenous hormone, by people's Anterior pituitary synthesis secretion, plays an important role aspect growing at human body.Grownup's plasma hGH level is 1-5ng/ml, and the transformation period is 15-20 minute.
Gene engineering expression rhGH starts from 1979, and people such as Goeddel place the hGH gene under the regulation and control of lactose operon, expresses the output that obtains 2.4mg/L in the intestinal bacteria system.Gray and he's colleague has realized secretion expression's (the rhGH gene connects the signal peptide sequence of escherichia coli alkaline phosphatase) of people rhGH-be secreted into colibacillary periplasmic space in 1985, but output is too low; After this, Becker utilizes different signal peptide sequences that the secreting, expressing level (being secreted into periplasmic space) of hGH is brought up to about 15mg/L respectively with people such as Chang.1987, people such as Kato were with killer's gene (kill gene) lotus root connection of hGH gene and bacterium, and the secreting, expressing level that obtains hGH reaches 20.5mg/L, and wherein direct secretion reaches 11.2mg/L to the level in the substratum, is trapped in about 8.6mg/L of periplasmic space.At present, the rhGH of listing all uses escherichia expression system production, and this production technique rhGH yield is lower, has much room for improvement.
1997, people such as Olazaran adopted pichia spp (Pichia pastoris) expression system to express rhGH first and have obtained success.But the expression level of the Pichia anomala expression rhGH that people such as Olazaran obtain is on the low side, and expression level also has only 13mg/L after optimizing, and realizes that the industrialization cost is higher.
We use pichia yeast expression system production recombinant human somatropin (rhGH): the gene order of the synthetic rhGH of at first full gene, it is cloned among the vector plasmid pPIC9K, subsequent transformation pichia spp strain GS115, by the high copy of G418 screening transformant, pass through screening then and obtain the high expression level engineering strain, and the engineering bacteria that obtains is carried out physiological and biochemical property evaluation and copy number of foreign gene mensuration.Set up main seed bank, and carried out the mitotic stability experiment.By classic methods, find that HGH can have certain expression, but expression level is lower, illustrates that this purpose zymotechnique also needs constantly to grope, the optimization for fermentation technology route improves expression level.
The invention provides a kind of recombinant human somatropin's novel fermentation method, adopt Pichia anomala expression system secreting, expressing, by optimization for fermentation technology, whole process stabilization, quick, easy, the recombinant human somatropin of acquisition high-density, high expression level.The present invention lays a good foundation for recombinant human somatropin's further study on the industrialization.
Summary of the invention
Purpose of the present invention just provides a kind of highdensity recombinant human somatropin's (rhGH) expressional scheme.Comprise the substratum and the inductive condition that are used for this scheme.
In a first aspect of the present invention, just provided the aminoacid sequence of a kind of novel recombinant human tethelin (rhGH), it is characterized in that its sequence is A-B, wherein A is a r-hGH 1-26 position signal peptide amino acid; B is that r-hGH 27-217 position is non-glycosylated amino acid, has removed A district signal peptide sequence.
In a second aspect of the present invention, a kind of engineering bacteria is provided, it is characterized in that it is integrated with the nucleotide sequence of the above-mentioned novel recombinant human tethelin of coding (rhGH).
In another preference, described engineering bacteria is a pichia spp.
In a third aspect of the present invention, a kind of recombinant human somatropin's pilot scale production method is provided, the method comprising the steps of:
A) under the expression condition that is fit to, in shaking bottle, cultivate host cell, thereby secreting, expressing goes out novel recombinant human somatropin;
B) under the expression condition that is fit to, the best pilot scale production decision of optimization in the 5L fermentor tank.
Description of drawings
Do not have.
Embodiment
The inventor is extensive studies by going deep into, by the system row optimization expression experiment to reorganization HGH, for the basic technology condition has been determined in last jar of fermentation; This technology is again by having carried out optimization constantly and checking in fermentor tank level (5L) then, finally determined the zymotechnique that this engineering bacteria is stable, by expression stability research, obtained can be in pichia spp and other yeast expression system the recombinant human somatropin (rhGH) of stability and high efficiency secreting, expressing.Finished the present invention on this basis.
The purpose of shake flask fermentation mainly is under close with the fermentor tank condition as far as possible condition; by a series of experiments as: the selection (table 1) of substratum, induce situations such as pH value, protective material type and concentration and feed supplement interpolation; grope and establish preliminary zymotechnique be: to adopt low salt culture medium, inducing temperature be 30 ℃, induce pH is 3.0～4.0, the feed supplement of selecting Yeast extract to express as HGH.Jar research that is optimized in the pilot scale of conditions of flask fermentation provides certain data refer.
Tentatively finish pilot experiment, obtain certain experimental data, stablizing high expression level technology for the later stage pilot scale provides certain data refer, provides foundation for further optimizing pilot process later on.With the 5L fermentor tank, working volume is 3L.Experimental subjects all adopts through the screening bacterial classification of stable high expression level.
After finishing preliminary pilot experiment, use the 5L fermentor tank, working volume 3L pilot scale fermentation is expressed HGH, determines and stable pilot scale fermentation technology, and this technology is through continuous many batches of pilot scales (5L volume), and data see Table 5, confirm process stabilizing.RhGH is gone up a jar technical study, and the expression amount that obtains target protein is more than 60%, and total amount reaches the 1g/L fermented liquid, and not tangible degraded band, and these all provide great convenience for purifying.Confirm that this zymotechnique is ripe and high-caliber.
In an example of the present invention, provide the preferred plan of tank body feed supplement.
In another example of the present invention, confirm this zymotechnique be stablize, ripe and high-caliber.
In another example of the present invention, provide pilot scale fermentation process optimization preferred plan.
Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, and every liter of fermented liquid is expressed the rhGH amount and can be reached 952mg, and 5L fermentor tank rhGH total amount can reach 4760mg, and the rhGH expression level can reach 63%, is fit to industrialization production.
The invention has the advantages that:
(1) this scheme has the advantages that high stable is expressed.This high-density high-efficiency expression method has been carried out the stable checking of zymotechnique, confirmed that this technology stability is good.
(2) by the crucial technological condition for fermentation of control, make expression level higher, apparently higher than the traditional classical method.
(3) zymotechnique is easy, the expression amount height.Owing to be secretory protein, can simplify purification procedures, make the scale operation recombinant human somatropin become possibility.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1 tank body supplying technics is optimized
Get mono-clonal, be inoculated in the BMGY primary seed solution, cultivate 17-20hr; In 1: 10 ratio two-stage inoculation in the 1L of 2 bottles of 250ml BMGY Erlenmeyer flask, cultivate about 4～8hr, the last jar of initial pH 5.0 of fermentation, induce pH 3.0,30 ℃ of temperature, DO＞35%, treat to add 50% glycerine 300ml with 10～15 commentaries on classics degree streams after dissolved oxygen rises, treat to begin to induce with commentaries on classics degree 1 with 100% methyl alcohol after dissolved oxygen rises once more, gradually speed-raising, induce the back keep sample every 4hr centrifugal-20 ℃ frozen, induce 48hr to finish.Sample detection SDS-PAGE, protein content.After inducing 8h, adding concentration with commentaries on classics degree 2 streams is that 10% Yeast extract solution is to inducing end.
Go up a jar checking based on shaking conclusion for a short time.The result shows, compares with not adding feed supplement, and adding Yeast extract can not be degraded by the better protecting target protein, therefore determines to add Yeast extract in last jar of technology.
The stable checking of embodiment 2 zymotechniques
Method is with embodiment 1.By this experiment, determine and verified the zymotechnique of HGH substantially.Under these processing condition, the expression level of HGH is stable, the expression amount height, the final sample scanning result shows that the expression amount of target protein is more than 60%, and total amount reaches the 1g/L fermented liquid, and the band of significantly not degrading, these all provide great convenience for purifying.Therefore, we can say this zymotechnique be stablize, ripe and high-caliber.
Embodiment 3 pilot scale fermentation process optimizations
Use the 5L fermentor tank, working volume 3L pilot scale fermentation is expressed rHGH,
1 engineering bacteria cultivation stage optimum result:
Substratum: less salt culture medium inoculated ratio: 1: 10
Culture temperature: 30 ℃ of cultivation stage pH:5.0
Initial DO
2:＞35% initial rotating speed: 250rpm
Air flow: 3L/min interlock rotating speed: 250～800rpm
Incubation time: 20～30h OD
600:＞150
2 engineering bacteria induction period optimum result:
Induction period pH:3.0 induction period DO:＞30%
Induction time: 48h induces and finishes OD
600:＞500
All quote in this application as a reference at all documents that the present invention mentions, just as each piece document quilt
Quote such as a reference separately.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 |
| Condition | BMGY | The less salt of sulfur acid ammonium (being adjusted to pH 6.0) with KOH | Less salt (being adjusted to pH 6.0) with ammoniacal liquor | Less salt+1%Yeast extract (being adjusted to pH 6.0) with ammoniacal liquor | Less salt+2% Peptone (being adjusted to pH 6.0) with ammoniacal liquor | Less salt+1% Peptone (being adjusted to pH 6.0) with ammoniacal liquor |
| Numbering | 7 | 8 | 9 | 10 | 11 | 12 |
| Condition | BMGY | The less salt of sulfur acid ammonium (being adjusted to pH 6.0) with KOH | Less salt (being adjusted to pH 6.0) with ammoniacal liquor | The less salt of sulfur acid ammonium+1%Yeast extract (being adjusted to pH 6.0) with ammoniacal liquor | Less salt+the 2%Peptone of sulfur acid ammonium (being adjusted to pH 6.0) with ammoniacal liquor | Less salt+the 1%Peptone of sulfur acid ammonium (being adjusted to pH 6.0) with ammoniacal liquor |
Table 1
| Lot identification mark | First | Second batch | The 3rd batch |
| The fermentation final volume | 5 | 5 | 5 |
| Results fermented liquid supernatant volume (L) | 2.8 | 3.1 | 3.0 |
| RhGH expression level (%) | 63 | 72 | 67 |
| HGH total amount (mg) | 4760 | 4935 | 5090 |
| Every liter of fermented liquid is expressed HGH amount (mg/L) | 952 | 987 | 1018 |
Table 2
Claims (6)
1. a recombinant human somatropin production method is characterized in that, the method comprising the steps of:
A) under the expression condition that is fit to, in shaking bottle, cultivate host cell, thereby secreting, expressing goes out novel recombinant human somatropin;
B) under the expression condition that is fit to, in fermentor tank, carry out large-scale production.
2. host cell as claimed in claim 1 is characterized in that it is a pichia spp.
3. host cell as claimed in claim 1 is characterized in that, it contains the nucleotide sequence of coding growth hormone protein (r-hGH).
4. host cell as claimed in claim 1 is characterized in that, the nucleotide sequence of the coding growth hormone protein (r-hGH) that it contains, and aminoacid sequence is A-B, the present invention has removed A district signal peptide sequence.Wherein,
A is a r-hGH 1-26 position signal peptide amino acid;
B is that r-hGH 27-217 position is non-glycosylated amino acid.
5. the method for claim 1 is characterized in that, the expression condition shown in the step (a) is: preferable substratum is No. 3 promptly low salt culture mediums of conditioned medium, and inducing temperature is 25-37 ℃, and preferable is 30 ℃; Inducing pH is 3-7, and preferable pH is 3-4.
6. the method for claim 1 is characterized in that, the expression condition shown in the step (b) is: preferable feed supplement is a yeast extract, and inducing temperature is 25-37 ℃, and preferable is 30 ℃; Induce the pH value to be 3.0-4.5, preferable is 3.0.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111662836A (en) * | 2019-03-05 | 2020-09-15 | 上海医药工业研究院 | Genetically engineered bacterium for expressing insulin precursor and preparation method and application thereof |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111662836A (en) * | 2019-03-05 | 2020-09-15 | 上海医药工业研究院 | Genetically engineered bacterium for expressing insulin precursor and preparation method and application thereof |
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Application publication date: 20080402 |