CN102911955A - Synthetic human enterokinase gene and expression and purification method thereof - Google Patents
Synthetic human enterokinase gene and expression and purification method thereof Download PDFInfo
- Publication number
- CN102911955A CN102911955A CN2012104101568A CN201210410156A CN102911955A CN 102911955 A CN102911955 A CN 102911955A CN 2012104101568 A CN2012104101568 A CN 2012104101568A CN 201210410156 A CN201210410156 A CN 201210410156A CN 102911955 A CN102911955 A CN 102911955A
- Authority
- CN
- China
- Prior art keywords
- hek
- light chain
- human enterokinase
- gene
- expression
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses a synthetic human enterokinase gene and an expression and purification method of a product of the synthetic human enterokinase gene. The method is used for designing and coding a human enterokinase light chain nucleotide sequence which is in line with a human enterokinase light chain amino acid sequence according to a reported human enterokinase light chain nucleotide sequence and in light of a using principle of escherichia coli preferred codons, and then synthesizing in vitro, so as to achieve efficient expression in the escherichia coli; and then affinity purification is carried out to obtain high-activity MBP-hEKL fusion protein. The method is applied to large-scale preparation of human enterokinase. The recombinant protein can specifically recognize and cut a substrate with an enterokinase cutting site, and can be used as tool protease for removing a fusion label during recombining the fusion protein; and the recombinant protein is applicable to research on aspects of bioengineering pharmacy, genetic engineering, biochemistry and molecular biology and the like.
Description
One, technical field
The present invention relates to gene engineering technology field, specifically belong to a kind of recombinant human Enterpeptidase light chain (Human Enterokinaselight chain, hEK
L) the preparation method, comprise the kinase whose expression of structure, recombined human intestine and the purifying of its engineering bacteria cell.
Two, background technology
Utilize amalgamation and expression often can realize high efficient expression and the separation and purification of target protein.But target protein can not use with the form of fusion rotein sometimes, therefore designs special proteolytic enzyme restriction enzyme site between target protein and label protein, and fusion rotein just obtains complete target protein through specific cutting or fracture.Common instrument proteolytic enzyme has zymoplasm, enteropeptidase or Xa factor etc. at present, and wherein the enteropeptidase specificity is best.
Adopted at present protokaryon or eukaryotic expression system successfully to obtain the Enterpeptidase light chain albumen such as bioactive recombinant bovine, mouse, but rarely found research about human enterokinase light chain genetically engineered aspect.
Enteropeptidase is the duodenal a kind of heterodimer serine protease of Mammals, is comprised of a heavy chain (82-140kD) and a light chain (35-62kD), connects by a pair of disulfide linkage.Heavy chain plays grappling intestinal cell film, identification of protein; Light chain then has the holoenzyme catalytic activity, is the catalytic subunit of this enzyme.People's enteropeptidase (Human Enterokinase, hEK) also comprises a mini small subunit except containing above two subunits, be heterotrimer.Principal feature be to Asp-Asp-Asp-Asp-Lys-↓-X (↓ expression cleavage site, X represents arbitrary amino acid) sequence has very high identification and cleavage specificity.Because enteropeptidase endonuclease reaction condition is relatively gentle wide in range, all has good enzyme to cut activity in pH value (4.5-9.5) and temperature (4-45 ° of C), therefore become parting tool commonly used in the expressing fusion protein system.It is limited that but natural people's enteropeptidase is not only originated, and cost is high, and yield is low, also easily polluted by other proteolytic enzyme, thereby cause the purpose product degradation.Therefore use engineered method to produce people's enteropeptidase and become trend.
Have the report of studying about Enteropeptidase in a large number both at home and abroad, but the research of relevant people's enteropeptidase also seldom.There are some researches show the K of people's enteropeptidase
Cat/ K
mValue is about 10 times of Enteropeptidase, has higher cutting efficiency, and therefore, if can utilize gene engineering method to obtain the recombined human intestine kinases, its using value will be higher than the restructuring Enteropeptidase.At present, recombinant expressed in pichia spp of human enterokinase light chain is 3.8mg/L, and vigor is the commercial enteropeptidase catalyzing subunit product E of Invitrogen KMax
TM3 times, and recombinant expressed in intestinal bacteria is inclusion body, but every liter of fermented liquid purifying obtains the activated human enterokinase light chain albumen of 10mg after becoming renaturation and processing, vigor is EKMax
TM5 times.Because Enterpeptidase light chain contains 9 Cys residues, wherein forms 4 pairs of disulfide linkage, only has a free Cys residue not participate in pairing, therefore when recombinant expressed, very easily form the disulfide linkage mispairing, cause product to occur with the inclusion body form.MBP has been proved has very strong hydrotropy effect, can be increased in solvability, the especially eukaryotic protein of the fusion rotein of overexpression in the bacterium.These characteristics are particularly useful for this experiment hEK
LExpression and purification.
Three, summary of the invention
The object of the present invention is to provide a kind of can be in the expression and purification method of E. coli people enteropeptidase gene and product thereof, the method is suitable for the extensive preparation of people's enteropeptidase.
The present invention is according to the using priciple of nucleotide sequence and the intestinal bacteria preference codon of the human enterokinase light chain of reporting (http://www.ncbi.nlm.nih.gov/), the nucleotide sequence of the human enterokinase light chain of design coding and human enterokinase light chain consensus amino acid sequence, carry out external synthetic, in intestinal bacteria, obtain high efficient expression, and utilize affinity purification to obtain the MBP-hEK of high vigor
LFusion rotein.
Human enterokinase light chain (the hEK of a kind of synthetic provided by the invention
L) gene, it is the nucleotide sequence of SEQ NO 1 in the sequence table.
A kind of expression vector, it contains the nucleotide sequence of SEQ NO 1, and contains the Tac promotor.
A kind of engineering bacteria, it contains above-mentioned expression vector.Described engineering bacteria is intestinal bacteria.
A kind of synthetic human enterokinase light chain gene Expression in Escherichia coli purification process comprises the steps:
(1) makes up the hEK that contains synthetic
LThe recombinant plasmid pMAL-s-hEK of gene
L
(2) with recombinant plasmid pMAL-s-hEK
LBe converted in e. coli bl21 (DE3) cell, obtain containing the engineering strain of recombinant plasmid;
(3) engineering bacteria is inoculated in the LB culture medium solution cultivates, as recombinant liquid concentration OD
600nmWhen reaching 0.6-0.8, adding final concentration is the IPTG of 0.1-1.0mM, 37 ℃ abduction delivering 3-7 hour, centrifugal collection thalline, centrifugal after the ultrasonication, get supernatant, obtain target protein MBP-hEK with the method purifying of Amylose affinity chromatography
L
Analyze the target protein of purifying with 10%SDS-PAGE, result's demonstration, the present invention has obtained electrophoretically pure target protein matter.Like product EKMax with Invitrogen company
TMBe the MBP-hEK of standard to the present invention's production
LActivity is demarcated, and 12%Tricine SDS-PAGE the analysis showed that, the MBP-hEK that the present invention produces
LEnzyme activity reach EKMax
TM7 times.
Compared with prior art, of the present invention having a few and effect:
The present invention has realized a large amount of soluble-expressions of people's enteropeptidase in intestinal bacteria first, and every liter of fermented liquid can obtain 40mg with the fusion rotein MBP-hEK of MBP label behind affinitive layer purification
L, the active detection shows that its vigor is EKMax
TM7 times, reach 6.0 * 10
5U/ μ M is the highest in currently reported at expression amount and vigor.We also are designed with the restriction enzyme site of human Rhinovirus 3C Protease between MBP-tag and target protein in addition, can when needed the MBP label be removed.And the fusion rotein MBP-hEK that contains the recombinant human enteropeptidase catalyzing subunit that obtains of purifying
LThere is cutting to contain the protein and peptide of people's enteropeptidase cleavage site and the activity of recombination fusion protein.The kinase whose production method of a kind of High-efficient Production recombined human intestine provided by the invention adopts escherichia expression system, adopts affinity purification method, obtains quick, easy, stable, highly active recombined human intestine kinases light chain protein.This enzyme is applicable to the research of the aspects such as biotechnology, genetically engineered, biological chemistry and molecular biology.
Four, description of drawings:
Fig. 1 synthetic hEK
LThe wild type gene nucleotide sequence of expressing in gene and the human body is compared, and the base of black shade is the base of codon optimized rear change among the figure.
Fig. 2 recombinant plasmid pMAL-s-hEK
LPCR and enzyme cut the evaluation electrophoretic analysis: swimming lane 1 is cut the evaluation electrophoretic analysis for DNA maker DL5000 enzyme; Swimming lane 2 is recombinant plasmid pMAL-s-hEK
LPCR identify; Swimming lane 3 is empty plasmid pMAL-s; Swimming lane 4 is recombinant plasmid pMAL-s-hEK
LSwimming lane 5 is recombinant plasmid pMAL-s-hEK
LThe electrophoretic analysis of BamH I single endonuclease digestion; Swimming lane 6 is recombinant plasmid pMAL-s-hEK
LBamH I/Hind III double digestion identify.
Fig. 3 recombinant plasmid pMAL-s-hEK
LMake up schematic diagram
The SDS-PAGE of the kinase catalytic subunit expression of Fig. 4 recombined human intestine and purifying analyzes collection of illustrative plates: swimming lane 1 is Protein maker; Swimming lane 2 is not induced for pMAL-s/BL21 (DE3); Swimming lane 3 is induced for pMAL-s/BL21 (DE3); Swimming lane 4 is pMAL-s-hEK
L/ BL21 (DE3) does not induce; Swimming lane 5 is pMAL-s-hEK
L/ BL21 (DE3) induces; Swimming lane 6 is ultrasonic supernatant; Swimming lane 7 is precipitation; Swimming lane 8 is the MBP-hEK through the Amylose affinitive layer purification
L
The SDS-PAGE of Fig. 5 recombinant enterokinase catalytic subunit cutting restructuring GST-melittin analyzes collection of illustrative plates: swimming lane 1 is Proteinmaker; Swimming lane 2 is GST albumen; Swimming lane 3 is the GST-melittin fusion rotein; Swimming lane 4-7 is MBP-hEK
LBe respectively in mol ratio under the ratio of 1:1000,1:3000,1:5000 and 1:7000 with GST-melittin and cut; Swimming lane 8 is EKMax
TMBe to cut under the ratio of 1:1000 in mol ratio with GST-melittin.
Five, embodiment
1. by searching people's enteropeptidase sequence U09860.1 among the GenBank, utilize colibacillary codon-bias that it is carried out sequence optimisation, the nucleotide sequence of the human enterokinase light chain of Enterpeptidase light chain consensus amino acid sequence in design coding and the human body, see the nucleotide sequence of SEQ ID NO1 in the sequence table, the wild-type Gene sequence comparison is seen Fig. 1 in its Nucleotide and the human body.This synthetic human enterokinase light chain mrna length is 708bp, 235 amino acid of encoding.
2. synthesize following PCR primer:
FW(hEK
L)：5′-CGGGATCCATTGTTGGAGGAAGTAAT-3′；
RV(hEK
L)：5′-GCAAGCTTCTCGAGCTAATGTAGAAAACTTTG-3′；
Primers F W (hEK wherein
L) and RV (hEK
L) introduced respectively the restriction enzyme site of BamH I and Hind III, with FW (hEK
L) and RV (hEK
L) be the PCR primer, take people's enteropeptidase gene of synthetic as template, carry out pcr amplification hEK with Prime STAR high-fidelity enzyme
LGene, program is as follows: 98 ℃ 10 seconds, 60 ℃ 10 seconds, 72 ℃ 1 minute, 30 circulations, 72 ° C10 minute, 4 ℃ of insulations.
3. cut hEK with restriction enzyme BamH I, Hind III enzyme
LThe PCR product, the same BamH I that uses, Hind III enzyme is cut prokaryotic expression plasmid pMAL-s(and is got by pMAL-p2X plasmid house of correction, change Factor Xa recognition sequence into the PreScissionProtease recognition sequence), enzyme is cut product through 1% agarose gel electrophoresis, reclaim the test kit recovery with glue and obtain corresponding nucleic acid fragment, utilize the T4DNA ligase enzyme to connect at 16 ℃, connect product and transform the bacillus coli DH 5 alpha competent cell, 37 ° of C cultivate, the PCR screening positive clone also extracts plasmid and carries out double digestion and PCR evaluation (see figure 2), and carries out dna sequence analysis and determine.
4. with the recombinant plasmid pMAL-s-hEK that builds
L(see figure 3) is converted in the host cell e. coli bl21 (DE3), obtains containing the engineering strain of recombinant plasmid.
5. picking engineering bacteria list bacterium colony is in 5mL LB substratum, and 37 ℃, 180 rev/mins shaking culture are spent the night, and go in the 1LLB substratum by 2% inoculum size, and 37 ℃ of shaking culture are to OD
600nmDuring=0.6-0.8, adding the IPTG(final concentration is 0.2mM), 37 ℃ were continued shaking culture 4 hours, and SDS-PAGE detects protein expression situation (see figure 4).
6.MBP-hEK separation and purification: 8000r/min, the centrifugal collection thalline of 10min (contains 500mM NaCl with 20mM Tris-HCl, pH8.0) wash thalline twice, be resuspended in again in the 25mL same buffer ultrasonication on ice, 11000r/min, 4 ° of centrifugal 30min of C.Collect supernatant, be splined on and use in advance level pad (20mM Tris-HCl, 500mM NaCl, pH8.0) the Amylose affinity column that balance is good, ice bath vibration 2h.Carry out wash-out except foreigh protein removing with the 30mL level pad, then use 10mL elution buffer (10mM Maltose, pH 8.0 for 20mM Tris-HCl, 500mM NaCl) that target protein is eluted.The Bradford method is measured protein concentration.With the concentrated target protein of PEG20000 to 3g/L, and with target protein at dialysis buffer liquid (20mM Tris-HCl, 50mM NaCl, 2mM CaCl
2, 0.1%Tween-20, pH 7.4) the middle dialysis 24h that stirs, all operations all carries out in 4 ° of C chromatography cabinets.The purified rear 40mg purity that finally can obtain of 1L fermented liquid gained thalline is at the MBP-hEK more than 97%
L(concentration is 3mg/mL) fusion rotein (see figure 4).
7.MBP-hEK
LActivation analysis: measure the kinase whose activity of recombined human intestine take the fusion rotein GST-Melittin that contains people's enteropeptidase restriction enzyme site as substrate.Adding 90 μ L GST-Melittin fusion roteins in 1mL Eppendorf pipe (concentration: 1mg/mL, purity:〉95%), MBP-hEK
LCut with the mol ratio of 1:1000,1:3000,1:5000,1:7000 with substrate, enzyme cutting buffering liquid is 20mM Tris-HCl pH 7.4,100mM NaCl, puts 25 ° of C waters bath with thermostatic control, and Tricine SDS-PAGE detected after enzyme was cut 20h.MBP-hEK
LActivity unit demarcates: with the product E KMax of Invirogen company
TM(be 2000U/mg than vigor, concentration is 0.5gL) is the MBP-hEK of benchmark to purifying
LActivity is demarcated, and it is quite active for the basically identical dilution enzyme sample of TricineSDS-PAGE collection of illustrative plates, in prediction on such basis the MBP-hEK of purifying
LActive (U/ μ M).Result such as Fig. 5, MBP-hEK
LWhen cutting with the mol ratio of 1:5000 with substrate, fusion rotein still can by complete enzymolysis, obtain two bands that molecular weight is respectively 26kD (GST) and 2.8kD (Melittin).When 1:7000, the enzymatic hydrolyzation of fusion rotein is also more than 90%, with EKMax
TMTo cut effect substantially suitable for enzyme when 1:1000, i.e. the MBP-hEK of purifying
LThe ratio vigor be EKMax
TM(Invitrogen company) 7 times.The above results shows that we have obtained having bioactive recombined human intestine kinases light chain protein by gene engineering method, and enzyme activity reaches 6.0 * 10
5U/ μ M.
Claims (6)
1. the human enterokinase light chain of a synthetic (hEKL) gene is characterized in that nucleotide sequence is SEQ NO 1.
2. an expression vector is characterized in that, it contains human enterokinase light chain gene claimed in claim 1.
3. expression vector as claimed in claim 2 is characterized in that, it contains the Tac promotor.
4. an engineering bacteria is characterized in that, it contains expression vector claimed in claim 3.
5. engineering bacteria as claimed in claim 4 is characterized in that, it is intestinal bacteria.
6. the expression and purification method of the human enterokinase light chain gene of synthetic as claimed in claim 1 is characterized in that, comprises the steps:
1) makes up the hEK that contains synthetic
LThe recombinant plasmid pMAL-s-hEK of gene
L
2) with recombinant plasmid pMAL-s-hEK
LBe converted in e. coli bl21 (DE3) cell, obtain containing the engineering strain of recombinant plasmid;
3) engineering bacteria is inoculated in the LB culture medium solution cultivates, as recombinant liquid concentration OD
600nmWhen reaching 0.6-0.8, adding final concentration is the IPTG of 0.1-1.0mM, 37 ℃ abduction delivering 3-6 hour, centrifugal collection thalline, centrifugal after the ultrasonication, get supernatant,
4) the method purifying with the Amylose affinity chromatography obtains target protein MBP-hEK
L
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210410156.8A CN102911955B (en) | 2012-10-24 | 2012-10-24 | Synthetic human enterokinase gene and expression and purification method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210410156.8A CN102911955B (en) | 2012-10-24 | 2012-10-24 | Synthetic human enterokinase gene and expression and purification method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102911955A true CN102911955A (en) | 2013-02-06 |
| CN102911955B CN102911955B (en) | 2014-01-29 |
Family
ID=47610535
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210410156.8A Expired - Fee Related CN102911955B (en) | 2012-10-24 | 2012-10-24 | Synthetic human enterokinase gene and expression and purification method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102911955B (en) |
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 |
| CN112795583A (en) * | 2020-11-16 | 2021-05-14 | 上海大学 | Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method |
-
2012
- 2012-10-24 CN CN201210410156.8A patent/CN102911955B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| 孙琳等: "重组鼠源性肠激酶轻链在大肠杆菌中的表达", 《吉林大学学报》 * |
| 李德华等: "牛肠激酶基因工程菌的构建，高密度发酵，纯化及活性鉴定", 《应用与环境生物学报》 * |
| 杜广营等: "人肠激酶轻链基因的克隆及其在大肠杆菌中的表达", 《烟台大学学报》 * |
Cited By (3)
| 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 |
| 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 |
| CN112795583A (en) * | 2020-11-16 | 2021-05-14 | 上海大学 | Preparation method of recombinant sialic acid exonuclease, expression gene, recombinant expression vector and construction method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102911955B (en) | 2014-01-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sriskanda et al. | Characterization of an ATP-dependent DNA ligase from the thermophilic archaeon Methanobacterium thermoautotrophicum | |
| CN112941094B (en) | Active mutant enzyme and method for producing soluble mutant protein | |
| Haag et al. | Crystal structures of the Arabidopsis thaliana organellar RNA editing factors MORF1 and MORF9 | |
| Zhu et al. | A simple and effective strategy for solving the problem of inclusion bodies in recombinant protein technology: His-tag deletions enhance soluble expression | |
| JP5308630B2 (en) | Polypeptide containing extracellular region of Fc receptor and method for producing the same | |
| CN102911955B (en) | Synthetic human enterokinase gene and expression and purification method thereof | |
| Gotthard et al. | Crystallization and preliminary X-ray diffraction analysis of the organophosphorus hydrolase OPHC2 from Pseudomonas pseudoalcaligenes | |
| CN102250846A (en) | Method for preparing enzyme through coexpression of recombinant protease and molecular chaperone | |
| WO2014177021A1 (en) | Strong secretory signal peptide enhancing small peptide motif and use thereof | |
| CN110573616A (en) | Phi29 DNA polymerase and coding gene and application thereof | |
| Esipov et al. | Production of thymosin α1 via non‐enzymatic acetylation of the recombinant precursor | |
| CN112301014B (en) | Esterase mutant with improved thermal stability and application thereof | |
| Niu et al. | Efficient expression and purification of recombinant human enteropeptidase light chain in Esherichia coli | |
| Li et al. | Characterization of the diatomite binding domain in the ribosomal protein L2 from E. coli and functions as an affinity tag | |
| Dobrovetsky et al. | A robust purification strategy to accelerate membrane proteomics | |
| CN105002150A (en) | Alpha-amylase gene and application thereof | |
| CN111705050A (en) | Preparation method and application of novel halophilic archaea extracellular protease | |
| CN101058805B (en) | Method of producing mutation procarboxypeptidase B and mutation carboxypeptidase B | |
| CN114181922B (en) | Recombinant esterase, gene, recombinant bacterium and application of recombinant esterase and recombinant bacterium in degradation of phthalate | |
| TWI712691B (en) | Dextran affinity tag and application thereof | |
| CN114990090B (en) | Bifunctional fusion protein PETase-MHETase variant | |
| CN114457062B (en) | Algin lyase for preparing alginate oligosaccharides and application thereof | |
| Wojtowicz et al. | Expression of yeast deubiquitination enzyme UBP1 analogues in E. coli | |
| US20090259035A1 (en) | Method for producing recombinant RNase A | |
| WO2012128661A1 (en) | Fusion protein, fusion protein-producing strain of escherichia coli bacteria and a method for producing methionine-free human interferon alpha-2b from said 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 | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140129 Termination date: 20211024 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |