CN101165179A - Method for preparing recombination human casein macropeptide - Google Patents
Method for preparing recombination human casein macropeptide Download PDFInfo
- Publication number
- CN101165179A CN101165179A CNA200710129893XA CN200710129893A CN101165179A CN 101165179 A CN101165179 A CN 101165179A CN A200710129893X A CNA200710129893X A CN A200710129893XA CN 200710129893 A CN200710129893 A CN 200710129893A CN 101165179 A CN101165179 A CN 101165179A
- Authority
- CN
- China
- Prior art keywords
- recombination human
- casein macropeptide
- human casein
- macropeptide
- rhcmp
- 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
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
By means of DNA recombination technology, humanized casein macropeptide (hCMP) coding gene is designed and synthesized according to the preferred code of colibacillus, and is cloned into pET28a(+) directionally through BamH I and Sac I. Through screening out positive clone, selecting one single colony and fermenting and separating in optimized conditions, adding inducer IPTG in 0.5 mM/l at 30 deg.c after the thallus density OD600 reaches 1.0 or so and inducing for 8 hr to reach the peak of the target protein, initially separating the coarse protein by means of Ni-chelation affinity chromatography, DEAE-sepherose ion exchange column purifying, and cleavage with recombinant enterokinase, recombinant humanized casein macropeptide (rhCMP) is prepared. The rhCMP with rich branched chain amino acids and essential amino acids is suitable for use as the nutrient solution for people with weak health, athletes, etc.
Description
Technical field
The present invention relates to biotechnology, food engineering, field of health care products; Molecular cloning, the genetic engineering bacterium that specially refers to recombination human casein macropeptide ferments, the preparation method of target peptide.
Background technology
(human caseinomacropeptide hCMP) comes from mammals Kappa type-milk casein C and holds a fragment (106-170), 65 amino-acid residues of total length human casein macropeptide.This fragment is rich in different sugar chains, so the molecular weight size has unhomogeneity.The pancreas Chymotrypsin is generally adopted in the preparation of natural CMP, and (chymosin, EC3.4.23.4) the enzymolysis milk casein obtains.But method is difficult to obtain human casein macropeptide thus, therefore is difficult to meet the need of market.According to the literature, this huge peptide has the various biological function.It can promote probiotic bacterium such as intestinal bifidobacteria growth, regulate food absorption, anticoagulant, can also suppress the harmful bacterium in oral cavity and adhere to cytolemma and suppress Toxins,exo-, cholera (cholera toxin) and its receptors bind.Except, this group analysis is compared its amino acid and is formed discovery, and this huge peptide has very high nutritive value.In 65 amino-acid residues, indispensable amino acid has 34, and totally 5 kinds, only scarce methionine(Met), tryptophane, leucine; Branched-chain amino acid has 16, accounts for nearly 25%; And phenylalanine only has 1, so the branched-amino acid number of this peptide (F value) is 12.2.As a kind of huge peptide of natural structure, be well suited for hepatitis B patient, critically ill patient, weak senior people and sportsmen and use.
This huge peptide of application yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) 2805 secreting, expressings had been reported in Korea S Sunghoon Park laboratory in 2005.Because yeast saccharomyces cerevisiae can cause the transition glycosylation to recombinant glycoprotein.For this reason, the present invention adopts the prokaryotic organism intestinal bacteria to be this huge peptide of host cell fermentative preparation, forms upward two high nutritive values in the hope of making full use of its amino acid.
Summary of the invention
1. gene design, synthetic and molecular cloning
1.1., the sequence of amino acid of people's CMP is transferred to the nucleotide sequence of DNA according to the intestinal bacteria preference codon.
Because the degeneracy of genetic code can change the 3rd bit base of above-mentioned some codon, but not cause the change of aminoacid sequence, be still the gene of expressing CMP.
And, design 6 dna fragmentations according to above-mentioned nucleotide sequence, after their annealing 15 Nucleotide complementations will be arranged between any two.
1.2. use the synthetic said gene of PCR instrument
With above-mentioned 6 dna fragmentations, fill up earlier with archaeal dna polymerase (Klenow), then with it as template, add two terminal primers and obtain goal gene by PCR, adopt the Sanger method to check order and guarantee that gene structure is correct.
1.3. it is the maternal carrier of construction of expression vector that the present invention adopts commercial already pET28a (+).Above-mentioned synthetic dna fragmentation is inserted pET28a (+).Also can adopt other model plasmids of this system even self-built carrier.
Identify positive colony 1.4. use antibiotic-screening.
2.rhCMP preparation
2.1. the fermentation of genetically engineered colibacillus
The general LB substratum that adopts also can adopt 2x YT, or M9, TB, M9ZB substratum.With the LB medium sterilization, add kantlex and reach 30 μ g/ML, single colony inoculation, 37 ℃ of shaken overnight were inoculated in overnight culture in fresh 2 * YT substratum (containing 30 μ g/ML kantlex), as bacterium liquid OD by 1: 100
600Reach 0.6-1.0, add inductor IPTG and reach 0.5mM/L, continue cultivation and stopped fermentation in about 8 hours.If last jar fermentation also will add additional carbon/nitrogenous source and regulation and control pH by stream and 7.5 reach high density fermentation.
2.2.rhCMP separation, purifying and sign
The rhCMP that the present invention relates to mainly appears at cell pericentral siphon district, has small part to appear in the inclusion body.
Because target protein N end has 6x His, so the general Ni-chelating affinity column that adopts earlier is used for rough segmentation.When crossing post, target protein and Ni ion chelating and be trapped.Contain the high density imidazoles elutriant target protein that dissociates down with what optimize, imidazoles and metal ion are removed in dialysis or ultrafiltration, sample is used the DEAE-sepharose purifying again, wash-out goes out target protein from the post again, adds recombinant enterokinase excision target protein N end after the desalination and concentration and contains 6x His fragment and obtain maturation protein.
The sign of maturation protein adopts SDS-PAGE, methods such as RP-HPLC and MALDI-TOF-TOF-MS.
3.rhCMP purposes
3.1. nutritive medium as the hepatopathy people.RhCMP includes branched-chain amino acid residue number and reaches 25% (16/65), and die aromatischen Aminosaeuren has only phenylalanine a kind of, only accounts for 1.7% (1/65); Indispensable amino acid has 5 kinds, and the residue number accounts for 34/65.Therefore this section is derived from natural little peptide, is fit to various hepatopathys, liver cancer and the old and the weak's body void person and uses.
3.2. oral liquid as quick Ginseng Extract such as sportsmen.The easier absorption of little peptide specific ionization amino acid, and the contained branched-chain amino acid of rhCMP is mainly in the muscle cell metabolism, therefore rhCMP can be separately or is made multiple oral liquid with other material such as Vc, citric acid, carbohydrate, collagen etc., make the sportsmen behind the high speed vigorous exercise quick Ginseng Extract, regain one's strength.
Description of drawings
Fig. 1 expression vector pET28a (+)-rhCMP makes up synoptic diagram
The influence that the different induction times of Fig. 2 are expressed target protein rhCMP.
From left to right, it is 0,2,3,4 that swimming lane 1-6 represents induction time respectively, 8,16 hours.The target protein band thickens gradually with the induction time prolongation as can be seen.
The electrophoretogram that Fig. 3 target protein rhCMP distributes in the host cell different zones.
From left to right, swimming lane 1 is an inclusion body sex change liquid, and swimming lane 2 is an inclusion body, and swimming lane 3 is a cell conditioned medium liquid, and swimming lane 4 is the pericentral siphon district, and swimming lane 5 is full bacterium lysate, and swimming lane 6 is a standard specimen.
Fig. 4 rhCMP peptide quality fingerprinting-mass spectrum (MALDI-TOF-TOF-MS) figure.766.5185 places are identical with theoretical value on the collection of illustrative plates, thereby learn that peptide section sequence herein is: IAIPPKK.
Embodiment
1. gene design, synthetic and molecular cloning
1.1. according to the intestinal bacteria preference codon, with the sequence of amino acid of people's CMP (65aa, Seq.1) transfer to gene nucleotide sequence (195bases, Seq.2).
Because the degeneracy of genetic code can change the 3rd bit base of above-mentioned some codon, but not cause the change of aminoacid sequence, be still the gene of expressing CMP.
1.2. in order above-mentioned encoding gene to be cloned on the expression vector and to consider that fusion rotein is cut into slaking required, add the encoding sequence (GGATCCGATG ACGATGACAA A) of BamHI (GGATCC) and recombinant enterokinase (Enterokinase) recognition sequence (DDDDK) at this gene 5 ' end, add a termination codon (TAA) and SacI (GAGCTC) at this gene 3 ' end again, need synthetic dna sequence dna (Seq.3).
1.3. 6 dna fragmentations of chemosynthesis are as PCR primer (seeing sequence page 5).
1.4. acquisition goal gene.With Primer2,5 ' the end T4 phosphokinase phosphorylation of Primer3, to remain 4 fragments then and be diluted to Primer2 with phosphorylation, the concentration that Primer3 is identical with the isoconcentration mixing of taking a sample, adds archaeal dna polymerase Klenow with these 6 fragments, the 10x reaction buffer, dNTP, T4Ligase, ddH
2O fills up reaction, obtains gene template thus.
Then carry out PCR and obtain an amount of target dna, and it is cloned on the pMD 18-T carrier, carry out the nucleotide sequence analysis of goal gene, select a correct sequence.
1.5 the structure of expression vector.With commercial Novagen prokaryotic expression system pET28a (+) is maternal carrier, is finished the structure of expression vector by BamH I and Sac I double digestion directed cloning.Adopt the microbiotic preliminary screening to identify positive colony then.
2. the fermentation of genetically engineered colibacillus
PET-28a (+) transforms expressive host bacterium E.coli BL21 (DE3).Picking list colony inoculation LB substratum (30 μ g/ml kan, same under the no specified otherwise), 37 ℃ of activation are spent the night.In 1: 100 ratio inoculation 2 * YT substratum, 30 ℃ of shaking tables (250r/min) were cultured to OD
600Be about 1.0, add IPTG, induce 8h, centrifugal collection thalline to final concentration 0.5mM/L.
3.rhCMP separation, purifying and sign
3.1. the distribution of recombinant protein rhCMP in bacterium E.coli BL21 (DE3)
With the method that the commercial working instructions of Novagen company provide, carry out the bacterial penetration shock respectively, ultrasonic treatment, rhCMP is at the cell pericentral siphon in research, the distribution in tenuigenin and the inclusion body.
3.1.1. osmotic shock
Induce bacterium (10ml volume) with method above-mentioned, survey bacterium OD
600, 10000r/min is centrifugal, and 0.5min removes supernatant, adds osmotic shock solution 1 (V
1=OD
600* V
2/ 5, V
1Be osmotic shock solution 1, V
2For cultivating bacterium liquid), ice bath 10min, the centrifugal 0.5min of 10000r/min removes supernatant.Add V
1Volume osmotic shock solution 2 is resuspended, shakes ice bath 10min, and the centrifugal 5min of 10000r/min preserves supernatant, bacterial precipitation.
Do following processing: the supernatant trichloroacetic acid precipitation adds 1/10 volume 100%TCA, vortex 15sec, ice bath 10min, the centrifugal 5min of 13000r/min, 100ul washing with acetone precipitation, dry 1h, add 0.5 volume, 1 * PBS dissolving,-20 ℃ of preservations are got 10ul and are added equal-volume 2 * SB, carry out the 15%SDS-PAGE electrophoretic analysis, carry out the protein band gray scale scanning, calculate the relative content that recombinant protein c MP accounts for total bacterial protein.
3.1.2. ultrasonic degradation
It is resuspended that ultrasonic treatment bacterial penetration shock bacterial precipitation adds 1/10 volume of culture 20mM Tris-HCl (pH7.5), ice bath, ultrasonic treatment, 20% work is selected, 1min is pulverized in pulse, and the centrifugal 5min of 13000r/min preserves supernatant, bacterial precipitation for-20 ℃ then.Supernatant carries out the TCA precipitation, handles the same.Precipitation provides the method repeated treatments by Novagen company commercial " pET System Manual ", the washing inclusion body.Inclusion body is by cultivating bacterium 100 * OD
600/ 3ul volume adds the 1%SDS dissolving, adds equal-volume 2 * SB, carries out the 15%SDS-PAGE electrophoretic analysis, carries out the protein band gray scale scanning, calculates the relative content that recombinant protein c MP accounts for total bacterial protein.
3.1.3 experimental result
By the pair cell pericentral siphon, tenuigenin and inclusion body carry out the 15%SDS-PAGE electrophoretic analysis, and the result shows that target protein mainly is present in cell pericentral siphon and the tenuigenin, exists a spot of target protein, as shown in Figure 3 in the inclusion body.
3.2. the purifying of recombinant protein c MP
PET-28a (+) transforms and expresses bacterium E.coli BL21 (DE3), and in the LB substratum, 30 ℃, shaking table (250r/min) is cultured to OD
600Be about 1.0, add IPTG to final concentration 0.5mM/L, 8h, the centrifugal collection thalline of 4000g.Use the recombinant protein in Ni-NTA HiBind Rasin separation and purification tenuigenin and the inclusion body.
3.2.1 the purifying of recombinant protein in the cell pericentral siphon
Use the osmotic shock and the TCA precipitator method, just can from the cell pericentral siphon, obtain purer recombinant protein.
3.2.2 the separation and purification of target protein in the tenuigenin
1. 0.5M Ni
2+The chelating sepharose.
2. use buffer A (25mM PBS, 0.5M Nacl, pH7.4) 5～10 bed volumes of balance, flow velocity 1ml/min.
3. with the buffer A fragmentation of a certain amount of cell, the centrifugal 10min of 10000r/min, supernatant is through 0.45 μ m membrane filtration, last sample, flow velocity 1ml/min.
4. wash 5～10 bed volumes, flow velocity 1ml/min with buffer A once more.
5. respectively with containing 20,50,100, the buffer B of 200mM imidazoles is carried out stepwise elution.Flow velocity, 2ml/min.
6. carry out the 15%SDS-PAGE electrophoretic analysis, can obtain purer recombinant protein at the 200mM place.
3.2.3. the purifying of target protein in the inclusion body
1. 0.5M Ni
2+The chelating sepharose.
2. use damping fluid C (25mM PBS, 0.5M Nacl, 8M Urea, pH8.0) 5～10 bed volumes of balance, flow velocity 1ml/min.
3. washed inclusion body is fully dissolved sex change with damping fluid C, last sample, flow velocity 1ml/min.
4. wash 5～10 bed volumes, flow velocity 1ml/min with damping fluid C once more.
5. respectively with containing 20,50,100, the damping fluid D of 200mM imidazoles carries out stepwise elution.Flow velocity, 2ml/min.
6. carry out the 15%SDS-PAGE electrophoretic analysis, can obtain purer recombinant protein at the 200mM place equally.
4.rhCMP MALDI-TOF-TOF-MS identify
4.1. enzymolysis prepares sample in the glue
To examine the protein site that dyes with blade and downcut, change in the 200 μ l centrifuge tubes from glue.
Add 100 μ l destainers (50% acetonitrile, 50mmol/L bicarbonate of ammonia) in the pipe, room temperature was placed 20 minutes, inhaled and removed destainer, repeated above step twice.
Every glue adds 100 μ l, 100% acetonitrile, and room temperature was placed 10 minutes, removed acetonitrile wherein, put into 37 ℃ of baking oven 5-10 minutes then to guarantee dried fully glue.
Adding 5 μ l-10 μ l concentration is the trypsin enzyme solution of 12.5ng/ μ l, in 4 ℃ of refrigerators about 30 minutes, can absorb enzyme liquid well to guarantee micelle, and take out back enzymolysis in 37 ℃ of baking ovens and spend the night.
Every glue adds 60 μ l, 0.1% trifluoroacetic acid/50% acetonitrile, and 30-40 minute, solution is transferred in the 96 new orifice plates, repeat above operation 2-3 time.With peptide section solution at N
2Flow down and dry up concentratedly, identify to be ready for use on mass spectrum
4.2.MALDI-TOF-TOF/MS identify:
With the peptide section of complete drying be dissolved in again 0.7uL 0.5g/LCHCA solution (solvent, 0.1%TFA+50%ACN) in, and it is all put on the stainless steel MALDI target plate, and seasoning at room temperature.
4.3. mass spectroscopy
Sample is with 4700 time-of-flight mass spectrometry instrument [4700Proteomics Analyzer (TOF/TOFTM) (AppliedBiosystems, USA)] carry out mass spectroscopy, laser source is the Nd:YAG laser apparatus of 355nm wavelength, acceleration voltage is 20kV, adopts positive ion mode and obtains the type collection data of data automatically.PMF mass scanning scope is 700-3500Da, and spectrogram carries out external standard with the myoglobin peptide hydrolysis and proofreaies and correct.
MS adopts Reflector Positive parameter: CID (OFF), mass rang (700-3200Da), Focus Mass (1200Da), Fixed laser intensity (6000), Digitizer:Bin Size (1.0ns)
With the proteic theoretical sequence of software PeptideMass analysis purposes, obtain the theoretical peptide section of this sequence Trypsin enzymolysis, then peptide section on the PMF figure and theoretical peptide section are compared, learn that 766.5185 places are identical with theoretical value on the PMF collection of illustrative plates, thereby learn that peptide section sequence herein is: IAIPPKK; The N end of this and target protein rhCMP is in full accord.
To sum up state, rhCMP can prepare with this method.
The sequence page
1.Seq.1 the sequence of amino acid of people's CMP (65amino acids)
IAIPPKKIQD?KIIIPTINTI?ATVEPTPAPA?TEPTVDSVVT?PEAFTESII?STTPETPTVA?VTSTPA
2.Seq.2 based on the inclined to one side hence nucleotide sequence (195bases) of the recombination human casein macropeptide of codon of intestinal bacteria
ATTGCGATTC?CGCCGAAAAA?GATTCAGGAT?AAAATCATTA?TCCCGACCAT
TAACACCATT?GCGACCGTGG?AACCGACCCC?GGCGCCGGCG?ACCGAACCGA
CCGTGGATAG?CGTGGTGACC?CCGGAAGCGT?TTACCGAAAG?CATTATTAGC
ACCACCCCGG?AAACCCCGAC?CGTGGCGGTG?ACCAGCACCC?CGGCG
3.Seq.3 the nucleotide sequence of the recombination human casein macropeptide of affix restriction enzyme site (225bases)
GGATCCGATG?ACGATGACAA?AATTGCGATT?CCGCCGAAAA?AGATTCAGGA?TAAAATCATT
ATCCCGACCA?TTAACACCAT?TGCGACCGTG?GAACCGACCC?CGGCGCCGGC?GACCGAACCG
ACCGTGGATA?GCGTGGTGAC?CCCGGAAGCG?TTTACCGAAA?GCATTATTAG?CACCACCCCG
GAAACCCCGA?CCGTGGCGGT?GACCAGCACC?CCGGCGTAAG?AGCTC
4. the nucleotide sequence of 5 primers of chemosynthesis
Primerl(5’-GCCGGATCCGATGACGATGACAAAATTGCGATTCCGCCGAAAAAGATTCA-3’)
Primer2(5’-CATTAACACCATTGCGACCGTGGAACCGACCCCGGCGCCGGCGACCGAACCGACC-3’)，
Primer3(5’-GAAGCGTTTACCGAAAGCATTATTAGCACCACCCCGGAAACCCCGACCGTGGCGG-3’)，
Primer4(5’-TCGCAATGGTGTTAATGGTCGGGATAATGATTTTATCCTGAATCTTTTTCGGCGG-3’)，
Primer5(5’-GCTTTCGGTAAACGCTTCCGGGGTCACCACGCTATCCACGGTCGGTTCGGTCGCCGGC-3’)，
Primer6(5’-GGCGAGCTCTTACGCCGGGGTGCTGGTCACCGCCACGGTCGGGGTTTCCG-3’)
Claims (9)
1. the preparation method of a recombination human casein macropeptide is characterized in that, adopt genetic engineering fungus fermentation method production, rather than the enzyme cutting is got from naturally occurring casein.
2. genetic engineering fungus fermentation method as claimed in claim 1 comprises: a, the design of recombination human casein macropeptide genes involved are with synthetic, b, structure recombination human casein macropeptide expression vector, c, transform the host with described expression vector and make up genetic engineering bacterium, d, utilize described genetic engineering bacterium fermentative production recombination human casein macropeptide.
3. as claim 1,2 described preparation methods, described genes involved structure is:
ATTGCGATTC?CGCCGAAAAA?GATTCAGGAT?AAAATCATTA?TCCCGACCAT?TAACACCATT
GCGACCGTGG?AACCGACCCC?GGCGCCGGCG?ACCGAACCGA?CCGTGGATAG?CGTGGTGACC
CCGGAAGCGT?TTACCGAAAG?CATTATTAGC?ACCACCCCGG?AAACCCCGAC?CGTGGCGGTG
ACCAGCACCC?CGGCG
4. method as claimed in claim 2, wherein the technical process of fermentative production recombination human casein macropeptide is as follows: select single bacterium colony engineering bacteria, carry out seed liquor and amplification culture, abduction delivering, collect thalline for following jar and carry out cytoclasis, centrifugal that born of the same parents contain body, broken born of the same parents contain body and target protein sex change and renaturation, and it is former that mistake Ni chelate column obtains target protein, cross the Ni chelate column behind the Enterokinase enzymolysis and obtain recombination human casein macropeptide, desalination and impurity elimination again, freeze drying example, quality inspection gets final sample.
5. recombination human casein macropeptide as claimed in claim 4, it is:
IAIPPKKIQD?KIIIPTINTI?ATVEPTPAPA?TEPTVDSVVT?PEAFTESII?STTPETPTVA?VTSTPA.
6. gene nucleotide as claimed in claim 3 order, and replace the gene that obtains not changing the base of being carried out under its amino acid sequence coded prerequisite.
7. amino-acid sequence as claimed in claim 5, and the amino acid that is carried out under the prerequisite that does not change its biological function is replaced the huge peptide that obtains.
8. the described carrier of claim 2 is commercial pET28a (+), and substitute.
9. the described host cell of claim 2 is commercial BL21 (DE3), and substitute.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200710129893XA CN101165179A (en) | 2007-08-01 | 2007-08-01 | Method for preparing recombination human casein macropeptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA200710129893XA CN101165179A (en) | 2007-08-01 | 2007-08-01 | Method for preparing recombination human casein macropeptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101165179A true CN101165179A (en) | 2008-04-23 |
Family
ID=39334035
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA200710129893XA Pending CN101165179A (en) | 2007-08-01 | 2007-08-01 | Method for preparing recombination human casein macropeptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101165179A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009040089A3 (en) * | 2007-09-11 | 2009-10-29 | Mondobiotech Laboratories Ag | Use of the peptides maippkknqdk (cow kappa casein 106-116) and/or ygfqna (serorphin) as therapeutic agents |
| CN113214378A (en) * | 2021-06-11 | 2021-08-06 | 新希望乳业股份有限公司 | Method for separating and extracting casein glycomacropeptide |
-
2007
- 2007-08-01 CN CNA200710129893XA patent/CN101165179A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009040089A3 (en) * | 2007-09-11 | 2009-10-29 | Mondobiotech Laboratories Ag | Use of the peptides maippkknqdk (cow kappa casein 106-116) and/or ygfqna (serorphin) as therapeutic agents |
| WO2009039976A3 (en) * | 2007-09-11 | 2009-11-12 | Mondobiotech Laboratories Ag | Use of a peptide as a therapeutic agent |
| CN113214378A (en) * | 2021-06-11 | 2021-08-06 | 新希望乳业股份有限公司 | Method for separating and extracting casein glycomacropeptide |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| DK2632944T3 (en) | PROCEDURE FOR PURIFICATION OF HUMAN GRANULOCYT COLONY STIMULATING FACTOR FROM RECOMBINANT E. COLI | |
| CN107245494A (en) | Solution expression with high efficiency and purification process of the A β 42 in Escherichia coli | |
| CN101144093B (en) | Recombination expression carrier and method for soluble expressing human I-type metallothionin | |
| CN103614386A (en) | Halostachys caspica metallothionein gene as well as recombinant protein and application thereof | |
| CN114181321B (en) | Lateolabrax japonicus FGF6A, FGF6B and FGF18 recombinant protein and preparation method and application thereof | |
| CN1119415C (en) | Clone and expression of anti-CA125 bifunctional genetically engineered antibody | |
| CN104195157A (en) | High-efficiency recombination expression and purification method of biological active peptide in prokaryotic cells | |
| CN101165179A (en) | Method for preparing recombination human casein macropeptide | |
| CN105463006A (en) | Novel preparation method for recombinant human epidermal growth factor | |
| EP1417237B1 (en) | Process for preparation of polypeptides of interest from fusion peolypeptides | |
| CN103966191A (en) | Preparation method of recombinant bovine trypsin | |
| CN102250938B (en) | Preparation method of cardiactide | |
| CN108484749B (en) | Recombinant soluble human bone-targeted interferon gamma-1 b and preparation method thereof | |
| CN101544692B (en) | Rousettus leschenaulti leptin and its cDNA sequence | |
| CN110357970A (en) | A kind of hEGF's fusion protein and its nucleic acid molecules, a kind of hEGF's preparation method for being easy to purify | |
| CN106008701A (en) | Rapid preparation method of high-purity superhelical structure type I collagen | |
| CN107746432A (en) | A kind of modified proteins of A β 42 and its expression and purification method | |
| CN101691402B (en) | Miniopterus fuliginosus leptin protein and cDNA sequence thereof | |
| ES2391457T3 (en) | Procedure for purification of recombinant polypeptides | |
| CN101619101B (en) | Rhinolophus ferrumequinum leptin protein and cDNA sequence thereof | |
| CN108165539A (en) | A kind of pears S7The vivoexpression method of-RNase albumen and its preparation method of polyclonal antibody | |
| CN108840934B (en) | Recombinant sheep long-acting interferon tau, fusion protein for preparing long-acting interferon tau and preparation method of fusion protein | |
| CN101134963A (en) | Cascade expression method of recombinant human glandulae parathyroideae (1 to 34 peptide) | |
| CN101089181A (en) | Production method of recombination human interleukin-4 | |
| CN101144080A (en) | Method for preparing recombination human VIP-Humanin fusion protein |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20080423 |