CN1807456A - Recombinant human parathormone PTH1-34 preparation method - Google Patents

Recombinant human parathormone PTH1-34 preparation method Download PDF

Info

Publication number
CN1807456A
CN1807456A CN 200510023428 CN200510023428A CN1807456A CN 1807456 A CN1807456 A CN 1807456A CN 200510023428 CN200510023428 CN 200510023428 CN 200510023428 A CN200510023428 A CN 200510023428A CN 1807456 A CN1807456 A CN 1807456A
Authority
CN
China
Prior art keywords
pth1
fusion rotein
trx
sequence
parathyroid hormone
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN 200510023428
Other languages
Chinese (zh)
Other versions
CN1807456B (en
Inventor
丁邦
黎军
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Saijin Biomedical Co., Ltd.
Original Assignee
SHANGHAI SAIJIN BIOMEDICAL CO Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by SHANGHAI SAIJIN BIOMEDICAL CO Ltd filed Critical SHANGHAI SAIJIN BIOMEDICAL CO Ltd
Priority to CN 200510023428 priority Critical patent/CN1807456B/en
Publication of CN1807456A publication Critical patent/CN1807456A/en
Application granted granted Critical
Publication of CN1807456B publication Critical patent/CN1807456B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

The invention provides the method of merged protein which contains recombined human parathormone, coding the merged protein's DNA sequence, the carrier which contains the DNA sequence, the host cell which contains the carrier, using genetic engineering to prepare the merged protein and then produce PTH1-34. Enzyme cutting the merged protein by enterokinase can produce the recombined human parathormone PTH (1-34) has high physiologically active. The expression product is high, purity technology is simple and the cost is depressed, it can be used to the production of recombined human parathormone in force.

Description

The preparation method of recombinant human parathyroid hormone PTH1-34
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, forms Pro PTH after rough surfaced endoplasmic reticulum removes 25 amino-acid residues of N end.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 conditioning agent 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, determined 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, 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 biologic activity of PTH all are positioned at these 84 amino acid whose proteinic N-terminal, and PTH1-34 has complete PTH biologic activity [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 may 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 of chemosynthesis is too high, is not suitable for mass production PTH1-34.Along with the development of genetically engineered recombinant technology, people can utilize this technology to produce recombined parathyroid hormone.
Yet present existing production technique is difficult to satisfactory on expression amount purity and physiologically active.Therefore, this area presses for exploitation expression amount height, purity height, technology scale operation simple and with low cost 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 intermediate that is used to produce Rat parathyroid hormone 1-34 PTH1-34, and it is the fusion rotein that contains the protein sequence of the fusion rotein sequence of carrier pET32b self coding and human parathyroid hormone PTH1-34.
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 PTH1-34 that step is easy.
In a 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 a second aspect of the present invention, provide a kind of isolated DNA molecule, its described fusion rotein of encoding.
In another preference, described dna molecular has the nucleotide sequence shown in the SEQ ID NO:1.
In a third aspect of the present 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 a 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.
In a fifth aspect of the present invention, 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 described 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.
In a sixth aspect of the present invention, the purposes of the above-mentioned fusion rotein of the present invention is provided, it is used as the intermediate of preparation parathyroid hormone PTH1-34.
Description of drawings
Fig. 1 is a preparation 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 as follows: 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 as follows: 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 as follows: A: molecular weight standard albumen; B: enzyme is cut the back sample; C: enzyme is cut precipitation; D: enzyme is cut preceding fusion rotein.
Fig. 9 has shown the electrophorogram to the pure product of PTH1-34.
Figure 10 has shown the HPLC figure to the pure product of PTH1-34.
Figure 11 has shown that serious osteoporosis is organized in the physiological saline treatment in rat excision ovary osteoporosis model.
Figure 12 has shown in rat excision ovary 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.Finished the present invention on this basis.
Definition
As used herein, term " fusion rotein of Trx and parathyroid hormone PTH1-34 ", " Trx-PTH1-34 fusion rotein " etc. are used interchangeably, and 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, described 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 described 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, as the aminoacid sequence of 1-109 position among the SEQID NO:2.
As used herein, " parathyroid hormone PTH1-34 element " or " PTH1-34 element " are used interchangeably in the term fusion rotein, the a part of aminoacid sequence of finger in described 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 " are used interchangeably, and 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 the aminoacid sequence of Trx element and PTH1-34 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 be with above-mentioned connection peptides in conjunction with forming new connection peptides.A kind of particularly preferred 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 particularly preferred fusion rotein is the fusion rotein that is made of Trx, connection peptides and PTH1-34, wherein connection peptides is made 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 and 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, cultivate the host cell after transforming, obtain new fusion rotein of the present invention by 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 express in eukaryotic cells such as eukaryotic cell such as CHO, COS series, the carrier that can express in yeast saccharomyces cerevisiae or pichia yeast 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 by 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, superly handle, the combination of super centrifugal, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Trx-PTH1-34 fusion rotein of the present invention can be used as the intermediate 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 by centrifugal collection thalline, add lysate carrying out ultrasonic bacteria breaking, dilution centrifugal, collect supernatant and cross leaching filtrate 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) go out PTH1-34 with conventional means separation and purification such as anion-exchange chromatography, cation-exchange chromatographies.
Major advantage of the present invention is as follows:
(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) the expression amount height, 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 and limit the scope of the 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: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The structure of Trx-PTH1-34 expression vector and host cell
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 1-34 cDNA.Primer sequence is as follows:
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
With the CaCl2 method expression vector pET32-Trx-PTH (1-34) is transformed conventional e. coli bl21 (DE3), screening positive clone, just obtain expressing the engineering bacteria of Trx-PTH (1-34) fusion rotein, i.e. intestinal bacteria recombinant bacterial strain B21 (DE3)/Trx-PTH1-34.
Embodiment 2
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 for 37 ℃, select the nutrient solution (contain Amp 100 μ g/mls) of well-grown single colony inoculation in LB+1% glucose, 37 ℃, 270rpm, overnight incubation.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 precipitation thalline adds sample-loading buffer, and 100 ℃ of broken bacterium of water-bath 5min are got SDS-PAGE electrophoresis on the soluble protein supernatant liquor (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 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).
Described 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.
Embodiment 3
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.
The following metal-chelating column chromatography affinity column chromatography purifying that carries out: 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 sample, 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 sample: 0.5N HCl regulates rhPTH (1-34) sample pH value to 6.4 of DEAE Sepharose FF column purification gained
Last sample: regulate sample 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 obtains behind anion-exchange chromatography and cation-exchange chromatography (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 retention time of sample, and purity is greater than 95% (Figure 10).
The peak title Retention time (branch) Area (V * second) Area % Highly (V) Height %
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 as follows: stimulate UMR 106 cells to produce the amount of cAMP by 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 osteoporosis model rat, PTH1-34 20 μ g/kg group is 0.322g/cm through the rat bone density for the treatment of 2, and physiological saline treatment group is 0.262g/cm 2(Figure 11 and 12).
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉Shanghai Saijin Biomedical Co., Ltd
＜120〉preparation method of recombinant human parathyroid hormone PTH1-34
<130>041425
<160>6
<170>PatentIn version 3.1
<210>1
<211>579
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉encoding sequence of parathyroid gland plain fusion protein
<400>1
atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60
gacggggcga tcctcgttga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgct 120
ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180
atcgatcaaa acccgggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240
ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300
aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360
catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420
ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caagtctgtg 480
agtgaaatac agcttatgca taacctggga aaacatctga actcgatgga gagagtagaa 540
tggctgcgta agaagctgca ggatgtgcac aatttttga 579
<210>2
<211>192
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉parathyroid gland plain fusion protein
<400>2
Met Arg Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp
1 5 10 15
Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp
20 25 30
Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp
35 40 45
Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn
50 55 60
Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu
65 70 75 80
Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser
85 90 95
Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly
100 105 110
Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro
115 120 125
Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln
130 135 140
His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ser Val
145 150 155 160
Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met
165 170 175
Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe
180 185 190
<210>3
<211>105
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>3
tctgtgagtg aaatacagct tatgcataac ctgggaaaac atctgaactc gatggagaga 60
gtagaatggc tgcgtaagaa gctgcaggat gtgcacaatt tttga 105
<210>4
<211>34
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>4
Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn
1 5 10 15
Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His
20 25 30
Asn Phe
<210>5
<211>49
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>5
atctgggtac cgacgacgac gacaagtctg tgagtgaaat acagcttat 49
<210>6
<211>25
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
＜223〉primer
<400>6
ttatcaaaaa ttgtgcacat cctgc 25

Claims (10)

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.
2. fusion rotein as claimed in claim 1 is characterized in that, 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.
3. fusion rotein as claimed in claim 1 is characterized in that described fusion rotein contains the aminoacid sequence shown in the SEQID NO:2.
4. an isolated DNA molecule is characterized in that, the described fusion rotein of its coding claim 1.
5. dna molecular as claimed in claim 4 is characterized in that, it has the nucleotide sequence shown in the SEQ ID NO:1.
6. a carrier is characterized in that, it contains the described dna molecular of claim 4.
7. a host cell is characterized in that, it contains the described carrier of claim 6.
8. 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 described fusion rotein, cultivate the described host cell of claim 7, thereby give expression to described fusion rotein; With separate described fusion rotein.
9. method as claimed in claim 8 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.
10. the purposes of the described fusion rotein of claim 1 is characterized in that, as the intermediate of preparation parathyroid hormone PTH1-34.
CN 200510023428 2005-01-19 2005-01-19 Recombinant human parathormone PTH1-34 preparation method Active CN1807456B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 200510023428 CN1807456B (en) 2005-01-19 2005-01-19 Recombinant human parathormone PTH1-34 preparation method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 200510023428 CN1807456B (en) 2005-01-19 2005-01-19 Recombinant human parathormone PTH1-34 preparation method

Publications (2)

Publication Number Publication Date
CN1807456A true CN1807456A (en) 2006-07-26
CN1807456B CN1807456B (en) 2012-09-05

Family

ID=36839567

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 200510023428 Active CN1807456B (en) 2005-01-19 2005-01-19 Recombinant human parathormone PTH1-34 preparation method

Country Status (1)

Country Link
CN (1) CN1807456B (en)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007112676A1 (en) * 2006-03-31 2007-10-11 Shenzhen Watsin Genetech Ltd. A human parathyroid hormone 1-34 fusion protein and expression vectors thereof
WO2009019715A1 (en) * 2007-08-09 2009-02-12 Usv Limited 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
CN110128542A (en) * 2018-02-08 2019-08-16 深圳市新产业生物医学工程股份有限公司 PTH fusion protein, preparation method, the detection reagent containing it, kit and application
CN110938151A (en) * 2019-12-30 2020-03-31 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
CN111004318A (en) * 2019-12-30 2020-04-14 北京博康健基因科技有限公司 Purification method of rhPTH (1-34) protein stock solution
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 Use of parathyroid hormone (1-34) in preparing medicine for treating 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

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1257916C (en) * 2001-01-19 2006-05-31 重庆富进生物医药有限公司 Parathyroid hormone analog and its preparing process by recombination
CN1212336C (en) * 2002-11-29 2005-07-27 西南生物工程产业化中试基地有限公司 Prepn of recombinant human parathyroid hormone PTH (1-34)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007112676A1 (en) * 2006-03-31 2007-10-11 Shenzhen Watsin Genetech Ltd. A human parathyroid hormone 1-34 fusion protein and expression vectors thereof
WO2009019715A1 (en) * 2007-08-09 2009-02-12 Usv Limited Novel orthogonal process for purification of recombinant human parathyroid hormone (rhpth) (1-34)
US8298789B2 (en) 2007-08-09 2012-10-30 Usv Limited 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
CN110128542A (en) * 2018-02-08 2019-08-16 深圳市新产业生物医学工程股份有限公司 PTH fusion protein, preparation method, the detection reagent containing it, kit and application
CN110938151A (en) * 2019-12-30 2020-03-31 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
CN111004318A (en) * 2019-12-30 2020-04-14 北京博康健基因科技有限公司 Purification method of rhPTH (1-34) protein stock solution
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 Use of parathyroid hormone (1-34) in preparing medicine for treating 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

Also Published As

Publication number Publication date
CN1807456B (en) 2012-09-05

Similar Documents

Publication Publication Date Title
CN1807456A (en) Recombinant human parathormone PTH1-34 preparation method
CN1178955C (en) Modified TNF alpha molecules, DNA encoding such modified TNF alpha molecules and vaccines comprising such modified TNF alpha molecules and DNA
CN101068833A (en) Method for producing carboxy-terminal amidified peptides
CN1483831A (en) Process for obtaining insulin and insulin derivatives having correctly bonded crystine bridges
CN1902221A (en) Neutralizing epitope-based growth enhancing vaccine
CN1582295A (en) Novel epidermal growth factor protein and gene, and methods of use therefor
CN1529757A (en) Fusion Protein for secretion of protein of interest in supernatant of bacterial culture
CN1318070A (en) Modified i(E.coli) enterotoxin II signal peptide and microorganism expressing fusion protein of said peptide and heterologous protein
CN1924006A (en) Anti-glioma peptide of scorpion, preparation method and application thereof
CN1402788A (en) Fibrinolytically active polypeptide
CN1854294A (en) Escherichia expression system of secretory expression recombinant human epidermal growth factor
CN1916172A (en) Method for preparing parathormone 1-34
CN1766107A (en) Recombinant human keratinized cell growth factor production method
CN1231259C (en) Generation of human insulin
CN1254375A (en) Motilin homologs
CN1597697A (en) Human parathyrin 1.34 peptide related peptide-Pro-Pro-[Arg11 hPTH (1.34)-Pro-Pro
CN1800398A (en) Prunus necrotic ring spot virus coat protein gene and its special primer for checking
CN1302337A (en) Method for controlling cleavage by ompT protease
CN1194087C (en) New plasmin and its coding sequence and use
CN1769456A (en) Recombinant a human peptide production method
CN1184232C (en) Human tumor necrosin relative death inducing ligand mutein and its prepn process and medicine composition
CN1225481C (en) Recombinant fusion resisting tomur attack and transfer
CN1020923C (en) Method for producing inhibin using dan recombinant technique using dan)
CN1137261C (en) Punctiform aerogenic monad prolyl endopeptidase and its preparation method and application
CN101058805A (en) Method of producing mutation procarboxypeptidase B and mutation carboxypeptidase B

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C41 Transfer of patent application or patent right or utility model
TA01 Transfer of patent application right

Effective date of registration: 20060728

Address after: Room 5, No. 666, 102 lane, long East Road, Shanghai

Applicant after: Ding Bang

Co-applicant after: Li Jun

Address before: Shanghai city Pudong New Area Chunxiao Road No. 300

Applicant before: Shanghai Saijin Biomedical Co., Ltd.

C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
ASS Succession or assignment of patent right

Owner name: SHANGHAI SAIJIN BIOMEDICAL CO., LTD.

Free format text: FORMER OWNER: DING BANG

Effective date: 20130516

Free format text: FORMER OWNER: LI JUN

Effective date: 20130516

C41 Transfer of patent application or patent right or utility model
TR01 Transfer of patent right

Effective date of registration: 20130516

Address after: 201203 Shanghai Zhangjiang High Tech Park of Pudong New Area Chunxiao Road No. 300

Patentee after: Shanghai Saijin Biomedical Co., Ltd.

Address before: 102, room 5, No. 666, Lane 201203, long East Road, Shanghai

Patentee before: Ding Bang

Patentee before: Li Jun

EE01 Entry into force of recordation of patent licensing contract

Application publication date: 20060726

Assignee: Shanghai Puchuang Longke Finance Leasing Co.,Ltd.

Assignor: SHANGHAI CELGEN BIOPHARMA Co.,Ltd.

Contract record no.: X2020980003139

Denomination of invention: Recombinant human parathormone PTH1-34 preparation method

Granted publication date: 20120905

License type: Exclusive License

Record date: 20200616

EE01 Entry into force of recordation of patent licensing contract
PE01 Entry into force of the registration of the contract for pledge of patent right

Denomination of invention: Recombinant human parathormone PTH1-34 preparation method

Effective date of registration: 20200616

Granted publication date: 20120905

Pledgee: Shanghai Puchuang Longke Finance Leasing Co.,Ltd.

Pledgor: SHANGHAI CELGEN BIOPHARMA Co.,Ltd.

Registration number: Y2020980003141

PE01 Entry into force of the registration of the contract for pledge of patent right
EC01 Cancellation of recordation of patent licensing contract

Assignee: Shanghai Puchuang Longke Finance Leasing Co.,Ltd.

Assignor: SHANGHAI CELGEN BIOPHARMA Co.,Ltd.

Contract record no.: X2020980003139

Date of cancellation: 20210809

EC01 Cancellation of recordation of patent licensing contract
PC01 Cancellation of the registration of the contract for pledge of patent right

Date of cancellation: 20210809

Granted publication date: 20120905

Pledgee: Shanghai Puchuang Longke Finance Leasing Co.,Ltd.

Pledgor: SHANGHAI CELGEN BIOPHARMA Co.,Ltd.

Registration number: Y2020980003141

PC01 Cancellation of the registration of the contract for pledge of patent right