CN1854299A - Production of recombinant insulinum primary C peptide - Google Patents
Production of recombinant insulinum primary C peptide Download PDFInfo
- Publication number
- CN1854299A CN1854299A CN 200510025594 CN200510025594A CN1854299A CN 1854299 A CN1854299 A CN 1854299A CN 200510025594 CN200510025594 CN 200510025594 CN 200510025594 A CN200510025594 A CN 200510025594A CN 1854299 A CN1854299 A CN 1854299A
- Authority
- CN
- China
- Prior art keywords
- peptide
- gly
- leu
- expression
- gln
- 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.)
- Pending
Links
Images
Landscapes
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention provides a nucleic acid of the recombination human insulinogen C peptide fused protein to produce the recombination human insulinogen C peptide and construct, express, purify the relate engineering cells. The recombination gene is proper to express in the Pichia pastoris. The expression amount can be improved by optimizing the expression plasmid, the host bacteria and the process of the fermentation and the purification. So it can get the pure peptide in a low cost.
Description
Technical field
The present invention relates to gene engineering technology field.More specifically, the invention provides a kind of method of High-efficient Production recombinant insulinum primary C peptide, and the structure of relevant engineering cell, recombinant insulinum primary C peptide expression and purifying process.
Background technology
Human insulinogen C peptide is insulinogenic integral part, by the beta Cell of islet secretion, contains 31 amino acid, the human insulinogen C peptide level is about 1ng/ml in normal people's fasting plasma, rising rapidly in the feed back, peaked after 1 hour, is about 8 times of empty stomach value.
Research shows, human insulinogen C peptide can be used for treating diabetic microvascular complications such as diabetic nephropathy, it gathers by minimizing messangial cell epimatrix and causes the mesentery expansion, under the medium control carbohydrate metabolism of Regular Insulin situation, diabetic microvascular complications such as diabetic nephropathy is had therapeutic action.The human research shows: reduce the early stage glomerular filtration rate(GFR of diabetes for type i diabetes patient infusion human insulinogen C Toplink, significantly reduce the urinary protein excretion rate, improve peripheral neuropathy, increase the utilization of skeletal muscle to glucose.Human insulinogen C peptide is an endogenous material, and the security of use promotes greatly.Therefore, human insulinogen C peptide is safe and effective product.
The acquisition of Insulinogen C peptide mainly contain chemosynthesis and. two kinds of methods of genetically engineered.Chemical synthesis process because. the cost height. productive rate is low, the production cycle is long, shortcoming such as generation environmental pollution etc., is replaced by gene engineering method gradually.
And the gene engineering expression system mainly contains these two kinds of pichia spp and intestinal bacteria, and the molecular weight that is fit to expression product is all about 5-200KD, can not express maybe can only hang down and express molecular weight at 1 to 5KD small peptide.And also there is the problem that is difficult for detection in low-molecular-weight small peptide expression product.Multiple copied is a kind of effective means that improves the small peptide expression level.Make up multi-copy gene and can express the fusion rotein of larger molecular weight, reach the purpose that efficiently expresses and easily detect.
At present, adopt the method for pichia spp and escherichia coli expression human insulinogen C peptide all to have report.PerJonasson etc. adopt escherichia coli expression C peptide, and its method is the C peptide fusion protein that obtains multiple copied earlier with series connection method, carry out that enzyme is cut, purifying again.The connected C peptide of 7 copies of this report, expression product is present in endochylema, and the last yield of albumen is 400mg/L.Patent application 01112929.8 discloses adopts pichia spp secretion polyphone to express the C peptide of 8 copies, and protein expression level reaches 1g/L.
The present invention adopts expressing human Insulinogen C peptide in the novel Pichia anomala expression system born of the same parents, by the optimized gene sequence, and optimizes fermentation and purifying process, obtains the production method of quick, easy, stable, that yield is high a kind of human insulinogen C peptide.
Summary of the invention
Purpose of the present invention just provides a kind of method of efficient and/or easy production recombinant insulinum primary C peptide.
Another object of the present invention just provides the encoding sequence of regrouped human proinsulin C peptide series protein and is used for the expression vector and the engineering cell of this method.
In a first aspect of the present invention, just provided a kind of nucleotide sequence of the regrouped human proinsulin C peptide series protein of encoding, it is characterized in that, aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and nucleotides sequence is shown 95% above homogeny shown in described nucleotide sequence coded district and the SEQ ID NO:1.
In another preference, described nucleotide sequence contains the nucleotide sequence shown in the SEQ ID NO:1.
In a second aspect of the present invention, just provided the optimum combination of a kind of expression vector and host bacterium, it is characterized in that described expression vector contains the described nucleotide sequence of claim 1.
In another preference, described expression vector is pHIL-D2/C-fusion, and the host bacterium is GS115.
In a third aspect of the present invention, a kind of engineering cell is provided, it is characterized in that it is integrated with the described expression vector of claim 3.
In a fourth aspect of the present invention, a kind of method of producing the recombinant insulinum primary C peptide fusion rotein is provided, the method comprising the steps of:
A) under the expression condition that is fit to, cultivate host cell as claimed in claim 4, thereby give expression to the fusion rotein of placed in-line recombinant insulinum primary C peptide;
B) divide isolated fusion protein.
In a fifth aspect of the present invention, a kind of method of producing recombinant insulinum primary C peptide is provided, the method comprising the steps of:
A) cut fusion rotein of the present invention with trypsinase and protaminase enzyme, produce recombinant insulinum primary C peptide;
B) purification of Recombinant human insulinogen C peptide.
Description of drawings
Fig. 1 is the structure synoptic diagram of recombinant plasmid pHIL-D2/C-fusion.
Fig. 2 is that recombinant plasmid pHIL-D2/C-fusion enzyme is cut the evaluation electrophorogram.Adopt EcoRI digestion recombinant plasmid pHIL-D2/C-fusion among the figure, the result shows that recombinant plasmid pHIL-D2/C-fusion has the C peptide to insert fragment.
Fig. 3 is the recombinant bacterial strain of the high copy of dot hybridization screening C peptide gene.Carry out the dot blot experiment with C peptide gene as probe.The result screens the positive colony that contains high copy C peptide gene, and pichia spp host cell (negative control) does not then detect C peptide gene.
Embodiment
The inventor is by deeply and extensive studies, by the optimization design of gene coded sequence, fusion rotein efficiently expressed in the pichia spp cell.Finished the present invention on this basis.
In another preference, also, not only improved the recombinant insulinum primary C peptide fusion protein expression, and simplified separation purifying technique by the optimization of zymotechnique.
According to the human insulinogen C peptide natural acid sequence, press codon-bias, under the condition that does not change aminoacid sequence, the target gene sequences of full gene synthesizing recombined human Insulinogen C peptide series protein, with this gene clone in the pUC19 after the sequence verification, with the ordinary method of molecular cloning, be cloned into expression vector pHIL-D2.Transform, be integrated into the P.pastoris host cell chromosome, filter out the transformant of multi-copy integration by dot blot, and then filter out engineering cell.Shake flask test shows, induce 24 hours after, more than the expression level 1mg/ml.According to its speed that consumes methyl alcohol, judge that this engineering cell strain is Mut+.
After obtaining engineering cell, just can be under the condition that is fit to the culturing engineering cell.In the present invention, the recombinant insulinum primary C peptide fermentation condition of engineering bacterium expression is not particularly limited.Can adopt the fermentation condition of this area routine.For example, suitable medium includes, but is not limited to following composition:
(i) nitrogenous source contains organic nitrogenous source and inorganic nitrogen-sourced, wherein organic nitrogen source such as peptone, yeast powder, or the mixture of the two, total concn 0.1-3%; Inorganic nitrogen-sourced as ammoniacal liquor or NH
4Cl, (NH
4)
2SO
4Deng ammonium salt, concentration 0-1.5%.
(ii) inorganic salt comprise phosphoric acid salt, Chinese holly hydrochloride, Mg
2+Salt etc.Preferably, phosphate buffer concentration 20-200mM, pH5-8; Mg
2+Salt 0-5mM.
(iii) in substratum, add VITMAIN B1 VITAMIN as a supplement, concentration 1～1000PPM.
(iv) according to M
9Trace element formula in the substratum, trace element can add 0.1-2ml/L training liquid.
(v) carbon source comprises glycerine, glucose, lactose etc.Can be single carbon source, also can be mixed carbon source.Preferably, carbon source is selected from down group: glycerol concentration is 0.1-3%; Lactose concn is 0.1-3%; Glucose concn is 0.1-1.5%.
For the extensive recombinant insulinum primary C peptide that obtains, need in fermentor tank, express cultivation.The present invention has studied pilot scale fermentation technology, and the expression level after the optimization reaches 10mg/ml:
Behind fermentation expression, the recombinant insulinum primary C peptide fusion rotein of expressing is separated.Usually, fermented sample is earlier removed fermented liquid supernatant in modes such as centrifugal, filtrations, obtains thalline.Behind the buffered soln suspension thalline, carry out broken fully bacterium, obtain broken bacterium liquid in modes such as ultrasonic disruption, high-pressure machinery fragmentations.Then broken bacterium liquid is carried out centrifugal acquisition supernatant, carry out preliminary purification then, carrying out trypsinase and the protaminase enzyme is cut.Enzyme is cut product and is carried out chromatography purification, comprises cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, affinity chromatography etc.
Through different chromatography processes relatively, the purification process of optimization comprises:
1. adopt the mode of the broken thalline of high-pressure compressing, obtain supernatant.
2. adopt hydrophobic chromatography to obtain purity greater than 85% human insulinogen C peptide fusion protein.
3. adopt trypsinase and protaminase enzyme to cut the fusion rotein of acquisition
4. carry out ion exchange chromatography, hydrophobic chromatography and reverse chromatography, obtain purity, removed other impurity such as nucleic acid simultaneously greater than 95% pure product of recombinant insulinum primary C peptide.
5. through above-mentioned purification step, can obtain the pure product of recombinant insulinum primary C peptide, the purifying yield is more than 40%, and purity is more than 95%, the about 4g/L fermented liquid of pure product yield.
Recombinant insulinum primary C peptide can be made various formulations with routine techniques behind the purifying.
In an example of the present invention, the acquisition of recombinant insulinum primary C peptide antigen-4 fusion protein gene and the structure of expression plasmid are provided, the gene after the optimization makes the expression amount of express recombinant human insulinogen C peptide improve.
In another example of the present invention, obtained the bacterial strain of high expression level C peptide by screening, improved the expression amount of recombinant insulinum primary C peptide.
In another example of the present invention, the optimization of technology has by fermentation further improved the expression amount of recombinant insulinum primary C peptide.
In another example of the present invention,, can obtain pure product 4g/L through purifying.
Stoste adds suitable auxiliary material behind the purifying, makes the powder ampoule agent for injection of recombinant insulinum primary C peptide.The preliminarily stabilised test shows that this powder injection is stable.
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 can obtain the pure product 4g of recombinant insulinum primary C peptide.Be fit to industrialization production.
The invention has the advantages that:
(1) the recombinant insulinum primary C peptide antigen-4 fusion protein gene after the optimization is highly suitable in the pichia spp and expresses, and has the characteristics of high expression level, high stable.
(2) improved C peptide expression amount greatly by screening the engineering cell that contains high copy C peptide gene,, made expression level reach 10g/L by controlling crucial technological condition for fermentation.
(3) purifying process is easy, and rate of recovery height makes the scale operation recombinant insulinum primary C peptide 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.
The acquisition of embodiment 1 goal gene and the structure of expression plasmid
Because human insulinogen C peptide has only 31 amino acid, molecular weight is too little, and simultaneously in order to improve the yield of final product, we plan a plurality of C peptide genes and are cascaded and express.According to the aminoacid sequence of human insulinogen C peptide and the codon bias of pichia spp, and the C peptide fusion protein gene of connecting for convenience inserts expression vector and further expresses the back purifying, we have introduced the EcoRI site at series connection C peptide gene two ends, and introduced six His at its 5 ' end, introduce terminator codon at its 3 ' end.We have designed and synthesized the human insulinogen C peptide antigen-4 fusion protein gene that is suitable for Pichia anomala expression,, its sequence is fragment shown in SEQ ID NO:1, contains 8 series connection C peptide genes in the right direction.
The construction of recombinant plasmid circuit carries out the C-fusion gene of synthetic after enzyme cuts processing with EcoRI as shown in Figure 1, and agarose gel electrophoresis reclaims about 1kb fragment; Simultaneously plasmid pHIL-D2 is carried out enzyme with EcoRI and cut, reclaim big fragment.Two fragments are connected with the T4DNA ligase enzyme, connect the product conversion and enter bacillus coli DH 5 alpha (available from Promega company).Prepare plasmid in a small amount, the method for cutting evaluation by enzyme filters out the positive colony (Fig. 2) that contains C-fusion.PHIL-D2 can efficiently express its multi-copy integration after inserting foreign gene to the karyomit(e) of host bacterium, itself carry the screening that a HIS gene can be used for transformant simultaneously.Promotor AOX1 from host's Pichia yeast alcohol oxidase is arranged before the carrier pHIL-D2 multiple clone site, with can be in host bacterium GS115 behind the methanol induction efficiently expressing exogenous gene.
The screening of embodiment 2 high expression level C peptide engineering strains
The recombinant plasmid pHIL-D2/C-fusion that builds is prepared in a large number, linearizing, electroporation transformed host cell P.pastoris GS115, coating MD plate screening positive colony, because the MD flat board do not contain Histidine, the positive colony after having only recombinant plasmid to change over to could be grown.The total DNA of extracting positive colony as probe, screens the positive colony of high copy with the C-fusion gene with the method for dot blot.As shown in Figure 3, color is deeply felt more and is shown that C-fusion gene contained in the genome is many more, and promptly copy number is high more.
Choose several high copy positive colonies, inoculation is gone in the BMGY substratum, grow into state of saturation after, change the substratum BMMY of abduction delivering, get cell pyrolysis liquid after 48 hours, behind the preliminary purification, SDS-PAGE detects, and therefrom obtains a high-expression clone.
Embodiment 3pH is to the influence of expression level
Get mono-clonal, be inoculated in the BMGY primary seed solution, cultivate 17-20hr; In 1: 10 ratio two-stage inoculation (each condition has looped pipeline) in the 500ml of 50ml substratum Erlenmeyer flask, to cultivate about 24hr, 1% methanol induction, the pH of induction period need according to the form below to regulate (directly regulating pH in the BMMY substratum).Induce the 24hr sampling, measure OD and pH simultaneously, add 1% methyl alcohol again and continue to induce.Coinduction 48hr.Induce preceding, induce different time sample detection SDS-PAGE and immunoblotting to determine the content of polyphone C peptide.
Experimental result shows, is shaking a bottle level, and the optimal pH of expressing the C peptide is between 5～7.Best pH6.
The optimization of embodiment 4 induction times
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 250ml BMGY Erlenmeyer flask; cultivate about 4～8hr last jar of fermentation, pH6.0,30 ℃ of temperature, DO＞35%; treat to add 50% glycerine 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, look the growth particular case and raise speed gradually, and add protective material in the regular hour; induce the back to keep sample every 4hr; it is centrifugal to put the 100ml fermented liquid every 8hr, and-20 ℃ frozen, induces 72hr to finish.Sample detection SDS-PAGE (and scanning), protein content.
Experimental result shows that to induce 40 hours electrophoresis result about (sample 5) more satisfactory, and main band is denseer, and assorted band (degraded band) is less, and induces 60 hours later samples, obvious degradation, and main band fogs.At the sample of inducing before 20 hours, expression amount is on the low side.Therefore, by this experiment, technological condition for fermentation from now on should be controlled at induction time between 20 to 60 hours as far as possible.And best induction time was at 40 hours.
Embodiment 5 recombinant insulinum primary C peptide purifying
The bacterial cell disruption that will ferment is got precipitation and is carried out enzyme cut the back and adopt column chromatography to carry out purifying research behind method preliminary purifications such as oversalting or ultrafiltration.
(1) ion exchange chromatography:
Chromatography media: Q Sepharose Fast Flow
Chromatography sample-loading buffer: 10mMPB, pH7.4;
Chromatography elution buffer: 10mMPB+1MNaCl, pH7.4.
Gradient elution: 0-20%B, 3CV
Last sample, cleaning and elution flow rate are 30cm/hr.
(2) ultrafiltration
The target protein elutriant of collecting the Q column chromatography carries out ultrafiltration.
Through above purifying process, can get the pure product 4g/L of albumen at last.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.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.
Sequence table
＜110〉Shanghai Xinshengyuan Medicine Research Co., Ltd.
＜120〉production method of recombinant insulinum primary C peptide
<160>2
<210>1
<211>999
<212>DNA
＜213〉artificial sequence
<221>misc_feature
<222>(1)…(999)
＜223〉the recombinant insulinum primary C peptide fusion gene of You Huaing
<400>1
atgcatcatc?atcatcatca?tagatctaag?gaagctgaag?atttgcaagt?tggtcaagtt 60
gaattgggtg?gtgggcccgg?tgctggttct?ttgcaaccat?tggctttgga?aggttctttg 120
caaaaggctg?gatctaagga?agctgaagat?ttgcaagttg?gtcaagttga?attgggtggt 180
gggcccggtg?ctggttcttt?gcaaccattg?gctttggaag?gttctttgca?aaaggctgga 240
tctaaggaag?ctgaagattt?gcaagttggt?caagttgaat?tgggtggtgg?gcccggtgct 300
ggttctttgc?aaccattggc?tttggaaggt?tctttgcaaa?aggctggatc?taaggaagct 360
gaagatttgc?aagttggtca?agttgaattg?ggtggtgggc?ccggtgctgg?ttctttgcaa 420
ccattggctt?tggaaggttc?tttgcaaaag?gctggatcta?aggaagctga?agatttgcaa 480
gttggtcaag?ttgaattggg?tggtgggccc?ggtgctggtt?ctttgcaacc?attggctttg 540
gaaggttctt?tgcaaaaggc?tggatctaag?gaagctgaag?atttgcaagt?tggtcaagtt 600
gaattgggtg?gtgggcccgg?tgctggttct?ttgcaaccat?tggctttgga?aggttctttg 660
caaaaggctg?gatctaagga?agctgaagat?ttgcaagttg?gtcaagttga?attgggtggt 720
gggcccggtg?ctggttcttt?gcaaccattg?gctttggaag?gttctttgca?aaaggctgga 780
tctaaggaag?ctgaagattt?gcaagttggt?caagttgaat?tgggtggtgg?gcccggtgct 840
ggttctttgc?aaccattggc?tttggaaggt?tctttgcaaa?aggctggatc?taaggaagct 900
gaagatttgc?aagttggtca?agttgaattg?ggtggtgggc?ccggtgctgg?ttctttgcaa 960
ccattggctt?tggaaggttc?tttgcaaaag?gctggatcc 999
<210>2
<211>333
<212>PRT
＜213〉homo sapiens
<400>2
Met?His?His?His?His?His?His?Arg?Ser?Lys?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
1 5 10 15
Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?Ala?Leu
20 25 30 35
Glu?Gly?Ser?Leu?Gln?Lys?Ala?Gly?Ser?Lys?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
40 45 50
Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?Ala?Leu
55 60 65 70
Glu?Gly?Ser?Leu?Gln?Lys?Ala?Gly?Ser?Lys?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
75 80 85 90
Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?Ala?Leu
95 100 105
Glu?Gly?Ser?Leu?Gln?Lys?Ala?Gly?Ser?Lys?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
110 115 120 125
Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?Ala?Leu
130 135 140
Glu?Gly?Ser?Leu?Gln?Lys?Ala?Gly?Ser?Lys?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
145 150 155 160
Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?Ala?Leu
165 170 175 180
Glu?Gly?Ser?Leu?Gln?Lys?Ala?Gly?Ser?Lys?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
185 190 195
Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?Ala?Leu
200 205 210 215
Glu?Gly?Ser?Leu?Gln?Lys?Ala?Gly?Ser?Lys?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
220 225 230
Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?Ala?Leu
235 240 245 250
Glu?Gly?Ser?Leu?Gln?Lys?Ala?Gly?Ser?Lys?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
255 260 265 270
Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?Ala?Leu
275 280 285
Glu?Gly?Ser?Leu?Gln?Lys?Ala?Gly?Ser?Lys?Glu?Ala?Glu?Asp?Leu?Gln?Val?Gly
290 295 300 305
Gln?Val?Glu?Leu?Gly?Gly?Gly?Pro?Gly?Ala?Gly?Ser?Leu?Gln?Pro?Leu?Ala?Leu
310 315 320
Glu?Gly?Ser?Leu?Gln?Lys?Ala?Gly?Ser
325 330
Claims (7)
1. nucleotide sequence of human insulinogen C peptide fusion protein of encoding, it is characterized in that, aminoacid sequence shown in the described nucleotide sequence coded SEQ ID NO:2, and nucleotides sequence is shown 95% above homogeny shown in described nucleotide sequence coded district and the SEQ ID NO:1.
2. nucleotide sequence as claimed in claim 1 is characterized in that, contains the aminoacid sequence shown in the SEQ ID NO:2.
3. the optimum combination of expression vector and host bacterium is characterized in that described expression vector contains the described nucleotide sequence of claim 1.
4. an engineering cell is characterized in that, it is integrated with the described expression vector of claim 3.
5. engineering cell as claimed in claim 4 is characterized in that it is a pichia spp.
6. method of producing the human insulinogen C peptide fusion protein is characterized in that the method comprising the steps of:
(a) under the expression condition that is fit to, cultivate engineering cell as claimed in claim 4, thereby give expression to the human insulinogen C peptide fusion protein;
(b) separation and purification goes out the human insulinogen C peptide fusion protein of expression.
7. method as claimed in claim 6 is characterized in that, the expression condition shown in the step (a) is: induction time is 20-60 hour, and preferable is 30-50 hour; Inducing pH is 4-8, and that preferable is 5-7.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510025594 CN1854299A (en) | 2005-04-29 | 2005-04-29 | Production of recombinant insulinum primary C peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510025594 CN1854299A (en) | 2005-04-29 | 2005-04-29 | Production of recombinant insulinum primary C peptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1854299A true CN1854299A (en) | 2006-11-01 |
Family
ID=37194750
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510025594 Pending CN1854299A (en) | 2005-04-29 | 2005-04-29 | Production of recombinant insulinum primary C peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1854299A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101029077B (en) * | 2007-02-02 | 2010-09-29 | 广东东阳光药业有限公司 | Method for purifying gene-recombinant insulin precursor |
-
2005
- 2005-04-29 CN CN 200510025594 patent/CN1854299A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101029077B (en) * | 2007-02-02 | 2010-09-29 | 广东东阳光药业有限公司 | Method for purifying gene-recombinant insulin precursor |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1238490C (en) | Stable recombinant yeasts for fermenting xylose to ethanol | |
| CN1155712C (en) | Process for preparing enzyme encoded by cephalosporin acetylthydrolase gene | |
| CN1348009A (en) | Process for producing basic amino acid | |
| CN1974601A (en) | New-type Fc fusion protein and its production process | |
| CN114853881B (en) | Recombinant humanized fusion collagen and efficient hydroxylation method and application thereof | |
| CN1279171C (en) | Use of fusion proteins whose N-terminal part is a hirudin derivative for the production of recombinant proteins via secretion by yeasts | |
| CN1793375A (en) | Yeast expressing system of recombined human nerve growth factor and process for preparing recombined human nerve grouth factor | |
| CN1873006A (en) | Method for producing recombined human proinsulin | |
| CN106337054A (en) | Method for preparing high activity recombinant porcine epidermal growth factor | |
| CN1766098A (en) | A kind of mannase and encoding gene thereof and application | |
| CN1304422C (en) | Non-amidated omega-conotoxin VIIA and its prepn process and application | |
| CN1680443A (en) | Recombinat protein containing albumin multimer | |
| CN1105727C (en) | Process for preparing recombined human serum albumin | |
| CN1958797A (en) | Nucleotide sequence of lipase of antarctic candida | |
| CN1861790A (en) | Preparation process of human recombined parathyroid hormone 1 84 | |
| CN1854299A (en) | Production of recombinant insulinum primary C peptide | |
| CN100344758C (en) | Antibacterial peptide gene of Chinese prawn containing single whey acidic protein structure domain and its coded antibacterial peptide and application | |
| CN1125081C (en) | Recombined natural and new-type human insulin and its preparation | |
| CN1181199C (en) | Lichenized bacillus L-25 keratinase and its encoding DNA | |
| CN1869236A (en) | Production method of recombination ox intestine kinase | |
| CN1916024A (en) | Constructing mutant sequence of high relative quick alphd-2b interferon, expression plasmid of yeast, strain filtration, and purification method | |
| CN1308452C (en) | Pichia yeast phytase expression system containing reorganized ethyl alcohol oxidase promotor | |
| CN1876817A (en) | Production method for human tumor necrosis factor TNF-alpha mutant | |
| CN1618968A (en) | Production method of recombination human growth hormone | |
| CN1888066A (en) | Production process of recombinant human horny cell growth factor-2 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Shen Lili Document name: Deemed not to advise |
|
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Shen Lili Document name: Notice of conformity |
|
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Shen Lili Document name: Notification before expiration of term |
|
| C57 | Notification of unclear or unknown address | ||
| DD01 | Delivery of document by public notice |
Addressee: Shen Lili Document name: Notification that Application Deemed to be Withdrawn |
|
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |