CN101967493A - Prokaryotic expression vector and application thereof - Google Patents
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Abstract
The invention relates to a prokaryotic expression vector and application thereof. The vector comprises: (a), a molecular chaperone for promoting the soluble expression of target protein or/and the correct folding of disulfide bonds; and (b), an encoding nucleotide sequence of intein which can be broken on peptide bonds at a C terminal. In the vector, the high-efficiency soluble expression of the target protein is promoted by the molecular chaperone, so that the target protein maintain natural bioactivity, and the self-shearing action of the intein is induced effectively under a certain condition to separate the target protein from fusion protein, and the target protein is separated and purified again to obtain the target protein with natural bioactivity economically and efficiently. The invention also relates to host cells containing the vector and application of the vector in the recombinant expression of bioactivity protein.
Description
Technical field
The invention belongs to technical field of bioengineering.Specifically, the present invention relates to a kind of prokaryotic expression carrier and comprise the host cell and the application of this carrier in recombinant expressed biological activity protein of this carrier.
Technical background
Intestinal bacteria are the major project bacterium that produce recombinant exogenous protein at present.Yet, utilize prokaryotic system express to produce recombinant protein at present, recombinant protein is normal problems such as form with insoluble inclusion body (inclusion body) exists, expression amount is low, biological activity is not high to occur.With molecular chaperones (molecular chaperone or chaperone; Also claim fusion partner, fusion partner or fusion tag, fusion tag) and target protein to be connected to become that a fusion rotein expresses be one of strategy commonly used that increases the target protein solubility expression.The most frequently used molecular chaperones has Thiadiazolidine isomerase (GST), Trx (Trx), maltose binding protein (MBP), NusA albumen, disulfide bond reduction enzyme and isomerase (Dsb family at present, as DsbA, DsbC), little ubiquitin modified protein (SUMO) and heat shock protein(HSP) (heat shock protein, (Esposito D such as HSP), Chatterjee DK.Curr Opin Biotechnol.2006,17:353-358; Betiku E.Biotechnology and Molecular Biology Review.2006,1:66-75).For producing recombinant protein, not only to obtain high yield solubility recombinant protein, the more important thing is the biologic activity that will guarantee recombinant protein.Special in many protein that contain disulfide linkage, correct disulfide linkage is folded in keeps proteinic structure and active aspect plays an important role.In prokaryotic expression system, amalgamation and expression not only can improve expressing quantity and solubility, and the part molecular chaperones can also help target protein correctly folding, thereby keeps better biological activity.As, disulfide bond reduction enzyme and isomerase (Dsb family, as DsbA, DsbC), Trx (Trx) and little ubiquitin modified protein (SUMO) etc. can promote the correct connection of the disulfide linkage of the target protein of expressing, thereby make the target protein of expression keep natural bioactive (Marblestone JG et al.Protein Sci.2006,15:182-189; Panavas T et al.Methods Mol Biol.2009,497:303-317; Yasukawa T et al, J Biol Chem.1995,270:25328-25331; Lauber T et al, Protein Expr Purif.2001.22:108-112).Convenience for purifying, can merge corresponding affinity purification label (affinity purification tags) at target protein, as 6 * his-tag, GST, MBP, FLAG, (Esposito D such as BAP, Strep-II and CBP, Chatterjee DK.Curr Opin Biotechnol.2006,17:353-358).Yet the amalgamation and expression weak point is often will pass through special proteolytic enzyme behind amalgamation and expression, cuts or chemical cracking is removed fusion tag and molecular chaperones as the enzyme of enteropeptidase, fXa etc.This just may need secondarily purified, and introduces the protease price costliness that enzyme is cut, the increase that can bring cost, and may cause non-specific cutting (Xie Hao etc. biological chemistry and biophysics are made progress .2009,36:1364-1369).In addition, also there is behind the excision fusion tag target protein formation deposited phenomenon that reassembles.
Intein (intein) is the one section sequence that is present in the precursor protein, be converted in the process of mature protein at precursor protein, rely on self-shearing action to discharge from precursor protein, the protein extein (extein) with two ends links together simultaneously.Intein can mediate N end or C holds one-sided peptide bond rupture through inducing.Aspect protein engineering, this specific character that intein has self-shear property can realize target protein and the isolating purpose of affinity tag.Link together as gene order coded affinity label, intein and target protein, in appropriate expression system, give expression to the triplet of an affinity tag (Tag)-intein-target protein, utilize affinity chromatography absorption label again and hold back fusion rotein, the oneself is taken place from the N-end of intein or C-end and is sheared and discharge target protein in (as the adding of pH, variation of temperature or sulfhydryl compound) this triplet under the chemical factors effect subsequently.(New England Biolabs NEB) has made up IMPACT according to this principle to New England's biology laboratory
TM, as the pTWIN1 expression vector, this system does not need to cut with the proteolytic enzyme enzyme.IMPACT
TMThe target protein of system expression and intein and chitin-binding protein form fusion rotein, by chitin post affinity purification fusion rotein.Induce the peptide bond lytic activity of the intein in the purified fusion protein then, on the chitin medium, target protein is discharged, and intein and chitin-binding protein (CBD) still are combined on the chitin medium, reach the proteic purpose of single-column separation and purification.Thisly shear affinity purification and avoided the adding of exogenous protease and later stage removing step proteolytic enzyme by introducing the intein oneself.And the cracking of intein mediation occurs over just splice site, and is irrelevant with the responsive site of the proteolytic enzyme on the target protein, also avoided degraded (Chong S et al.Gene.1997, the 192:277-281 of proteolytic enzyme to target protein; Sharma SS et al.J Biotechnol.2006,125:48-56).
But producing recombinant protein by the intein mediation at present all is first separation, purified fusion protein, and then to the fusion rotein on affinity media (as fusion rotein with the affine band of nickel post his-tag, the fusion rotein of the chitin medium is affine band CBD) induces shearing, to obtain target protein (Chong S et al.Gene.1997,192:277-281; Sharma SS et al.J Biotechnol.2006,125:48-56).This method often causes in the expressing fusion protein process and the target protein of shearing in advance that takes place in the separation and purification process loses in a large number.Simultaneously, intein can not effectively improve output, the solubility expression of target protein and promote the effectively folding of its disulfide linkage.
Hookworm coagulate peptide resistant is the polypeptide that has remarkable anticoagulating active from the class that the parasitic hookworm of sucking blood is found, usually form by 80 amino acid, contain 5 pairs of disulfide linkage, may be developed as anti-freezing new anti-embolism medicine or reagent (Peng Lifei etc. the journal .2006 of Guangdong Medical College, 24:624-627).Wherein, the anticoagulant peptide AcaNAP10 that is derived from the dog hookworm that inventor seminar finds has very strong anticoagulating active, highly efficient depressor (the Li Det al.Biochem Biophys Res Commun.2010 of crucial thrombin-tissue factor and activatory proconvertin mixture (TF/fVIIa) and coagulation pathway amplification stage key factor-activatory plasma thromboplastin antecedent (fXIa) unloading phase of being coagulation pathway, 392:155-159), be at present uniquely can suppress the anticoagulant substances that TF/fVIIa can suppress fXIa again, it is worth expectation as anti-freezing new anti-embolism medicine or reagent exploitation.
For recombinant expressed commercial albumen, particularly wish to express solubility efficiently by prokaryotic expression system, the target protein that keeps natural bioactive, production cost that minimizing separate targets albumen and fusion tag and molecular chaperones are brought and relatively large acquisition target protein, the inventor has developed a kind of prokaryotic expression carrier, and provide the fusion rotein of expressing at first effectively induced to shear target protein is separated with fusion rotein, and then method to carrying out separation and purification with the isolating target protein of fusion rotein, and further disclosed this expression vector contains disulfide linkage in expression albumen, the particularly application of hookworm coagulate peptide resistant AcaNAP10.
Summary of the invention
The object of the present invention is to provide a kind of prokaryotic expression carrier and application thereof.This carrier can promote highly-soluble ground expression target protein and make target protein that the target protein that particularly contains disulfide linkage keeps natural bioactive by molecular chaperones.Use the target protein of this vector expression and can realize that by the self-shearing action of intein target protein separates with fusion rotein economical and efficient ground, the further separation and purification of the target protein after the separation, but economical and efficient obtains a large amount of target proteins.
The invention provides a kind of prokaryotic expression carrier, the carrier that sets out that makes up expression vector described in the present invention can be various prokaryotic expression carriers described in the genetically engineered field, as pET series expression vector.By the genetically engineered ordinary method can be promoting the target protein solubility expression or/and the correctly folding molecular chaperones of disulfide linkage and connecting in the carrier that sets out at the intein coding nucleotide sequence that C end oneself shears.This carrier is expressed target protein or/and the disulfide linkage of target protein correctly folds with raising target protein output and keeps natural radioactivity by molecular chaperones promotion highly-soluble, and in the self-shearing action of the C-terminal of the intein of molecular chaperones target protein is separated by fusion with fusion rotein.Can be connected by long or short one section sequence (the flexible amino acid district of encoding) between described molecular chaperones and the intein.This carrier also can be based on designing affinity tag according to the purifying needs in fusion partner, as 6 * his-tag, GST, MBP, FLAG, (Esposito D such as BAP, Strep-II, CBD and CBP, Chatterjee DK.Curr Opin Biotechnol.2006,17:353-358), be used for the simple and fast affinity chromatography and remove and also do not have the fusion rotein sheared, as the affine fusion rotein sheared of not having of removing band CBD of available chitin post.Common can promote the target protein solubility expression or/and the correctly folding local fusion partner that merges of disulfide linkage has Thiadiazolidine isomerase (GST), Trx (Trx), maltose binding protein (MBP), NusA albumen, disulfide bond reduction enzyme and isomerase (Dsb family, as DsbA, DsbC), little ubiquitin modified protein (SUMO) and heat shock protein(HSP) (heat shock protein, but be not limited only to above-mentioned albumen HSP) etc..Can comprise mini Ssp DnaB (Mathys S et al.Gene.1999 at the intein that C end oneself shears, 231:1-13) and Sce VMA (Chong S et al.Gene.1997,192:277-281) etc., be good with mini Ssp DnaB, but be not limited only to above-mentioned albumen.
The oneself that passes through of the target protein that utilizes invention carrier high-efficiency amalgamation and expression and fusion rotein shears and separates, can be under 0 ℃～40 ℃, and preferably under cold condition, under 0 ℃～20 ℃, pH carries out in 6.0～8.0 the damping fluid.Carry out self-shearing at low temperatures and can avoid or reduce degraded target protein.Have with the separating of target protein after fusion rotein separates, purification process: can carry out separation and purification with corresponding conventional purification technique according to physico-chemical properties such as the size of target protein, electrically charged and iso-electric points; Convenient for purifying, also can in the target protein of expressing, design affinity tag, as behind the N-terminal or C-terminal fusion His-tag of target protein, available nickel affinity chromatography carries out quick and convenient purification of target albumen; Also can utilize the antibody of target protein to carry out affinity purification.Need remove for the fusion rotein that does not also have to have cut, can in fusion partner, design the affinity tag that is different from the affinity tag that target protein has, removing by affinity chromatography does not have the fusion rotein that cut, also can be according to physico-chemical properties such as the size of molecular chaperones and fusion rotein, electrically charged and iso-electric points, removing with conventional chromatographic technique, sieve technology etc. does not have the fusion rotein that cut.
An application of the present invention is that the expression vector economical and efficient ground of application invention obtains to keep the target protein of natural bioactive, particularly contains the biological activity protein of disulfide linkage, as r-hirudin, hookworm coagulate peptide resistant, proteinase inhibitor etc.Hookworm coagulate peptide resistant AcaNAP10 (SEQ ID NO.1) be at present unique to coagulation pathway the unloading phase crucial thrombin-tissue factor and activatory proconvertin mixture (TF/fVIIa) and coagulation pathway amplification stage key factor-activatory plasma thromboplastin antecedent (fXIa) inhibiting highly efficient depressor all arranged.Use vector expression AcaNAP10 of the present invention or its conservative property variation polypeptide or their active fragments or their reactive derivative, can be used as anti-freezing medicine for treating thrombus thing or preparation and develop and apply.
Others of the present invention are because disclosing of this paper technology contents is easy to understand and enforcement to those skilled in the art.For example, utilize linear interconnection technique or round pcr etc., connect into carrier of the present invention, can obtain not target protein with any amino acid residue sequence outside self only containing the target protein coding nucleotide sequence; A kind of method that relates to the target protein after separation and purification oneself shears can be separated according to target protein character, purifying etc.; Utilize characteristics of the present invention can relate to a kind of prokaryotic expression carrier that sets out and make up based on other prokaryotic expression carrier outside the pET expression vector with characteristics of the present invention; A kind of usefulness contains the genetically engineered host cell of expression vector expression biological activity protein of the present invention etc.
Therefore, carrier of the present invention improves output, the solubility of expressing target protein and keeps the target protein natural bioactive by molecular chaperones on the one hand; In addition on the one hand, the oneself by intein shears and realizes that target protein separates with fusion rotein economical and efficient ground, and the enzyme that reduces use enteropeptidase, fXa etc. cuts or chemical cracking is removed fusion tag and molecular chaperones and carried out the secondarily purified cost that brings; The third aspect, self-shearing action by intein realizes target protein and after fusion rotein economical and efficient ground separates, again to carrying out further separation and purification, can reduce in the expressing fusion protein process and the target protein of shearing in advance that takes place in the separation and purification process loses in a large number with target protein after fusion rotein separates.
Description of drawings
Fig. 1 expression vector pICET32 plasmid figure.
The complete nucleotide sequence of Fig. 2 expression vector pICET32.
Fig. 3 in e. coli bl21 (DE3) host in, express the SDS-PAGE electrophorogram of the hookworm coagulate peptide resistant AcaNAP10 (SEQ ID NO.1) contain 5 pairs of disulfide linkage with pICET32.
Fig. 4 in e. coli bl21 (DE3) host in, express the SDS-PAGE electrophorogram of the Kunitz type serpin AduKuI4 contain 3 pairs of disulfide linkage with pICET32.
Embodiment
Below in conjunction with specific examples, further set forth the present invention.Should be understood that these to implement 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 such as molecular cloning experiment guide (the 3rd edition): ([U.S.] J. Sa nurse Brooker, D.W Russell work. Huang Peitang etc. translate, Science Press, 2002), fine works molecular biology experiment guide (the 5th edition) ([U.S.] F.M. Ao Sibai, R. Brunt is compiled. Jin Youxin, wrap intelligent insidely, and Zhao Liyun translates. Science Press, 2008) described condition, or the condition of advising according to manufacturer.
The structure of embodiment 1 expression vector pICET32
With the pET32a carrier that sets out, pET32a has the T7 efficient promoter, contains Trx TrxA, is one of the albumen solubility efficient expression vector that promotes commonly used at present.Design upstream primer DP1:5 '-CA
TGGCCATATGAAAATCGAAGAAGGTAAAC-3 ' (underscore is that italicized item is a restriction enzyme Msc I restriction enzyme site) and downstream primer DP2:5 '-GTCCATGGCCGTTGTGTACAATGATGTCATTC-3 ' obtains chitin binding domain (CBD) and intein mini SspDnaB coding nucleotide sequence with the pfu enzyme from expression vector pTWIN1 (NEB company product) amplification, with this product is template, obtains second with upstream primer DP1 with new design primer 5 '-GTACACAACGGCCATGGACATCATCATCATCATCATGGATCC-3 ' amplification and takes turns product.Take turns amplification by this and introduce restriction enzyme NcoI restriction enzyme site and 6 * his tag encoding sequence.Taking turns amplification with second is template, with primer DP1 and new design primer 5 '-T
GCTCGAGTAAGCTTTGAATTCGGATCCATGATGATGATG-3 ' (underscore is that italicized item is a restriction enzyme Xho I restriction enzyme site) amplification obtains the third round product.Introduce restriction enzyme BamH I, EcoR I, Hind III and Xho I restriction enzyme site by the third round amplification.The third round product connects into equally the pET32a carrier of cutting through Msc I and Xho I enzyme behind Msc I and Xho I double digestion.The carrier that connects changes E.coli DH5 α over to, makes up plasmid and cuts and check order through enzyme and identify correctly, successfully obtains establishing target carrier pICET32.
PICET32 plasmid figure sees accompanying drawing 1, and nucleotide sequence is seen accompanying drawing 2.This carrier is based on the carrier of the structure that pET32a sets out, and has the required base components of prokaryotic expression system.Has the T7 efficient promoter, contain molecular chaperones Trx (TrxA) and promote the expression of target protein highly-soluble and make target protein keep natural bioactive, can realize that target protein separates with fusion rotein in shearing by the oneself who contains peptide mini Ssp DnaB.For purifying needs, in intein, merged affinity tag chitin binding domain (CBD), Trx and Ssp DnaB each other jointing the respective flexible district is all arranged.3 ' end at mini Ssp DnaB encoding sequence is followed successively by multiple clone site, 6 * his tag encoding sequence, multiple clone site and 6 * his tag encoding sequence.Also can realize the seamless link of target protein and intein by PCR, the target protein that makes acquisition is not with the aminoacid sequence outside itself.
According to Ancylostoma caninum anticoagulant peptide AcaNAP10 (SEQ ID NO.1) encoding sequence design primer amplification coding nucleotide sequence from dog hookworm adult cDNA, design primer sequence and be: N10-3:5 '-GAGGATCCAATCCAAGCTGTGGTGAG-3 ' (containing restriction enzyme site BamH I); N11-2:5 '-CGAAGCTTGGTCATTTTCTATTAGGG-3 ' (containing restriction enzyme site Hind III).The PCR product carries out double digestion with BamH I and Hind III after reclaiming purifying, and spending the night with the expression plasmid pICET32 of the same double digestion of warp is connected.Connect product and be converted in the E.coli DH5 α competent cell, in containing the substratum of penbritin, cultivate.Recombinant clone plasmid called after AcaNAP10/pICET32 through PCR and order-checking evaluation.Recombinant expression plasmid is converted into e. coli bl21 (DE3) competent cell.Cultivation contains recombinant expression plasmid AcaNAP10/pICET32 host bacterium BL21 (DE3) in containing the LB nutrient solution of penbritin, and the IPTG of 1.0mmol/L final concentration induces.Centrifugal collection thalline, after ultrasonication, collect supernatant, (supernatant: 10 * phosphate buffered saline buffer that ratio damping fluid) adds different pH values (being respectively pH6.0, pH6.4, pH6.8, pH7.2, pH7.6, pH8.0) placed 4 ℃, 16 ℃, 30 ℃, 40 ℃ cracking with 1: 4, carry out SDS-PAGE respectively at the sampling in 6 hours of every interval in 48 hours and observe lytic effect, with software Quantity One 4.52 analytical pyrolysis efficient.Get corresponding cracking supernatant Ni-IDA affinity purification, also do not have the cracked fusion rotein through affine the removing of chitin post, stream is worn liquid and is target protein, can be standby except that the concentrated back of imidazoles.(method is with reference to Li D et al.Biochem Biophys Res Commun.2010,392:155-159) with anticoagulating active outside PT and the aPTT method detection bodies for purification of target albumen.Simultaneously, with pTWIN1 vector expression AcaNAP10, prepare reorganization AcaNAP10 to specifications, the albumen for preparing is with comparing.With Bradford determination of protein concentration kit measurement purifying protein concentration.
The result shows: recombinant plasmid pICET32/AcaNAP10 obtains highly-soluble and expresses in intestinal bacteria, 95% above target protein (fusion rotein) is a soluble protein, and the fusion rotein amount can reach about 40% (Fig. 3 swimming lanes 1) of host's total protein.In every liter of LB substratum, obtaining the soluble fusion protein amount can reach more than the 250mg.Solubility and quantum of output with the AcaNAP10 of pICET32 preparation all significantly improve than pTWIN1.Purifying has obtained the AcaNAP10 of N-terminal band 6 * his tag, the molecular weight of albumen size is about 20Kda (having formed dimer) (Fig. 3 swimming lane 3), do not have the fusion rotein size after shearing and target protein are sheared to be about 49kDa respectively, 39kDa (Fig. 3 swimming lane 1) conforms to theoretical prediction.In the LB substratum, the final purifying rAcaNAP10 amount that obtains can reach more than the 70mg/L.Expressing and the host bacterium separates, the cracking shattering process has taken place all in advance that the oneself shears with pICET32 and pTWIN1 expressed proteins.As Fig. 2 swimming lane 1, pICET32 carries out in the broken cracked process at abduction delivering and collection host bacterium, has the fusion rotein above 1/3rd that oneself's shearing has taken place.4 ℃ place 12 hours after, there is the fusion rotein more than 80% to finish oneself's shearing cracking, basically, finish the oneself at 4 ℃ of fusion roteins of placing 18 hours and shear cracking (Fig. 3 swimming lane 2), almost all finish the oneself at 4 ℃ of fusion roteins of placing 36 hours and shear cracking.Carry out the oneself and shear in 4 ℃, the target protein and the fusion partner that are placed in 48 hours all do not have obvious degradation.Carry out 5 protein expression purifying at random and actively detect contrast and show, with (4 ℃ of the reorganization AcaNAP10 of pICET32 preparing carriers, the pH6.4 phosphate buffered saline buffer, the oneself shears purifying protein after 36 hours) to prolong 2 times of PT desired concns be 30.5 ± 4.2nM, and prolong 2 times of PT desired concns with the reorganization AcaNAP10 of pTWIN1 preparing carriers is 58.3 ± 3.8nM, promptly significantly wants high with the reorganization AcaNAP10 of pICET32 preparing carriers is active.
Expression and the separation and purification thereof of embodiment 3 serpin AduKuI4
AduKuI4 is a kind of Kunitz type serpin that contriver seminar separates from Ancylostoma duodenale, contain 3 pairs of disulfide linkage, its mature peptide aminoacid sequence is: RNPHRKGRCGDDPAETGGECPDPETKYTYKFGDCHEVKYCGEQETRNLFDSYEKCS GKCVIF.According to the AduKuI4 mature polypeptide coding sequence, design upstream primer: 5 '-TAGGATCCCGCAATCCTCACAGAAAG-3 ' and downstream primer: 5 '-CCAAGCTTAGAAGATCACGCACTTTCC-3 ', amplification obtains mature polypeptide coding sequence from Ancylostoma duodenale adult cDNA.Amplified production connects into expression vector and successfully makes up the pICET32/AduKuI4 expression plasmid.Plasmid is converted into e. coli bl21 (DE3), through inducing culture, centrifugal collection thalline, collect supernatant after the ultrasonication, (supernatant: 10 * phosphate buffered saline buffer that ratio damping fluid) adds different pH values (being respectively pH6.0, pH6.4, pH6.8, pH7.2, pH7.6, pH8.0) placed 4 ℃, 16 ℃, 30 ℃, 40 ℃ cracking, carries out SDS-PAGE respectively at the sampling in 6 hours of every interval in 48 hours and observes lytic effect with 1: 4.Get corresponding cracking supernatant Ni-NTA affinity purification, also do not have the cracked fusion rotein through affine the removing of chitin post, stream is worn liquid and is target protein.
The result shows: AduKuI4 obtains efficient soluble-expression, and target protein (fusion rotein) is almost all solvable, and melt-moldable hop protein total amount can reach the total bacterial protein amount more than 50%.The fusion partner that gets off from cracking as can be seen, the oneself shears (Fig. 4 swimming lane 2) promptly to have 50% above soluble protein to take place in advance in abduction delivering and bacteria breaking process.All can induce shearing at each temperature, after 24 hours, the fusion rotein all overwhelming majority is sheared (Fig. 4 swimming lane 3,4 is respectively host bacterium supernatant in the cracking situation after the intein oneself shears under 4 ℃ and 40 ℃), get corresponding cracking supernatant Ni-IDA affinity purification, through the affine fusion rotein that does not also have cracking intact of removing of chitin post, obtain target protein, size is about 8kDa, and (Fig. 4 swimming lane 5,6,7) conforms to theoretical prediction.In the LB substratum, the final purifying rAduKuI4 amount that obtains can reach more than the 80mg/L, and promptly AduKuI4 obtains efficient soluble-expression.
Sequence table
＜110〉Guangdong Medical College
＜120〉a kind of prokaryotic expression carrier and application thereof
<160>1
<210>1
<211>80
<212>PRT
＜213〉dog hookworm (Ancylostoma caninum)
<400>2
Asn?Pro?Ser?Cys?Gly?Glu?Asn?Glu?Arg?His?Asp?Glu?Cys?Ser?Arg
5 10 15
Lys?Glu?Cys?Asp?Pro?Lys?Cys?Lys?Tyr?Asp?Gly?Thr?Glu?Glu?Lys
20 25 30
Asp?Asp?Glu?Lys?Pro?Val?Val?Cys?Leu?Thr?Arg?Val?Cys?Tyr?Gly
35 40 45
Asp?Cys?Ile?Cys?Arg?Asp?Gly?Phe?Leu?Arg?Asn?Lys?Asn?Gly?Ala
50 55 60
Cys?Val?Lys?Ala?Glu?Asp?Cys?Glu?Leu?Asp?Asn?MET?Glu?Phe?Ile
65 70 75
Tyr?Pro?Asn?Arg?Lys
80
Claims (10)
1. prokaryotic expression carrier is characterized in that containing:
(a) promote the target protein solubility expression or/and the coding nucleotide sequence of the molecular chaperones that disulfide linkage correctly folds;
(b) can be at the coding nucleotide sequence of the intein of C-terminal peptide bond rupture.
2. according to the described a kind of prokaryotic expression carrier of claim 1, it is characterized in that host cell is intestinal bacteria.
3. according to the described a kind of prokaryotic expression carrier of claim 1, it is characterized in that described molecular chaperones can promote the target protein solubility expression or/and disulfide linkage is correctly folding, makes the target protein of expression maintain natural bioactive; Describedly include the peptide bond cracking that Toplink is effectively induced C-terminal under certain condition, target protein is separated with fusion rotein.
4. according to the described a kind of prokaryotic expression carrier of claim 3, it is characterized in that described molecular chaperones comprises Trx, disulfide bond reduction enzyme and isomerase, little ubiquitin modified protein, NusA and glutathione-S-transferase; Described intein comprises mini Ssp DnaB, Sce VMA etc., is good to select mini Ssp DnaB for use.
5. according to the application of the described a kind of prokaryotic expression carrier of claim 1, it is characterized in that, the target protein of expressing is the albumen or the polypeptide of biologically active, the biological activity protein or the polypeptide that particularly contain disulfide linkage comprise hookworm coagulate peptide resistant and conservative property thereof variation polypeptide or their active fragments or their reactive derivative.
6. according to the described a kind of prokaryotic expression carrier of claim 2, it is characterized in that a kind of host cell includes the described carrier of claim 5.
7. according to the application of the described a kind of prokaryotic expression carrier of claim 5, it is characterized in that it is under 0 ℃～40 ℃ that target protein effectively separates with fusion rotein, preferable under 0 ℃～20 ℃, pH carries out in 6.0～8.0 the damping fluid.
8. according to the application of the described a kind of prokaryotic expression carrier of claim 7, it is characterized in that, the separation of the target protein of separating from fusion rotein, purification process comprise with the affinitive layer purification fusion affinity tag, as target protein with His-tag, or with the antibody affinity purification target protein of preparation, or carry out chromatography purification according to the physico-chemical property of target protein.
9. according to the application of the described a kind of prokaryotic expression carrier of claim 5, it is characterized in that,
(a) in suitable medium, cultivate the described host cell of claim 6;
(b), carry out effectively separating of target protein and fusion rotein according under the described condition of claim 7;
(c) carry out the isolation and purification of target protein according to the described method of claim 8.
10. according to the application of claim 5 prokaryotic expression carrier, it is characterized in that target protein comprises polypeptide or its conservative property variation polypeptide or their active fragments or their reactive derivative of being made up of SEQ ID NO.1 aminoacid sequence.
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| EP2698378A1 (en) * | 2011-04-15 | 2014-02-19 | Guangdong Medical College | Anticoagulant polypeptide and applications thereof |
| CN104099362A (en) * | 2014-07-11 | 2014-10-15 | 中国人民解放军国防科学技术大学 | Expression vector and preparation method of HNTX (Hainantoxin)-IV analogue rHNIV-01 |
| CN105255932A (en) * | 2015-10-22 | 2016-01-20 | 江苏大学 | Conotoxin variant GMVIIA as well as preparation method and application thereof |
| CN106399352A (en) * | 2016-11-09 | 2017-02-15 | 华东理工大学 | Folding factors for adjusting expression of target protein and application of folding factors |
| CN107267539A (en) * | 2016-04-06 | 2017-10-20 | 沈阳药科大学 | A kind of efficient EHEC solubility expression carrier for obtaining recombinant protein |
| CN110655584A (en) * | 2018-06-29 | 2020-01-07 | 苏士哲 | Activity control of autocleavage proteins and uses thereof |
| CN115093470A (en) * | 2022-06-30 | 2022-09-23 | 广州市乾相生物科技有限公司 | Intein Mtu RecA mutant and application thereof in production of glutathione GSH |
| CN117327681A (en) * | 2022-12-31 | 2024-01-02 | 义翘神州(泰州)科技有限公司 | Chaperone plasmid for promoting correct folding of mammalian cell expression protein and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| EP2698378A1 (en) * | 2011-04-15 | 2014-02-19 | Guangdong Medical College | Anticoagulant polypeptide and applications thereof |
| EP2698378A4 (en) * | 2011-04-15 | 2014-02-19 | Guangdong Medical College | Anticoagulant polypeptide and applications thereof |
| CN104099362A (en) * | 2014-07-11 | 2014-10-15 | 中国人民解放军国防科学技术大学 | Expression vector and preparation method of HNTX (Hainantoxin)-IV analogue rHNIV-01 |
| CN105255932A (en) * | 2015-10-22 | 2016-01-20 | 江苏大学 | Conotoxin variant GMVIIA as well as preparation method and application thereof |
| CN105255932B (en) * | 2015-10-22 | 2018-12-14 | 江苏大学 | A kind of conotoxin variant GMVIIA and its preparation method and application |
| CN107267539A (en) * | 2016-04-06 | 2017-10-20 | 沈阳药科大学 | A kind of efficient EHEC solubility expression carrier for obtaining recombinant protein |
| CN106399352A (en) * | 2016-11-09 | 2017-02-15 | 华东理工大学 | Folding factors for adjusting expression of target protein and application of folding factors |
| CN110655584A (en) * | 2018-06-29 | 2020-01-07 | 苏士哲 | Activity control of autocleavage proteins and uses thereof |
| CN115093470A (en) * | 2022-06-30 | 2022-09-23 | 广州市乾相生物科技有限公司 | Intein Mtu RecA mutant and application thereof in production of glutathione GSH |
| CN117327681A (en) * | 2022-12-31 | 2024-01-02 | 义翘神州(泰州)科技有限公司 | Chaperone plasmid for promoting correct folding of mammalian cell expression protein and application thereof |
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