CN1869236A - Production method of recombination ox intestine kinase - Google Patents
Production method of recombination ox intestine kinase Download PDFInfo
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- CN1869236A CN1869236A CN 200510026133 CN200510026133A CN1869236A CN 1869236 A CN1869236 A CN 1869236A CN 200510026133 CN200510026133 CN 200510026133 CN 200510026133 A CN200510026133 A CN 200510026133A CN 1869236 A CN1869236 A CN 1869236A
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Abstract
The invention supplies a nucleotide sequence of reconstructing cattle enterokinase and the method to produce, and the technology to express the carrier, and the construct, express and purification to the engineer cell. It optimizes the fermenting and purifying technology, improves the expression quantity and purification yield. The invention has the advantages of high expression, good stability, high activity and simple producing technology. It could make reconstructing cattle enterokinase in high efficiency, convenient and low cost.
Description
Technical field
The present invention relates to gene engineering technology field.More specifically, the invention provides a kind of High-efficient Production recombination ox intestine kinase (Enterokinase or Enteropeptidase, production method EK), and the expression and the purifying process of the structure of relevant engineering cell, recombination ox intestine kinase.
Background technology
At present, in field of biology, many times require scinderin, target protein and transporter are cut, therefore need be in the special proteolysis site of design, the joining region of target protein and transporter, many commercialization carriers have been arranged before its multiple clone site and added the sequence in proteolysis site between the coding transporter sequence.Common proteolytic enzyme mainly contains Xa factor, zymoplasm, enteropeptidase (Enterokinase orEnteropeptidase) or the like.But it is worthy of note that these enzymes can not be realized cutting fully many times in the actually operating.In addition, the specificity of proteolytic enzyme neither be absolute, has two kinds as the optimum Cutting site of zymoplasm, and as seen its possibility that non-special cutting takes place in actual system is very big.
Based on above various reasons, in numerous alternative enzyme solutions, enteropeptidase as a kind of to (Asp)
4The serine protease of high degree of specificity is revealed in-Lys sequence table, and is relatively loose because of its reaction conditions, possesses activity fully between 4 ℃-45 ℃ of pH4.5-9.5, temperature; Have or not denaturing agent also all not influence enzyme and cut effect.The possibility that fracture takes place at substrate protein white matter other irrelevant positions in the reaction process is low, and its cleavage site is after whole recognition sequences, the first amino acid of the target protein that cuts out can complete faithful to native protein etc. characteristics, one of first-selected toolenzyme of downstream purification when becoming amalgamation and expression in the genetic engineering pharmaceutical field.
Enteropeptidase is a kind of proteolytic enzyme of taking charge of food digestion of vertebrates duodenal wall mucous membrane excretory, also finds to have analogue in recent years in microorganism, blood, insect, starfish.The enteropeptidase of ox is made up of two subunits of size, wherein small subunit has the essential characteristic of activity center, thereby be named as catalytic subunit, form by 235 amino acid, theoretical molecular 26,262 dalton, theoretical iso-electric point (pI) 5.1, its enzyme of possessing holoenzyme is cut high degree of specificity active and to substrate, lays a good foundation for Enteropeptidase is widely studied with application in the genetic engineering pharmaceutical field.
Abroad the expression study to recombination ox intestine kinase catalytic subunit (EKL) starts from 1993, in mammalian cell COS, carried out functional expression by LaVallie of U.S. GI Company Inc. etc., verified that with six peptide substrates Gly-(Asp) the 4-Lys-beta-naphthylamines and the natural substrate trypsinogen of synthetic this recombinant protein subunit by the 705bp coding is keeping the activity of holoenzyme, has drawn back the prelude of producing recombination ox intestine kinase catalytic subunit (rEKL) with engineered method subsequently.Nineteen ninety-five Lisa is amalgamation and expression rEKL in intestinal bacteria, and introduces the enteropeptidase recognition site before the target protein encoding sequence, and fusion rotein discharges active rEKL through the autocatalysis cutting.The recombinant protein enzyme is lived and the report basically identical in COS, but productive rate increases to the wet bacterium of 1mg/125mg.Compared to the natural Enteropeptidase holoenzyme (EKn) that obtains with the method for purifying, rEKL will hang down tens times to the activity of natural substrate protrypsin, and the enzyme of opposing artificial substrates such as white Jie 11 fusion roteins is mutually cut specific activity EKn and will be exceeded hundreds of times.
Laura in 1996 etc. attempt using yeast expression system secreting, expressing rEKL first, and behind ion-exchange and affinitive layer purification, target protein output reaches the 6.3mg/L fermented liquid supernatant, and its activity is also apparently higher than intestinal bacteria and mammalian cell expression system.And then this product is applied to U.S. at that time just in the downstream purification of the recombination human interleukin 11 using in new drug development stage, has obtained gratifying result.Subsequently, the Invitrogen company under the research group is with this recombinase preparation commercialization, and being widely used in the genetic engineering pharmaceutical field is the fusion rotein of recognition site with EK with other.
A Yugoslavic scientific research group had successfully advanced efficient secretory expression with EKL filamentous fungus (filamentousfungus Aspergillus niger) in 2000, activated protein yield 1.9mg/L nutrient solution supernatant.Calendar year 2001 Korea S scientist Seong introduces the Histidine affinity labelling the 3 ' end of rEKL first, expresses in yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) and is secreted in the substratum effectively.REKL-(His) 6 output are the 1mg/L fermented liquid supernatant, and it is active fair with escherichia expression system.
Domestic at present also seldom to the research of recombinant enterokinase, only open report is that utilization Trx-EKL amalgamation and expression systems such as YUAN in 2002 have been carried out prokaryotic expression to enteropeptidase catalyzing subunit, production of enzyme 4.3mg/100mL fermented liquid behind the purifying, enzyme is than 720U/mg albumen alive.Chinese patent 2004100148607 adopts to have expressed in the pichia and has added histidine-tagged reorganization cattle enteropeptidase catalyzing subunit, yield 5mg/L endways.
Therefore at present there is complex process in the production of recombination ox intestine kinase, the cost height, and efficiency of pcr product is low, active problem such as weak.The tissue-derived enteropeptidase holoenzyme activity of present commercial animal intestine is not high, and often accompanies the appearance of contaminative serine protease.And expensive because of import zymin itself, make the amalgamation and expression one that adopts this enzyme to be known as " the expensive expression system of a cover ".
Along with increasing gene engineering product adopts the amalgamation and expression strategy, rEKL has important market utility value, and promotes the development of China's albumen integration technology and application also to be extremely important to further.
The invention provides a kind of production method of High-efficient Production recombination ox intestine kinase, adopt novel Pichia anomala expression system secreting, expressing Enteropeptidase catalytic subunit, by optimizing its gene order, and optimize fermentation and purifying process, obtain the production method of quick, easy, stable, that activity is high a kind of Enteropeptidase.
Summary of the invention
Purpose of the present invention just provides a kind of method of production recombination ox intestine kinase of high-efficient simple.
Another object of the present invention just provides the encoding sequence of recombination ox intestine kinase 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 recombination ox intestine kinase 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 SEQID 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, a kind of expression vector is provided, it is characterized in that described expression vector contains the described nucleotide sequence of claim 1.
In another preference, described expression vector is pGAPZA/ α-EKL.
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 another preference, described engineering cell is a pichia spp.
In a fourth aspect of the present invention, a kind of method of producing recombination ox intestine kinase is provided, the method comprising the steps of:
A) under the expression condition that is fit to, cultivate host cell as claimed in claim 5, thereby secreting, expressing goes out recombination ox intestine kinase;
B) separation and purification excretory recombination ox intestine kinase albumen.
Description of drawings
Fig. 1 is the structure synoptic diagram of fusion gene α-EKL.
Fig. 2 is that recombinant plasmid pGAPZA/ α-EKL makes up synoptic diagram.
Embodiment
The inventor is extensive studies by going deep into, optimization design by gene coded sequence, outside born of the same parents, made up the recombination ox intestine kinase antigen-4 fusion protein gene that contains yeast saccharomyces cerevisiae α-factor leader peptide sequences, made its middle efficient secretory expression in the pichia spp cell.Finished the present invention on this basis.
According to recombination ox intestine kinase catalytic subunit natural acid sequence, press codon-bias, under the condition that does not change aminoacid sequence, the target gene sequences of the synthetic recombination ox intestine kinase catalytic subunit of full gene, with this gene clone in the pUCl9 after the sequence verification, use molecular biology method, the fusion sequence of external structure fusion rotein α-EKL is cloned into expression vector pGAPZA then.Transform, be integrated into the P.pastoris host cell chromosome,, screen the strongest clone of resistance, and then filter out the high expression level engineering cell by applying the different concns resistance.Shake flask test shows, grows after 48 hours, more than the expression level 5mg/L.
After obtaining engineering cell, just can be under the condition that is fit to the culturing engineering cell.In the present invention, the recombination ox intestine kinase 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, (NH4)
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 recombination ox intestine kinase that obtains, need in fermentor tank, be optimized cultivation.The present invention has studied pilot scale fermentation technology, and the expression level after the optimization reaches 30mg/L.
Behind the fermentation expression recombination ox intestine kinase, the rEKL that expresses is separated.
Usually, fermented sample earlier obtains fermented liquid supernatant in modes such as centrifugal, filtrations, removes thalline.Fermented liquid supernatant can by saltout, method such as ultrafiltration carries out carrying out chromatography purification again behind the preliminary purification, also can directly carry out chromatography purification.
Be applicable to that chromatographic technique of the present invention comprises cation-exchange chromatography, anion-exchange chromatography, gel permeation chromatography, affinity chromatography etc.
Through 2-3 step purifying, can obtain the pure product of rEKL, the purifying yield is more than 40%, and purity is more than 95%, the about 12mg/L fermented liquid of pure product yield.
Behind the purifying enzyme of rEKL while still alive property measure with fusion rotein substrate method, after measured, it is about 2.5 * 10 than living
6IU/mg.
In an example of the present invention, the acquisition of recombination ox intestine kinase 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 Enteropeptidase improve.
In another example of the present invention, obtained the bacterial strain of high expression level recombination ox intestine kinase by screening, improved the expression amount of recombination ox intestine kinase.
In another example of the present invention,, select suitable expression vector express recombinant Enteropeptidase albumen by the comparison between the different promoters.
In another example of the present invention, the optimization of technology has by fermentation further improved the expression amount of recombination ox intestine kinase.
In another example of the present invention,, can obtain pure product 12mg/L through purifying process optimization.
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 12mg of recombination ox intestine kinase.Be fit to industrialization production.
The invention has the advantages that:
(1) optimized Enteropeptidase catalytic subunit gene order, and imported yeast saccharomyces cerevisiae α-factor leader peptide sequences before this sequence, this antigen-4 fusion protein gene is highly suitable for secreting, expressing in the pichia spp, has the characteristics of high expression level, high stable.
(2) expression process is simple; expression vector contains the promotor that does not need methanol induction; can composing type efficiently express Enteropeptidase; need not to change substratum during the fermentation or add inductor; simplified operating procedure; have higher security than methyl alcohol as the expression bacterium of inductor, help large-scale production.
(3) by the crucial technological condition for fermentation of control, expression level is further improved.
(4) purifying process is easy, rate of recovery height.Owing to be secretory protein, therefore simplified purification procedures, the purifying rate of recovery is improved greatly, make the scale operation recombination ox intestine kinase become possibility.
(5) adopting pichia spp is engineering cell, has kept the activity of recombination ox intestine kinase well.
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 fusion gene and the structure of expression plasmid
According to the aminoacid sequence of Enteropeptidase catalytic subunit, consider the codon-bias of pichia spp, the target gene sequences of full gene composite coding recombination ox intestine kinase catalytic subunit is cloned among the pUC19 this gene order and sequence verification.The structure of fusion rotein α-EKL is seen Fig. 1, is template with the plasmid pUC19/rEKL that contains the rEKL encoding sequence, obtains the EKL gene that can merge with correct framework with yeast saccharomyces cerevisiae α-factor leader peptide sequences by pcr amplification.Simultaneously, be template with the plasmid pPIC9K (available from Invitrogen company) that contains yeast saccharomyces cerevisiae α-factor leading peptide encoding sequence, pcr amplification obtains α-factor leader peptide sequences.Above-mentioned two kinds of PCR products behind the purifying are digested with restriction enzyme XhoI respectively, two kinds of enzymes are cut product connect.To connect product is that template is carried out pcr amplification, obtains can be used for the fusion gene α-EKL of secreting, expressing.
The construction of recombinant plasmid circuit carries out the α-EKL gene of synthetic after enzyme cuts processing with EcoRI as shown in Figure 2, and agarose gel electrophoresis reclaims about 1kb fragment; Simultaneously plasmid pGAPZA (available from Invitrogen company) is carried out enzyme with EcoRI and cut, reclaim big fragment.Two fragments are connected with the T4 dna ligase, connect the product conversion and enter bacillus coli DH 5 alpha (available from Promega company).Prepare plasmid in a small amount, cut by enzyme and identify the positive colony that contains α-EKL.PGAPZA can efficiently express its multi-copy integration after inserting foreign gene to the karyomit(e) of host bacterium, itself carry a Zeocin resistant gene simultaneously, can be used for the screening of transformant.The GAP promotor that the constructive expression is arranged before the carrier pGAPZA multiple clone site, do not need inductor can be in host bacterium GS115 efficiently expressing exogenous gene.
The screening of embodiment 2 high expression level recombination ox intestine kinase engineering strains
The recombinant plasmid pGAPZA/ α-EKL that builds is prepared in a large number, linearizing, electroporation transformed host cell P.pastorisGS115, coating contains the high anti-positive colony of YPDS plate screening of different concns microbiotic Zeocin, and carries out little megger and reach the engineering yeast strain that experiment screening obtains the recombination ox intestine kinase high expression level and (contain α-EKL).
Embodiment 3 selects suitable expression vector
When construction recombination plasmid pGAPZA/ α-EKL, we have made up recombinant plasmid pPIC9K/rEKL simultaneously, and by the process similar to embodiment 2, we have obtained the engineering yeast strain (containing rEKL) of recombination ox intestine kinase high expression level.The expression amount of these two engineering yeast strains under the prerequisite of expressing unanimities such as environment, expression time done a comparison, found to contain the expression amount of Enteropeptidase in α-EKL engineering yeast strain apparently higher than the engineering yeast strain that contains rEKL.According to the result, we select GAP as the first-selected promotor of expressing enteropeptidase.
| Bacterial strain | The expression amount of time (mg/L) | ||
| 12 | 24 | 36 | |
| The high expression level engineering yeast strain that pGAPZA/ α-EKL transforms | 7 | 19 | 30 |
| The high expression level engineering yeast strain that pPIC9K/rEKL transforms | 1 | 4 | 7 |
Embodiment 4 adds the influence of different protein protective agents to expression level
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, pH 5.0,20 ℃ of temperature, DO＞35%, treat that dissolved oxygen rising back stream adds 50% glycerine and includes 10%CA, or the nutrient solution of 10%Peptone or 10%Tryptone, take a sample behind the 36hr.Sample detection SDS-PAGE and protein content are to determine the optimum protein protective material of induction period.
Embodiment 5 recombinant enterokinase purifying
Fermented liquid is carried out ultrafiltration and concentration, the buffer system of fermented liquid is replaced with phosphate solution PB.Ultrafiltration and concentration and exchange buffering liquid: use the Millipore ultra-fine filter, the ultra-filtration membrane molecular weight that dams is 10KD, leaves and takes concentrated solution (effect is to remove small molecular weight impurity and salt) during ultrafiltration.Fermented liquid supernatant ultrafiltration to volume is left to add PB about 500ml, continues ultrafiltration; This program repeatedly flushes closely until the conductivity water of the gentle PB damping fluid of the conductivity water of sample.
Chromatography 1 (anion-exchange chromatography):
Chromatography media: Q Sepharose FF
Damping fluid: solution A: PB
Solution B: PB+1M NaCl
Last sample: with sample on the hK5 solution of ultrafiltration and concentration.
Clean: clean chromatography column with the solution A of 6CV after going up sample.
Gradient: clean the back and solution B is risen to 100% from 0% with 10CV.
Collect: collect EKL sample peak.
Chromatography 2 (hydrophobic chromatography):
Chromatography media: phenyl Sepharose FF
Damping fluid: solution A: PB+1M NaCl
Solution B: PB
Last sample: with sample on the sample peak of chromatography 1.
Clean: clean chromatography column with the solution A of 6CV after going up sample.
Gradient: clean the back and solution B is risen to 100% from 0% with 10CV.
Collect: collect EKL sample peak.
Chromatography 3 (sieve chromatography):
Chromatography media: Sephadex 75
Damping fluid: PB
Last sample: the sample master branch that chromatography 1 is collected.
Sample is through this three steps purifying, and promptly after anion-exchange chromatography, hydrophobic chromatography, the sieve chromatography, purity is increased to more than 95%, can get the pure product 12mg/L of albumen.
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〉a kind of production method of recombination ox intestine kinase
<160>2
<210>1
<211>705
<212>DNA
＜213〉artificial sequence
<221>misc_feature
<222>(1)…(705)
＜223〉the recombination ox intestine kinase sequence of You Huaing
<400>1
attgtcggag gaagtgactc cagagaagga gcctggcctt gggtcgttgc tctgtatttc 60
gacgatcaac aggtctgcgg agcttctctg gtgagcaggg attggctggt gtcggccgcc 120
cactgcgtgt acgggagaaa tatggagccg tctaagtgga aagcagtgct aggcctgcat 180
atggcatcaa atctgacttc tcctcagata gaaactaggt tgattgacca aattgtcata 240
aacccacact acaataaacg gagaaagaac aatgacattg ccatgatgca tcttgaaatg 300
aaagtgaact acacagatta tatacagcct atttgtttac cagaagaaaa tcaagttttt 360
cccccaggaa gaatttgttc tattgctggc tggggggcac ttatatatca aggttctact 420
gcagacgtac tgcaagaagc tgacgttccc cttctatcaa atgagaaatg tcaacaacag 480
atgccagaat ataacattac ggaaaatatg gtgtgtgcag gctatgaagc aggaggggta 540
gattcttgtc agggggattc aggcggacca ctcatgtgcc aagaaaacaa cagatggctc 600
ctggctggcg tgacgtcatt tggatatcaa tgtgcactgc ctaatcgccc aggggtgtat 660
gcccgggtcc caaggttcac agagtggata caaagttttc tacat 705
<210>2
<211>235
<212>PRT
＜213〉homo sapiens
<400>2
Ile Val Gly Gly Ser Asp Ser Arg Glu Gly Ala Trp Pro Trp Val Val
1 5 10 15
Ala Leu Tyr Phe Asp Asp Gln Gln Val Cys Gly Ala Ser Leu Val Ser
20 25 30
Arg Asp Trp Leu Val Ser Ala Ala His Cys Val Tyr Gly Arg Asn Met
35 40 45
Glu Pro Ser Lys Trp Lys Ala Val Leu Gly Leu His Met Ala Ser Asn
50 55 60
Leu Thr Ser Pro Gln Ile Glu Thr Arg Leu Ile Asp Gln Ile Val Ile Asn
65 70 75 80
Pro His Tyr Asn Lys Arg Arg Lys Asn Asn Asp Ile Ala Met Met His
85 90 95
Leu Glu Met Lys Val Asn Tyr Thr Asp Tyr Ile Gln Pro Ile Cys Leu
100 105 110
Pro Glu Glu Asn Gln Val Phe Pro Pro Gly Arg Ile Cys Ser Ile Ala
115 120 125
Gly Trp Gly Ala Leu Ile Tyr Gln Gly Ser Thr Ala Asp Val Leu Gln
130 135 140 145
Glu Ala Asp Val Pro Leu Leu Ser Asn Glu Lys Cys Gln Gln Gln Met
150 155 160
Pro Glu Tyr Asn Ile Thr Glu Asn Met Val Cys Ala Gly Tyr Glu Ala
165 170 175
Gly Gly Val Asp Ser Cys Gln Gly Asp Ser Gly Gly Pro Leu Met Cys
180 185 190
Gln Glu Asn Asn Arg Trp Leu Leu Ala Gly Val Thr Ser Phe Gly Tyr
195 200 205
Gln Cys Ala Leu Pro Asn Arg Pro Gly Val Tyr Ala Arg Val Pro Arg
210 215 220 225
Phe Thr Glu Trp Ile Gln Ser Phe Leu His
230 235
Claims (8)
1. coding Enteropeptidase catalytic subunit proteic nucleotide sequence, 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. an expression vector is characterized in that, it contains the described nucleotide sequence of claim 1.
3. expression vector as claimed in claim 2 is characterized in that, it is to contain the GAP promotor.
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. the production method of an Enteropeptidase 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 5, thereby secreting, expressing goes out Enteropeptidase albumen;
(b) separation and purification goes out the Enteropeptidase albumen 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 12-120 hour, and preferable is 24-100 hour; Inducing pH is 3-9, and that preferable is 5-7.
8. method as claimed in claim 6 is characterized in that, the expression condition shown in the step (b) is:
(1). fermented liquid obtains to contain the supernatant liquor of target protein by simple centrifugal or ultrafiltration;
(2). by easy steps such as simple ion exchange chromatography, hydrophobic chromatographies, can obtain purity at the pure product more than 95%.
Priority Applications (1)
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|---|---|---|---|
| CN 200510026133 CN1869236A (en) | 2005-05-24 | 2005-05-24 | Production method of recombination ox intestine kinase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510026133 CN1869236A (en) | 2005-05-24 | 2005-05-24 | Production method of recombination ox intestine kinase |
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| Publication Number | Publication Date |
|---|---|
| CN1869236A true CN1869236A (en) | 2006-11-29 |
Family
ID=37443014
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|---|---|---|---|
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059896A (en) * | 2014-07-02 | 2014-09-24 | 杨霞 | Preparation method for recombinant bovine enterokinase catalytic subunit protein |
| US9611466B2 (en) | 2011-12-23 | 2017-04-04 | Novo Nordisk A/S | Modified enterokinase light chain |
-
2005
- 2005-05-24 CN CN 200510026133 patent/CN1869236A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9611466B2 (en) | 2011-12-23 | 2017-04-04 | Novo Nordisk A/S | Modified enterokinase light chain |
| CN104059896A (en) * | 2014-07-02 | 2014-09-24 | 杨霞 | Preparation method for recombinant bovine enterokinase catalytic subunit protein |
| CN104059896B (en) * | 2014-07-02 | 2016-06-29 | 山西锦波生物医药股份有限公司 | The method preparing recombination ox intestine kinase catalytic subunit albumen |
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