CN101736022B - High-efficiency preparation method of human soluble guanylate cyclase - Google Patents
High-efficiency preparation method of human soluble guanylate cyclase Download PDFInfo
- Publication number
- CN101736022B CN101736022B CN200810202926A CN200810202926A CN101736022B CN 101736022 B CN101736022 B CN 101736022B CN 200810202926 A CN200810202926 A CN 200810202926A CN 200810202926 A CN200810202926 A CN 200810202926A CN 101736022 B CN101736022 B CN 101736022B
- Authority
- CN
- China
- Prior art keywords
- sgc
- guanylate cyclase
- soluble guanylate
- expression
- human soluble
- 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.)
- Expired - Fee Related
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a preparation method of human soluble guanylate cyclase (sGC), belonging to the technical field of biological engineering. The method of the invention comprises the following steps of: (1) packaging the nucleotide coded sequence of the human sGC in an expression vector, and converting an Escherichia coli strain for expression; (2) inoculating the strain obtained in the step (1) into an improved TB culture solution, and carrying out IPTG induction expression overnight; and (3) treating the thallus obtained in the step (2) by breaking, centrifugation, Ni-NTA column chromatography, tev enzyme digestion, Ni-NTA column chromatography and heme recombination to obtain human sGC protein the purity of which reaches more than 90% and the yield reaches more than 20mg/L. The method can overcome the defects that the traditional method has lower yield of human sGC and higher cost and can not meet the application requirement for sGC, and is an expression method with highest yield and low cost at present.
Description
Technical field
The invention belongs to genetically engineered and protein expression field, relate to the method for a kind of soluble guanylate cyclase (sGC) with biotechnology efficient production people.
Background technology
Endogenous chemistry small molecules NO, CO, H in the life entity
2S, H
2O
2Deng very important physiological function is arranged in cardiovascular system is unified neural system as cellular signal transduction molecule.Based on these chemical micromolecular signal transducers mechanism and with medical diagnosis on disease treatment and original new drug development research be a forward position focus studying of chemicobiology and biomedical sector in recent years.Soluble guanylate cyclase (sGC) is as the acceptor of messenger molecule NO, the heterodimer of being made up of α and two subunits of β that contains protoheme.When NO with can activate sGC catalysis GTP after the heme of sGC combines and be converted into secondary messager molecule cGMP, and then mediate a series of biological cascade reaction.
How to activate sGC for NO and proposed more model; But its exact mechanism is still unclear; Reason mainly is owing to can not obtain the highly purified sGC albumen of capacity; SGC whole protein bigger (about 80kDa of α and the about 70kDa of β) on the other hand, thus the further investigation of people stoped for its structure-function-character-reaction.
The proteic acquisition approach of sGC mainly contains two kinds at present: the one, and from organizing central extraction, the amount that still this method extraction obtains is (～2mg/kg ox lung) seldom, for people's sGC albumen, hardly maybe owing to tissue-derived restriction becomes especially.Second kind of approach is from eukaryotic cell, to express: this method albumen productive rate low (～130 μ g/25ml cell extract), cost is quite high.Though the report of the sGC protoheme structural domain of bacterial body at expression in escherichia coli arranged, they and Eukaryotic protoheme structural domain homology are very low.For Eukaryotic sGC albumen, have only people such as Marletta at expression in escherichia coli the protoheme structural domain and the catalyst structure domain of β 1 subunit of mouse, but the output of their resulting soluble proteins is quite low.The sGC albumen of up to the present also having no talent is at the report of expression in escherichia coli.
Summary of the invention
The objective of the invention is to set up a kind of simple to operate, cost is low, and productive rate is high, the preparation method of the people's that purity is high soluble guanylate cyclase (sGC).
The invention provides a kind of high efficiency preparation method of human soluble guanylate cyclase, this method may further comprise the steps:
(1), and transforms to express and use coli strain with the human soluble guanylate cyclase nucleotide coding sequence expression vector of packing into;
(2) (1) obtained strains is inoculated in the TB nutrient solution after the improvement, the IPTG abduction delivering spends the night;
(3) with the broken bacterium of (2) gained thalline, centrifugal, cross the Ni-NTA post, tev enzyme enzyme is cut, and crosses the Ni-NTA post, and the heme reorganization gets final product.
In the method for the present invention, expression vector described in (1) can be pMAL-c2x, pET28b or pET22b.
In the method for the present invention, said expression vector can be the MBPHT-mCherry2 plasmid.
In the method for the present invention, coli strain described in (1) can be BL21 (DE3) pLysS, Rosetta (DE3) pLysS or Rosetta (DE3).
In the method for the present invention, broken bacterium method can be ultrasonic, multigelation or N,O-Diacetylmuramidase enzymolysis in (3).
In the method for the present invention; " human soluble guanylate cyclase nucleotide coding sequence " be meant the coding have human soluble guanylate cyclase nucleotide sequence (the GENBANK number of landing: NM_000857), like ORFs position nucleotide sequence and degenerate sequence thereof in the sequence.This degenerate sequence is meant, is arranged in the encoder block ORFs position Nucleotide of NM_000857 sequence, and having one or more codons to be encoded, the degenerate codon of same amino acid replaces the back and the sequence that produces.Because the degeneracy of codon, so be low to moderate about 70% the degenerate sequence described sequence of NM_000857 of also encoding out with the encoder block ORFs position nucleotide sequence homology of NM_000857 sequence.
In the present invention, term " human soluble guanylate cyclase nucleotide coding sequence " refers to have the corresponding polypeptide of the active as above-mentioned sequence of human soluble guanylate cyclase.This term also comprises having the variant form that has identical function with above-mentioned albumen.These variant forms comprise: several (are generally 1-50; Preferably 1-30; 1-20 more preferably, 1-10 best) amino acid whose disappearance, insertion and/or replacement, and add one or several at C-terminal and/or N-terminal and (be generally in 20; Preferably being in 10, more preferably is in 5) amino acid.For example, in the art, when replacing, can not change proteinic function usually with the close or similar amino acid of performance.Again such as, add one or several amino acid at C-terminal and/or N-terminal and also can not change proteinic function usually.This term also comprises the active fragments and the reactive derivative of human soluble guanylate cyclase.
The variant form of this polypeptide also comprises: the fusion rotein of homologous sequence, conservative property varient, allelic variant, natural mutation, induced mutation body, human soluble guanylate cyclase Nucleotide and the polypeptide or the albumen that utilize anti-human soluble guanylate cyclase Nucleotide serum to obtain.
Expression vector that adopts in the method for the present invention and bacterial strain can adopt this field expression vector and host bacterium commonly used; For example MBPHT-mCherry2 carrier and intestinal bacteria Rosetta (DE3) pLysS; PMAL-c2x and BL21 (DE3) pLysS; PET28b and Rosetta (DE3) pLysS, pET22b and Rosetta (DE3).
Pack into carrier and transform bacterial strain and can adopt conventional method of human soluble guanylate cyclase encoding sequence.For example, increase in the carriers such as the pBluescriptR that earlier the human soluble guanylate cyclase encoding sequence packed into, and then construction of expression vector; For another example, human soluble guanylate cyclase encoding sequence 5 ' with 3 ' add existing restriction enzyme site on the carrier, thereby encoding sequence is inserted the MCS place of carrier.Transform and adopt heat shock to transform.
TB substratum after the improvement comprises: 5-10g/L peptone, 5-10g/L yeast extract, 2-10ml/L glycerine, 0.01-0.02mol/L KH
2PO
4, 0.1-0.2mol/L K
2HPO
4, 1-2g/L MgSO
4, 1-4g/LNaNO
3, 10-30mM Glucose (glucose).
IPTG induces and can adopt commercially available IPTG and conventional inductive condition.Inducing spends the night is meant that induction time was at 10-16 hour.
In the method for the present invention, step (3) mainly is a separation and purification albumen.Can adopt the ordinary method separation and purification, the present invention also adopts effect technological separation and purification such as Ni-NTA post preferably.Because the fusion rotein of expressing has SANMALT-S fusion rotein (MBP) label and Histidine (His) label; The present invention comes purifying with the Ni-NTA post; The Ni-NTA post is the affinity column of Histidine (His) label, and its affinity and specificity are all than higher, so purification effect is relatively good.And Amylose (starch-polysaccharides) post is the affinity column of SANMALT-S fusion rotein (MBP) label, can come purifying protein with it, and is relatively poor but its avidity compares, and post is imitated lower.The used tev enzyme of the present invention is a kind of enzyme of specific recognition ENLYFQG peptide section, and we have been added in this seven peptide sequence between MBP and the target protein sequence when making up plasmid, so can come enzyme to cut with the tev enzyme.Can cut with Factor-Xa, but somewhat expensive.
Among the present invention, used the heme reorganization.This is that reduced hematin is meant a big proteinoid of being made up of protoheme (heme) prothetic group and apoprotein (apo-protein) because sGC is a kind of reduced hematin.General protoheme prothetic group is its active site.SGC exists with heme bonded form in human body; But because we are at expression in escherichia coli with plasmid; So occur only expressing apoprotein through regular meeting, and heme can not recombinate in intestinal bacteria, in addition; In the protein purification process, also occur heme easily and from proteic protoheme chamber, spin off, become apoprotein.So need after obtaining pure apo-protein, again heme be recombinated to the protoheme chamber.
In one embodiment of the present of invention, adopt following method specifically to prepare people sGC.At first, with people sGC-β 1 gene (can buy from reactivation genome company, can search) from genbank.Be cloned into the MBPHT-mCherry2 carrier, and extracting obtains containing the expression plasmid of goal gene.Then, plasmid is transformed into expression uses genetic engineering bacterium, be inoculated in improved TB (containing 50 μ g/mL penbritins) nutrient solution, 37 ℃ are cultured to OD
600Receive bacterium after adding IPTG after～0.6 and being cooled to 20 ℃ of abduction delivering～12h.Above-mentioned thalline carrying out ultrasonic bacteria breaking is centrifugal, crosses the Ni-NTA post, and tev enzyme enzyme is cut, and crosses the Ni-NTA post, and the heme reorganization can obtain the target protein of 90% above purity.
Expression vector of the present invention is the MBPHT-mCherry2 plasmid; Expression is intestinal bacteria Rosetta (DE3) pLysS with genetic engineering bacterium.
The nutrient solution that the present invention uses is the TB nutrient solution after improving, and is specially in the TB nutrient solution, to add 1.25g/L MgSO
4, 2g/L NaNO
3, 20mM Glucose.This improvement can increase substantially protein yield.
The present invention first from expression in escherichia coli people's the sGC albumen of β 1 subunit, and the albumen solubility is fine.Get brokenly last cleer and peaceful deposit sample behind the bacterium, use the SDS-PAGE detected through gel electrophoresis, its result shows that target protein is basically all in supernatant.Utilize people sGC protein yield that the present invention obtains up to more than the 20mg/L, purity reaches the human soluble guanylate cyclase albumen more than 90%, is the highest low-cost expression method of current production rate.The present invention has major application at biomedicine field and is worth.
Embodiment
At first, reactivation genome company buys people sGC-β 1 gene from Guangzhou for we, through genetic engineering means it successfully is cloned in the MBPHT-mCherry2 carrier then.Gene sequencing result shows that we have successfully made up human sGC-β 1 expression plasmid.
With Rosetta (DE3) pLysS is that the host bacterium transforms, and chooses spot and spends the night in the LB culture medium culturing, according to the 1:100 ratio bacterium liquid is forwarded to 250ml modified TB substratum then and (adds 1.25g/LMgS0 among the TB
4, 2g/L NaNO
3, 20mM Glucose), 37 ℃, 250rpm is cultured to OD
600~ 0.6, add IPTG (final concentration 0.1mM) then, simultaneous temperature is received bacterium after reducing to 20 ℃ of expression～12h.Thalline is resuspended in buffer A (50mM Na-Pi, 500mM NaCl, 5mM beta-mercaptoethanol, 20% glycerine by the wet bacterium of 5ml/g; PH8.0), add a spot of N,O-Diacetylmuramidase, DNase and PMSF (final concentration 1mM), 4 ℃ of back carrying out ultrasonic bacteria breaking that stir; Then 14,000rpm, 4 ℃ of centrifugal 15min; Take out supernatant and cross the Ni-NTA post,, use buffer C (adding the 200mM imidazoles among the buffer A) wash-out then with buffer B (adding the 10mM imidazoles among the bufferA) washing; Collect elutriant, the DTT that adds tev enzyme and final concentration 5mM behind the imidazoles is removed in the dialysis of albumen elutriant, cuts in the room temperature enzyme and spends the night.Mistake G-25 post was removed DTT after enzyme was cut the product ultrafiltration and concentration, and then crossed a Ni-NTA post, collected the protein stream fluid, so just can obtain human sGC-β 1 apoprotein of 90% above purity.
Through the heme reorganization, successfully obtained high purity, the whole protein that contains heme of high yield then.
The reagent and the biological material source that use among the present invention are following:
MBPHT-mCherry2 Carrier: by Fudan University School of Life Sciences Dr. Ding Yu gift
Human sGC-β 1 gene is available from Guangzhou reactivation genome company
Host bacterium Rosetta (DE3) pLysS is available from Novagen company
Hemin: available from sigma company
Ni-NTA resin, plasmid extraction test kit and gel reclaim test kit available from Qiagen company
N,O-Diacetylmuramidase (lysozyme), DNaseI, Ampicillin Trihydrate (Ampicillin), PMSF (PMSF), sec.-propyl-β-D thiogalactoside (IPTG), beta-mercaptoethanol, DTT available from Shanghai give birth to worker company (Sangon, Shanghai, China)
The Pfu archaeal dna polymerase, the T4 dna ligase, dNTPs and restriction enzyme BamHI, EcoRI, HindIII, XhoI is available from New England Biolabs company;
Sephadex G-25 sephadex column available from Pharmacia Biotech company (Uppsala, Sweden).
Peptone, yeast extract is available from OXOID company.
Claims (2)
1. the high efficiency preparation method of human soluble guanylate cyclase is characterized in that, this method may further comprise the steps:
With human soluble guanylate cyclase sGC-β 1 gene clone to expression vector MBPHT-mCherry2, transformed into escherichia coli bacterial strain Rosetta (DE3) pLys S;
⑵ be inoculated in the TB nutrient solution after the improvement with the ⑴ obtained strains, and described TB nutrient solution contains the 5-10g/L peptone, 5-10g/L yeast extract, 2-10ml/L glycerine, 0.01-0.02mol/L KH
2PO
4, 0.1-0.2 mol/L K
2HPO
4, 1-2g/L MgSO
4, 1-4g/L NaNO
3, 10-30mM glucose; Adding the IPTG abduction delivering spends the night;
⑶ with the broken bacterium of ⑵ gained thalline, centrifugal, crosses the Ni-NTA post, and tev enzyme enzyme is cut, and crosses the Ni-NTA post, and the heme reorganization promptly gets people sGC-β 1heme whole protein.
2. preparation method according to claim 1 is characterized in that, broken bacterium method is ultrasonic, multigelation or N,O-Diacetylmuramidase enzymolysis among the ⑶.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810202926A CN101736022B (en) | 2008-11-18 | 2008-11-18 | High-efficiency preparation method of human soluble guanylate cyclase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810202926A CN101736022B (en) | 2008-11-18 | 2008-11-18 | High-efficiency preparation method of human soluble guanylate cyclase |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101736022A CN101736022A (en) | 2010-06-16 |
| CN101736022B true CN101736022B (en) | 2012-09-05 |
Family
ID=42460051
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810202926A Expired - Fee Related CN101736022B (en) | 2008-11-18 | 2008-11-18 | High-efficiency preparation method of human soluble guanylate cyclase |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101736022B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103834676B (en) * | 2014-02-28 | 2016-12-07 | 中国科学院福建物质结构研究所 | A kind of E. coli secretion expresses plasmid vector and the construction method thereof of foreign protein |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1381579A (en) * | 2001-04-18 | 2002-11-27 | 上海博德基因开发有限公司 | Polypeptide-guanylyl cyclase-13.53 and polynucleotide for coding it |
-
2008
- 2008-11-18 CN CN200810202926A patent/CN101736022B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1381579A (en) * | 2001-04-18 | 2002-11-27 | 上海博德基因开发有限公司 | Polypeptide-guanylyl cyclase-13.53 and polynucleotide for coding it |
Non-Patent Citations (4)
| Title |
|---|
| Dmitry N. Kosarikov等.Human Soluble Guanylate Cyclase: Functional Expression,Purification and Structural Characterization.《Archives of Biochemistry and Biophysics》.2001,第388卷(第2期),第185-197页. * |
| DmitryN.Kosarikov等.HumanSolubleGuanylateCyclase:FunctionalExpression Purification and Structural Characterization.《Archives of Biochemistry and Biophysics》.2001 |
| Jonathan A. Winger等.Expression and Characterization of the Catalytic Domains of Soluble Guanylate Cyclase: Interaction with the Heme Domain.《Biochemistry》.2005,第44卷第4083-4090页. * |
| Yu-Chen Lee等.Human recombinant soluble guanylyl cyclase:Expression,purification,and regulation.《PNAS》.2000,第97卷(第20期),第10763-10768页. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101736022A (en) | 2010-06-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107245494A (en) | Solution expression with high efficiency and purification process of the A β 42 in Escherichia coli | |
| CN111117977B (en) | Recombinant polypeptide linked zymogen, preparation method, activation method and application thereof | |
| CN116333097A (en) | High-activity recombinant human fibronectin and preparation method and application thereof | |
| CN114853881A (en) | Recombinant human fusion collagen and efficient hydroxylation method and application thereof | |
| CN112980865A (en) | Construction method of recombinant human-like collagen engineering bacteria | |
| CN110964096B (en) | Preparation method of recombinant human C-reactive protein | |
| CN114107353B (en) | Plasmid for efficiently expressing polypeptide toxin and preparation method and application thereof | |
| CN117126754A (en) | Recombinant type I collagen pichia pastoris engineering bacteria, construction method and application thereof | |
| CN101429519A (en) | Process for producing recombinant insulin-like growth factor-1(IGF-1) amalgamation protein | |
| CN113004375B (en) | Small molecular protein for efficiently mediating recombinant polypeptide to form inclusion body | |
| CN101736022B (en) | High-efficiency preparation method of human soluble guanylate cyclase | |
| JP4088584B2 (en) | A method for separating a target protein from a fusion protein. | |
| CN114410558B (en) | ACE inhibitory peptide-ACEIP with blood pressure reducing function and genetic engineering preparation method thereof | |
| CN115093470B (en) | Intein Mtu RecA mutant and application thereof in production of glutathione GSH | |
| CN102167733A (en) | Construction, expression and application of acidly cleavable high-copy antihypertensive peptide tandem gene | |
| RU2453604C1 (en) | Hybrid protein (versions), bacterial strain escherichia coli - hybrid protein producer (versions) and method for producing methionine-free human interferon alpha-2 | |
| CN108753758B (en) | Hyperthermophilic lipase LipL and related biological material and application thereof | |
| CN104725485B (en) | One kind restructuring active peptide and its synchronic preparation method | |
| CN113025599A (en) | Recombinant Clostridium histolyticum type I collagenase and preparation method and application thereof | |
| CN108841804B (en) | Hyperthermophilic lipase LipH and related biological material and application thereof | |
| RU2441072C1 (en) | FUSION PROTEIN, ESCHERICHIA COLI STRAIN BEING FUSION PROTEIN PRODUCER AND METHOD FOR PRODUCING METHIONINE-FREE HUMAN INTERFERON ALPHA-2b OF SUCH FUSION PROTEIN | |
| CN117466992B (en) | Fibronectin mutant and preparation and application thereof | |
| TWI712691B (en) | Dextran affinity tag and application thereof | |
| CN112941058B (en) | Recombinant clostridium histolyticum type II collagenase as well as preparation method and application thereof | |
| KR102466926B1 (en) | Method for soluble expression and purification of hydrophobin |
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: 20120905 Termination date: 20201118 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |