CN1807456B - Recombinant human parathormone PTH1-34 preparation method - Google Patents
Recombinant human parathormone PTH1-34 preparation method Download PDFInfo
- Publication number
- CN1807456B CN1807456B CN 200510023428 CN200510023428A CN1807456B CN 1807456 B CN1807456 B CN 1807456B CN 200510023428 CN200510023428 CN 200510023428 CN 200510023428 A CN200510023428 A CN 200510023428A CN 1807456 B CN1807456 B CN 1807456B
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- Prior art keywords
- pth1
- fusion rotein
- trx
- parathyroid hormone
- sequence
- 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 - Lifetime
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Abstract
本发明提供了含重组人甲状旁腺激素的融合蛋白Trx-PTH(1-34)(简称为“Trx-PTH1-34融合蛋白”)，编码该融合蛋白的DNA序列，含该DNA序列的载体，含该载体的宿主细胞，用基因工程制备该融合蛋白进而生产PTH1-34的方法。用肠激酶对该融合蛋白酶切，可产生具有高度生理活性的重组人甲状旁腺激素PTH(1-34)。本发明的表达产量高、纯化工艺简单、成本降低，可用于重组人甲状旁腺激素PTH(1-34)的大规模、工业化生产。The present invention provides a fusion protein Trx-PTH(1-34) containing recombinant human parathyroid hormone (abbreviated as "Trx-PTH1-34 fusion protein"), a DNA sequence encoding the fusion protein, and a vector containing the DNA sequence A method for preparing the fusion protein and then producing PTH1-34 by using the host cell containing the vector. Recombinant human parathyroid hormone PTH(1-34) with high physiological activity can be produced by digesting the fusion protein with enterokinase. The invention has high expression yield, simple purification process and reduced cost, and can be used for large-scale and industrialized production of recombinant human parathyroid hormone PTH (1-34).
Description
Technical field
The present invention relates to a kind of method that contains fusion rotein (abbreviating " Trx-PTH1-34 fusion rotein " as) and then the production PTH1-34 of human parathyroid hormone PTH1-34 with the gene engineering method preparation.The invention also discloses the preparation method and the purposes of dna sequence dna, the recombinant expression vector that contains this sequence and intestinal bacteria recombinant strains and this fusion rotein of this fusion rotein of coding.
Background technology
Human parathyroid hormone (PTH) is the single chain polypeptide chain hormone by the synthetic justacrine of chief cell of human parathyroid; Initial synthetic is to contain 115 amino acid whose Pre Pro PTHs, after rough surfaced endoplasmic reticulum removes 25 amino-acid residues of N end, forms Pro PTH.Preparathyroid hormone removes one six peptide from the N end in Golgi complex, form 84 amino acid whose Rat parathyroid hormone 1-34s, molecular weight 9500.
PTH and parafollicular cells of thyroid gland excretory thyrocalcitonin, 1,25-dihydroxy VD3 are regulated and control the calcium phosphorus level in the blood plasma jointly.PTH is the main regulator of calcium, phosphorus metabolism in kidney and the bone.Its physiological action comprises: the regulation and control of bone metabolism, uriniferous tubules are to the heavily absorption of calcium, phosphorus and absorption [the Potts JT et al of intestines calcium; Parathyroid hormone:Chemistry; Biosynthesis, and mode of action.Adv ProteinChem.1982; 35:323-396].
The cDNA sequence of people PTH equals definite [Hendy GNET AL in 1981 by Hendy GN the earliest; Nucleitide sequence of cloned cDNAs encoding humanpreproparathyroid hormone; PNAS USA 1981,78:7365-7369].Nineteen eighty-three, confirmed the gene order [Tomas J et al, Nucleitide sequence of human preproparathyroidhormone gene, PNAS USA 1983,80:2127-2131] of PTH.
Traditional physiology thinks that PTH is the katabolism effect to bone; But just think that as far back as nineteen twenty-nine parathyroid peptide extract can produce anabolic effect to bone; Produce increase [the Bauer W et al of osseous tissue in animal body; Studies of phosphorus metabolism:study of bonetrabeculae as ready available reserve supply of calcium, 1929, J.Exp.Med 49:145-162].Up to the present research is thought; All express the functional receptor of PTH on scleroblast and the osteoclast; Therefore, PTH has the dual regulation effect to bone, and PTH has direct repression to osteoclast; And has an indirect regulation effect [David W et al Anabolic Actions ofparathyroid hormone on bone 1993, Endocrine Reviews 14:690-709] by scleroblast mediation.Discover that the administering mode of PTH can influence it to osteoplastic effect,, can suppress bone forming with the PTH continued stimulus; And with the intermittent stimulation of PTH; Then can stimulate bone forming [Canalis E et al; Insulin-like growthfactor I mediates selective anabolic effects of parathyroid hormone in bonecultures; J clin Invest 1989,83:60-65].
All known extracellular BAs of PTH all are positioned at these 84 amino acid whose proteinic N-terminal, and PTH1-34 has complete PTH BA [Ute CM et al, Structure-activityrelation of NH
2-terminal human Parathyroid Hormone fragments, 1998, TheJournal of Biological Chemistry, 273:4308-4316].Therefore; PTH1-34 can induce osteosis and effectively treat osteoporosis [Felicia C et al Parathyroid responsivity inpostmenopausal women with osteoporosis during treatment with parathyroidhormone; J.Clin.Endocrinol.Metab., Mar 1998; 83:788-790; Etah S.et al; Parathyroid hormone as a therapy for idiopathic osteoporosis in men:effects onbone mineral density and bone markers; J.Clin.Endocrinol.Metab., Sep 2000; 85:3069-3076.].
PTH1-34 is very unstable in vivo, and content is very low, and separation and Extraction hardly maybe from natural tissues.For hPTH1-34 is used widely clinically, must find a kind of method that can obtain large-tonnage product reliably.The cost and risk property of chemosynthesis is too high, is not suitable for mass production PTH1-34.Along with the development of genetically engineered recombinant technology, people's this technology capable of using is produced recombined parathyroid hormone.
Yet present existing production technique is difficult to satisfactory on expression amount purity and physiologically active.Therefore, this area presses for the scale operation high, that purity is high, technology is simple and with low cost of exploitation expression amount and has the production technique of the PTH1-34 of complete natural Rat parathyroid hormone 1-34 physiologically active.
Summary of the invention
One object of the present invention just provides a kind of midbody that is used to produce Rat parathyroid hormone 1-34 PTH1-34, and it is the fusion rotein of protein sequence that contains fusion rotein sequence and the human parathyroid hormone PTH1-34 of carrier pET32b self coding.
Another object of the present invention provide encoding said fusion protein DNA, contain the carrier of this dna sequence dna, contain the host cell of this carrier.
Another object of the present invention provides the method for a kind of with low cost and/or production PTH 1-34 that step is easy.
In first aspect of the present invention, a kind of fusion rotein is provided, it is characterized in that it comprises following element:
(a) Trx element, this element has the aminoacid sequence of Trx or its active fragments;
(b) parathyroid hormone PTH1-34 element, this element has the aminoacid sequence of human parathyroid hormone PTH1-34 or its active fragments; And
(c) the connection peptides sequence between Trx element and parathyroid hormone PTH1-34 element, described connection peptides contains 6 histidine-tagged sequences and enteropeptidase restriction enzyme site sequence.
In another preference, described fusion rotein constitutes by the element (a) and (b) with (c).
In another preference, described Trx element has the aminoacid sequence of 1-109 position among the SEQ ID NO:2;
Described parathyroid hormone PTH1-34 element has the aminoacid sequence of 1-34 position among the SEQ ID NO:4;
Perhaps, described joint peptide sequence has the aminoacid sequence of 110-158 position among the SEQ ID NO:2.
In another preference, described fusion rotein contains the aminoacid sequence shown in the SEQ ID NO:2.
In second aspect of the present invention, a kind of isolated DNA molecule is provided, its described fusion rotein of encoding.
In another preference, described dna molecular has the nucleotide sequence shown in the SEQ ID NO:1.
In the third aspect of the invention, a kind of carrier is provided, it is characterized in that it contains the dna molecular of above-mentioned code book invention fusion rotein.More preferably, described carrier is expression vector pET32-Trx-PTH1-34.
In fourth aspect of the present invention, a kind of host cell is provided, it contains the above-mentioned carrier of the present invention.Preferably, described host cell is intestinal bacteria.More preferably, described host cell is intestinal bacteria recombinant bacterial strain B21 (DE3)/Trx-PTH1-34.
Aspect the of the present invention the 5th, a kind of the present invention of generation is provided the method for above-mentioned fusion rotein, it comprises step:
Under the condition that is fit to the said fusion rotein of expression, cultivate above-mentioned host cell, thereby give expression to described fusion rotein; With separate described fusion rotein.
In another preference, described method also comprises step: isolated fusion protein is cut with the enteropeptidase enzyme, separate the parathyroid hormone PTH1-34 that downcuts then.
Aspect the of the present invention the 6th, the purposes of the above-mentioned fusion rotein of the present invention is provided, it is used as the midbody of preparation parathyroid hormone PTH1-34.
Description of drawings
Fig. 1 is a preparing method's of the present invention schematic flow sheet.
Fig. 2 has shown the cDNA sequence (SEQ ID NO:3) of PTH1-34.
Fig. 3 has shown the aminoacid sequence (SEQ ID NO:4) of PTH1-34.
Fig. 4 has shown Trx-rhPTH1-34 fusion gene sequence (SEQ ID NO:1) and the fusion rotein aminoacid sequence (SEQ ID NO:2) of deriving.Be the 1-109 position among the Trx element SEQ ID NO:2 among the figure; Parathyroid hormone PTH1-34 element is the 159-192 position; Connection peptides is the 110-158 position, comprises 6His (117-122 position), zymoplasm (thrombin) restriction enzyme site (126-131 position), STag (134-148 position), enteropeptidase restriction enzyme site (154-158 position).
Fig. 5 is the electrophorogram of expression vector.Wherein each swimming lane is following: 1.1kb DNA Ladder standard substance; 2.pET32-Trx-PTH (1-34) plasmid XhoI single endonuclease digestion; 3.pET32-Trx-PTH (1-34) plasmid KpnI single endonuclease digestion; 4.pET32-Trx-PTH (1-34) plasmid XhoI/KpnI double digestion; 5.pET32-Trx-PTH (1-34) plasmid; 6.100bp DNA Ladder standard substance.
Fig. 6 has shown the SDS-PAGE electrophorogram of Trx-rhPTH fusion rotein abduction delivering.Wherein each swimming lane is following: 1. standard molecular weight albumen; 2. before inducing; 3. after inducing.
Fig. 7 has shown the affinity column chromatography result of Trx-rhPTH1-34 fusion rotein.
Fig. 8 has shown the electrophorogram of Trx-rhPTH1-34 fusion rotein after 37 ℃, 24 hours enzymes are cut.Wherein each swimming lane is following: A: molecular weight standard albumen; B: enzyme is cut the back sample; C: enzyme is cut deposition; D: enzyme is cut preceding fusion rotein.
Fig. 9 has shown the electrophorogram to the pure article of PTH1-34.
Figure 10 has shown the HPLC figure to the pure article of PTH1-34.
Figure 11 has shown that serious osteoporosis is organized in the saline water treatment in rat excision ovary property osteoporosis model.
Figure 12 has shown in rat excision ovary property osteoporosis model, 20 μ gKg
-1D
-1PTH1-34 treatment group rat osteosis, bone structure recovers.
Embodiment
The inventor finds for the such small peptide of PTH1-34 that through extensive and deep research directly there is suitable difficulty in expression, because PTH1-34 is vulnerable to the hydrolysis of host cell endoproteinase.For the ease of high expression level PTH1-34 and purifying PTH1-34 easily, the inventor is in the same place Trx gene and PTH1-34 gene fusion, produces the fusion rotein of the Trx-PTH1-34 that is connected by amino acid connecting peptides (linker).In fusion rotein, Trx is extremely important to the solubility and the stability of this fusion rotein.In addition, 6 continuous Histidines in the connection peptides can greatly make things convenient for the purifying of fusion rotein; DDDDK pentapeptide in the connection peptides is a connection peptides, is again Enteropeptidase specificity cut point, therefore can conveniently downcut PTH1-34 from fusion rotein.Accomplished the present invention on this basis.
Definition
As used herein; Interchangeable uses such as term " fusion rotein of Trx and parathyroid hormone PTH1-34 ", " Trx-PTH1-34 fusion rotein " all refer to merge the albumen that forms by the aminoacid sequence of Trx element and the aminoacid sequence of parathyroid hormone PTH1-34 element.In addition, said fusion rotein can have or not have initial methionine(Met) or signal peptide.
As used herein; " Trx element " refers to a part of aminoacid sequence in said fusion rotein in the term fusion rotein; This sequence has substantially the same aminoacid sequence with total length Trx or its active fragments natural or variation, and has and the natural sulphur oxygen substantially the same biological activity of albumen also.Preferred Trx element is Trx or its active fragments of total length, like the aminoacid sequence of 1-109 position among the SEQID NO:2.
As used herein; " parathyroid hormone PTH1-34 element " or " PTH1-34 element " interchangeable use in the term fusion rotein; The a part of aminoacid sequence of finger in said fusion rotein; This sequence has substantially the same aminoacid sequence with parathyroid hormone PTH1-34 or its active fragments natural or variation, and has the biological activity substantially the same with natural parathyroid PTH1-34.Preferred PTH1-34 element is human parathyroid hormone PTH1-34, more preferably has the aminoacid sequence of 1-34 position among the SEQ ID NO:4, the protein sequence (Fig. 2 and Fig. 3) that 1 to 34 amino acids of promptly sophisticated human parathyroid hormone N-terminal is formed.
The sequence of Trx and parathyroid hormone PTH1-34 can be derived from the people, also can be derived from inhuman animal.Yet, people's native sequences preferably.
As used herein, term " connection peptides " or " amino acid connecting peptides " interchangeable use refer to small peptide between the aminoacid sequence of the aminoacid sequence of Trx element and PTH1-34 element, that play ligation.The length of connection peptides is generally 5-50 amino acid, preferably is 10-40 amino acid.The technician can be according to this area ordinary method (as referring to PNAS 1998; 95:5929-5934; ProteinEng, 2000; 13 (5): 309-312; Protein Eng, 2003; 15 (11): design connection peptides documents such as 871-879).Usually, connection peptides does not influence or the aminoacid sequence that has a strong impact on aminoacid sequence and the PTH1-34 element of Trx element forms correct folding and space conformation.
Preferred connection peptides example comprises (but being not limited to): becoming separate structural domain in order to help protein folding, is suitable (154-158 position among the SEQ ID NO:2) with sequences such as DDDDK as connection peptides.Similar, the restriction enzyme site of Chymotrypsin 1, papoid, Tryptase, Taka-proteinase, trypsinase etc. also can design as amino acid connecting peptides.In order to help purifying, can be 6His as connection peptides, so that with metal affinity chromatography purifying Trx-PTH1-34 fusion rotein.In addition, also can above-mentioned connection peptides be combined to form new connection peptides.A kind of preferred especially connection peptides has the aminoacid sequence of 110-158 position among the SEQ IDNO:2; This connection peptides has merged protease cutting site (enteropeptidase) and metal affinity chromatography site 6His; In addition; Also merged zymoplasm (thrombin) restriction enzyme site and STag, helped identifying and purifying with additive method.
A kind of special preferred fusion protein is the fusion rotein that is made up of Trx, connection peptides and PTH1-34; Wherein connection peptides is made up of following element: histidine-tagged (HisTag), zymoplasm (thrombin) restriction enzyme site, STag and enteropeptidase (enterokinase, EK) restriction enzyme site.More preferably, this fusion rotein has the aminoacid sequence shown in the SEQ ID NO:2.
The dna sequence dna of code book invention fusion rotein, all synthetic.Also available pcr amplification or synthetic method obtain Trx with or the DNA sequences encoding of parathyroid hormone PTH1-34, then it is stitched together, form the dna sequence dna of code book invention fusion rotein.
Having obtained it to be connected into suitable expression vector after code book invents the dna sequence dna of new fusion rotein, change proper host cell again over to.At last, the host cell after the culture transformation obtains new fusion rotein of the present invention through separation and purification.
As used herein, term " carrier " comprises plasmid, clay, expression vector, cloning vector, virus vector etc.Representational state comprises (but being not limited to): the carrier that can in eukaryotic cells such as eukaryotic cell such as CHO, COS series, express; The carrier that can in yeast saccharomyces cerevisiae or pichia yeast, express can be at the carrier and the prokaryotic expression carrier of expressed in insect cells such as silkworm.
In the present invention, can select various carrier known in the art such as commercially available carrier for use.Such as, select commercially available carrier for use, the nucleotide sequence of then code book being invented new fusion rotein operationally is connected in expression regulation sequence, can form protein expression vector.
As used herein, " operationally being connected in " refers to a kind of like this situation, and promptly some part of linear DNA sequence can influence the activity of same other parts of linear DNA sequence.For example, if signal peptide DNA as precursor expression and participate in the secretion of polypeptide, signal peptide (secretion leader sequence) DNA operationally is connected in polypeptid DNA so; If transcribing of promotor control sequence, it is operationally to be connected in encoding sequence so; When if ribosome bind site is placed in the position that can make its translation, it is operationally to be connected in encoding sequence so.Generally, " operationally being connected in " means adjoining, then means in reading frame adjacent for the secretion leader sequence.
In the present invention, term " host cell " comprises prokaryotic cell prokaryocyte and eukaryotic cell.The example of prokaryotic host cell commonly used comprises intestinal bacteria, Bacillus subtilus etc.Eukaryotic host cell commonly used comprises yeast cell, insect cell and mammalian cell.Preferably, this host cell is an eukaryotic cell, more preferably is bombyx mori cell.
After obtaining transformed host cells, can under the condition that is fit to expression fusion rotein of the present invention, cultivate this cell, thereby give expression to fusion rotein.Can utilize its physics, the separating through various separation methods with other characteristic and the albumen of purification of Recombinant of chemistry.These methods are well-known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation handles, with protein precipitant handle (salt analysis method), centrifugal, the broken bacterium of infiltration, ultraly handle, ultra centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) is technological with other various LCs and the combination of these methods.
Trx-PTH1-34 fusion rotein of the present invention can be used as the midbody of producing PTH1-34.Therefore, the present invention also provides the novel method of a kind of PTH1-34 of preparation.This method comprises:
(1) cultivates intestinal bacteria recombinant bacterial strain B21 (DE3)/Trx-PTH1-34, add inductor abduction delivering Trx-PTH1-34 fusion rotein.
(2) from culture, extract the Trx-PTH1-34 fusion rotein.For example can through centrifugal collection thalline, add lysate carrying out ultrasonic bacteria breaking, dilution centrifugal, collect supernatant and cross leaching filtrating and obtain.
(3) affinity column chromatography purified fusion protein for example uses chelating Sepharose FF post and changes liquid with Sephadex G25 (fine).
(4) use enzyme (for example when using DDDDK, cutting) enzyme to cut the Trx-PTH1-34 fusion rotein, thereby downcut PTH1-34 with enteropeptidase (EK) enzyme corresponding to cleavage site.
(5) dissolve PTH1-34 with conventional means separation of pure such as anion-exchange chromatography, cation-exchange chromatographies.
Major advantage of the present invention is following:
(1) in fusion rotein, Trx is extremely important to the solubility and the stability of this fusion rotein, thereby helps proteic expression.
(2) be positioned at 6 continuous Histidines at fusion rotein middle part, can greatly make things convenient for the purifying of fusion rotein.
(3) the DDDDK pentapeptide is a connection peptides, is again Enteropeptidase specificity cut point, therefore can conveniently downcut PTH1-34 from fusion rotein.
(4) expression amount is high, purifying process is easy, productive rate is high, with low cost, be fit to scale operation.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to normal condition; People such as Sambrook for example; Molecular cloning: the condition described in the laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or the condition of advising according to manufacturer.
The structure of Trx-PTH1-34 expression vector and host cell
The preparation process is referring to Fig. 1.
(a) preparation of PTH1-34
Two primers have been synthesized in design, and they are corresponding PTH 1-8 amino-acid residue and 29-34 amino-acid residue respectively, are used for PCR human cloning PTH 1-34cDNA.Primer sequence is following:
5 ' primer:
5′ATCTG
GGTACCGACGACGACGACAAGTCTGTGAGTGAAATACAGCTTAT 3′(SEQ ID NO：5)
3 ' primer:
5′TTATCAAAAATTGTGCACATCCTGC 3′(SEQ ID NO：6)
5 ' primer comprises the N end group of enteropeptidase point of contact DDDDK and PTH (1-34) because of sequence, and introduces the KpnI point of contact; 3 ' primer contains the C end group of PTH (1-34) because of sequence and terminator codon.Use above-mentioned primer, the human parathyroid hormone cDNA for preparing with routine is that template is carried out PCR.With the PCR2.1-TOPO clone test kit of Invitrogen company the PCR product directly is cloned in the PCR2.1 carrier (Invitrogen company) then, obtains PCR2.1-rhPTH (1-34) recombinant vectors, transform DH5 α and carry out dna sequencing.Sequencing result show PTH (1-34) gene order that obtains of cloning consistent with expected sequence.
(b) preparation of expression vector pET32-Trx-PTH1-34
PCR2.1-rhPTH (1-34) carrier is cut rear clone to the middle bacillus coli DH 5 alpha that transforms routine of pET32b (+) carrier of cutting through same enzyme (available from Novagen company) through KpnI and XhoI enzyme; Obtain recombinant expression vector pET32-Trx-PTH (1-34), check order.Enzyme is cut the evaluation collection of illustrative plates and is seen Fig. 5.
(c) structure of intestinal bacteria recombinant bacterial strain B21 (DE3)/Trx-PTH1-34
Use CaCl
2Method transforms conventional e. coli bl21 (DE3) with expression vector pET32-Trx-PTH (1-34), and screening positive clone just obtains expressing the engineering bacteria of Trx-PTH (1-34) fusion rotein, i.e. intestinal bacteria recombinant bacterial strain B21 (DE3)/Trx-PTH1-34.
The expression of Trx-PTH1-34
Picking transforms positive intestinal bacteria B21 (the DE3)/Trx-PTH1-34 on the flat board; Line is on the agar plate of the LB+1% glucose that contains 100 μ g/ml Amp; Cultivated 16 hours, and selected the nutrient solution (contain Amp 100 μ g/mls) of well-grown single colony inoculation, 37 ℃ for 37 ℃ in LB+1% glucose; 270rpm, overnight cultures.The bacterium that will spend the night is inoculated in the nutrient solution of LB+1% glucose (containing Amp 100 μ g/ml), 37 ℃ of shaking culture at 1: 50.Work as OD
600Reach at 0.6～0.8 o'clock, add IPTG, induced 3 hours to final concentration 1mM.Bacterium liquid is through the centrifugal supernatant that goes of 4000rpm 10min, and the deposition thalline adds sample-loading buffer, and 100 ℃ of broken bacterium of water-bath 5min are got SDS-PAGE electrophoresis on the soluble protein supernatant (spacer gel 5%, separation gel 12%), dyeing, decolouring, scanning analysis after centrifugal.
Because of Trx-PTH (1-34) fusion rotein contains 192 amino acid, theoretical molecular should be 22KD, and structure is as shown in Figure 4.The SDS-PAGE electrophoresis result shows: obvious abduction delivering band is arranged between 14KD and 31KD, conform to the expection of molecular weight size, fusion protein expression accounts for bacterial protein about 25%, is expressed as soluble proteins (Fig. 6).
Said cDNA in Bacillus coli cells with the Trx amalgamation and expression.Expression product is that PTH1-34 is connected by the DDDDK pentapeptide with Trx (Thioredoxin), and this sequence is an Enteropeptidase specificity cut point.Trx is extremely important to the solubility and the stability of this fusion rotein, and 6 continuous Histidines at fusion rotein middle part are extremely helpful to the purifying of fusion rotein.
The separation and purification of fusion rotein and PTH1-34
(a) separation and purification of fusion rotein
After the fermentation of engineering bacteria under condition of ice bath the ultrasonication thallus suspension liquid, behind the broken bacterium, 4 ℃, the centrifugal 20min of 9000rpm, the Trx-EK-PTH fusion rotein is mainly in supernatant.
Carry out metal-chelating column chromatography affinity column chromatography purifying as follows: 60mM imidazoles, 500mM NaCl, pH8.0,3 column volumes of 20mM PB sample-loading buffer balance.Behind ultrasonic supernatant 0.45 μ m membrane filtration upper prop, use the 60mM imidazoles, 500mM NaCl, pH8.0,20mM PB damping fluid balance is steady to baseline, removes the part foreign protein.With the 160mM imidazoles, 500mM NaCl, pH8.0,20mM PB buffer solution elution fusion rotein is collected the fusion rotein peak, and through the SDS-PAGE electrophoresis detection, molecular weight is about 22KD, and is consistent with theoretical value, purity about 80% (Fig. 7).
(b) enzyme is cut and is formed PTH1-34
Enteropeptidase (EK) enzyme is cut fusion rotein: 37 ℃ of constant temperature enzymes were cut 24 hours, visible clear rhPTH of electrophoresis and Trx degraded band (Fig. 8).
(c) anion-exchange chromatography and cation-exchange chromatography
Anion-exchange chromatography
DEAE Sepharose FF anion-exchange chromatography purifying rhPTH (1-34)
Trx-HisTag-EK-PTH (1-34) fusion rotein is in the 20mM PB buffer system of pH8.0 behind the enteropeptidase enzymolysis.After adopting identical buffer system balance DEAE Sepharose FF post, sample is directly gone up appearance, collects the liquid that penetrates at PTH (1-34) place, the 20mM PB buffer solution elution of the pH8.0 of the 1M NaCl peak of mixing.
Cation-exchange chromatography
CM Sepharose FF column chromatography
Balance: use level pad pH6.4,20mM PB balance columns.
Sample pH value is regulated before the last appearance: 0.5N HCl regulates rhPTH (1-34) sample pH value to 6.4 of DEAE Sepharose FF column purification gained
Last appearance: regulate appearance on rhPTH (1-34) sample of pH.
The drip washing reequilibrate: use level pad pH6.5,20mM PB drip washing equilibrates to baseline.
Stepwise elution: A liquid: 20mM NaCl, pH6.4,20mM PB
B liquid: 120mM NaCl, pH6.4,20mM PB
C liquid: 1M NaCl, pH6.4,20mM PB
Wherein B liquid elution peak is rhPTH (1-34) target protein peak.
Through the purifying of CM Sepharose FF post, the rhPTH that obtains (1-34) purity can reach more than 95%, and yield is greater than 70%, and specific activity is greater than 8.0 * 10
3IU/mg, pyrogen is less than 2.5EU/mg
The rhPTH that behind anion-exchange chromatography and cation-exchange chromatography, obtains (1-34) purity can reach (Fig. 9) more than 95%.
(d) HPLC purity.
Key instrument: Waters2487 high performance liquid chromatograph, Waters C18 performance liquid chromatographic column.
Method: moving phase, trifluoroacetic acid aqueous solution (containing 0.08%TFA, gradient 0-75%).Detect wavelength 280nm, flow velocity 0.8ml/min.Albumen applied sample amount 10-20 μ g.
The result: standard substance are consistent with the main peak RT of sample, and purity is greater than 95% (Figure 10).
| The peak title | RT (branch) | Area (V * second) | Area % | Highly (V) | |
|
| 1 | Peak1 | 15.135 | 11813 | 0.29 | 1667 | 0.40 |
| 2 | Peak2 | 15.313 | 14891 | 0.36 | 2016 | 0.49 |
| 3 | Peak3 | 16.514 | 46693 | 1.13 | 2651 | 0.64 |
| 4 | Peak4 | 16.772 | 3992187 | 96.86 | 400614 | 97.18 |
| 5 | Peak5 | 17.280 | 24035 | 0.58 | 2563 | 0.62 |
| 6 | Peak6 | 26.611 | 31830 | 0.77 | 2724 | 0.66 |
Embodiment 4
The active testing of PTH1-34
In the present embodiment, measure prepared PTH1-34 among the embodiment 3 with ordinary method.In brief, method is following: stimulate UMR 106 cells to produce the amount of cAMP through detecting PTH1-34.Adopt chemical luminescence reagent kit test sample and standard substance to stimulate UMR 106 cells to produce the amount of cAMP, the data that obtain are carried out " S " type curvilinear regression, the EC50 value that obtains sample and standard substance respectively compares, and draws the specific activity of sample.
The result: the specific activity of sample is greater than 8.0 * 10
3IU/mg.This shows that the PTH1-34 that produces with the inventive method has the physiologically active of natural Rat parathyroid hormone 1-34 completely.
In addition, also carried out conventional animal experiment.Extract in the ovary property osteoporosis model rat, PTH1-3420 μ g/kg group is 0.322g/cm through the rat bone density of treating
2, and saline water treatment group is 0.262g/cm
2(Figure 11 and 12).
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
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Claims (8)
1. a fusion rotein is characterized in that, it comprises following element:
(a) Trx element, this element has the aminoacid sequence of Trx or its active fragments;
(b) parathyroid hormone PTH1-34 element, this element has the aminoacid sequence of human parathyroid hormone PTH1-34 or its active fragments; And
(c) the connection peptides sequence between Trx element and parathyroid hormone PTH1-34 element, described connection peptides contains 6 histidine-tagged sequences and enteropeptidase restriction enzyme site sequence;
The aminoacid sequence of described fusion rotein is shown in SEQ ID NO:2.
2. an isolated DNA molecule is characterized in that, the described fusion rotein of its coding claim 1.
3. dna molecular as claimed in claim 2 is characterized in that its nucleotide sequence is shown in SEQ IDNO:1.
4. a carrier is characterized in that, it contains the described dna molecular of claim 2.
5. a host cell is characterized in that, it contains the described carrier of claim 4.
6. method that produces the described fusion rotein of claim 1 is characterized in that it comprises step:
Being fit to express under the condition of said fusion rotein, cultivate the described host cell of claim 5, thereby give expression to described fusion rotein; With separate described fusion rotein.
7. method as claimed in claim 6 is characterized in that, also comprises step: isolated fusion protein is cut with the enteropeptidase enzyme, separate the parathyroid hormone PTH1-34 that downcuts then.
8. the purposes of the described fusion rotein of claim 1 is characterized in that, as the midbody of preparation parathyroid hormone PTH1-34.
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| CN100484958C (en) * | 2006-03-31 | 2009-05-06 | 东莞太力生物工程有限公司 | Fusion protein containing human parathyroid hormone 1-34 and its expression vector |
| MX2010001612A (en) * | 2007-08-09 | 2010-07-01 | Usv Ltd | Novel orthogonal process for purification of recombinant human parathyroid hormone (rhpth) (1-34). |
| CN102718848A (en) * | 2012-06-01 | 2012-10-10 | 上海交通大学 | Recombinant protein of mycobacterium tuberculosis Rv 3120, preparation method and application in cellular immunological diagnosis thereof |
| CN105131094A (en) * | 2015-09-17 | 2015-12-09 | 上海交通大学 | Mycobacterium tuberculosis Rv 3248 c recombinant protein, preparation method and application of mycobacterium tuberculosis Rv 3248 c recombinant protein |
| CN105131095A (en) * | 2015-09-17 | 2015-12-09 | 上海交通大学 | Mycobacterium tuberculosis Rv 2991 recombinant protein, preparation method and application of mycobacterium tuberculosis Rv 2991 recombinant protein |
| CN105294844A (en) * | 2015-09-28 | 2016-02-03 | 上海交通大学 | Mycobacterium tuberculosis Rv3457c recombinant protein as well as preparation method and application thereof |
| CN110128542B (en) * | 2018-02-08 | 2020-12-29 | 深圳市新产业生物医学工程股份有限公司 | PTH fusion protein, preparation method thereof, detection reagent containing PTH fusion protein, kit and application |
| CN111004318B (en) * | 2019-12-30 | 2022-03-04 | 北京博康健基因科技有限公司 | Purification method of rhPTH (1-34) protein stock solution |
| CN110938151B (en) * | 2019-12-30 | 2023-03-17 | 重庆艾力彼生物科技有限公司 | Fusion protein for expressing parathyroid hormone PTH, recombinant plasmid and recombinant engineering bacteria |
| CN111529691A (en) * | 2020-06-03 | 2020-08-14 | 温州医科大学附属第二医院、温州医科大学附属育英儿童医院 | Application of parathyroid hormone (1-34) in the preparation of medicaments for the treatment of male hypogonadism |
| CN112646826A (en) * | 2020-12-23 | 2021-04-13 | 无锡和邦生物科技有限公司 | Gene sequence for coding Trx-hPTH (1-34) fusion protein, recombinant expression plasmid, engineering bacterium and application |
| CN118126197B (en) * | 2024-03-19 | 2025-02-25 | 康众(广东)生物医药有限公司 | A recombinant PTH fusion protein and its construction method and application |
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