CN1916172A - Method for preparing parathormone 1-34 - Google Patents

Method for preparing parathormone 1-34 Download PDF

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CN1916172A
CN1916172A CNA2006100742168A CN200610074216A CN1916172A CN 1916172 A CN1916172 A CN 1916172A CN A2006100742168 A CNA2006100742168 A CN A2006100742168A CN 200610074216 A CN200610074216 A CN 200610074216A CN 1916172 A CN1916172 A CN 1916172A
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fusion rotein
enteropeptidase
enzyme
rhpth
preparation
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杨立明
张仁怀
刘社际
阳勇
何凯
黄碧莲
刘德林
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Dongguan Taili Biotech Co., Ltd.
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HUASHENGYUAN GENE ENGINEERING DEVELOPMENT Co Ltd SHENZHEN CITY
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0036—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on NADH or NADPH (1.6)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
    • C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

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Abstract

This invention provides a method for preparing recombinant human parathyroid hormone 1-34 (rhPTH (1-34)). The method comprised: (1) expressing with an expression vector capable of expressing a fusion protein with an amino acid sequence (from N-terminal to C-terminal) containing thioredoxin, (His) 6, enterokinase recognition site and parathyroid hormone 1-34 peptide; (2) purifying the fusion protein by nickel ion complexation affinity chromatography; (3) cutting the purified fusion protein with enterokinase to release the rhPTH (1-34) from the fusion protein. By this method, rhPTH (1-34) can be purified rapidly, simply and efficiently, and the recovery yield is largely increased.

Description

The method for preparing human parathyroid hormone 1-34
Technical field
The present invention relates to proteinic technology of preparing, specifically, relate to the method for utilizing genetic engineering technique to prepare human parathyroid hormone 1-34 (hPTH (1-34)).
Background technology
(Parathyroid Hormone is by Rat parathyroid hormone 1-34 chief cell synthetic PTH) to Rat parathyroid hormone 1-34, is made up of 84 amino acid.People recognize that gradually PTH can be increased in osteoblastic quantity very active in the synthetic new ground substance of bone, and can change the genetic expression in the bone in vivo.PTH also has the active effect that brings high blood pressure down, regulates Vitamin D Receptor (VDR) expression, regulates alkaline phosphatase.Tregear etc. (Tregear GW etc., Endocrinology, 1973,93:1349) with experimental results show that PTH performance calcium phosphorus adjusting molecularity only needs an aminoterminal 1-34 amino-acid residue.But PTH (1-34) does not contain halfcystine, and is unstable in vivo.
The genetically engineered research of PTH starts from the eighties in 20th century.The gene engineering method of preparation PTH (1-34) mainly contains two classes at present, and a class is the form preparation with inclusion body, another kind of form preparation with soluble protein.1997, Japanese scientist Yuji Suzuki etc. were with human parathyroid hormone (hPTH (1-34)) and beta-galactosidase enzymes amalgamation and expression, but fusion rotein exists with the inclusion body form.Chinese patent publication number CN 1417231A also discloses the method that a kind of form with inclusion body prepares hPTH (1-34).In the method for this class, the fusion rotein that gives expression to needs to cut through inclusion body extraction, dilution refolding, enzyme, carry out means such as ion-exchange, reversed-phase HPLC chromatography then and carry out purifying, just can obtain hPTH (1-34), thereby the preparation process of these class methods is comparatively complicated.
Chinese patent application publication number CN1424325A discloses the preparation technology of a kind of recombinant human parathyroid hormone precursor peptide and recombinant human parathyroid hormone 1-34 peptide.In order to prepare this parathyroid hormone 1-34 peptide, need make engineering bacterium expression go out this fusion rotein of GST-Gly-Ser-Pro-PTH (1-34) earlier, successively the gained fusion rotein is carried out enzyme again and cut, after separation and purification, obtain required parathyroid hormone 1-34 peptide again with zymoplasm and proline(Pro) endopeptidase.Owing to need to use double digestion, the disclosed preparation technology of this patent application is also comparatively complicated.
People know Trx (Thiroedoxin already, hereinafter abbreviate " Trx " sometimes as) be a kind of yeast, bacterium, animal, intravital protein of plant of being prevalent in, this protein also is for example yeast or colibacillary intrinsic protein of present PTH (1-34) expressive host commonly used, utilizes this albumen and PTH (1-34) fusion to prepare hPTH (1-34) easily but also have no talent so far.
Summary of the invention
One aspect of the present invention provides a kind of new preparation hPTH (1-34) method.This method comprises the steps: that the expression vector that (1) enables expressed fusion protein expresses, and it is Trx-(His) 6-enteropeptidase recognition site-Rat parathyroid hormone 1-34 1-34 peptide that described fusion rotein is held the sequence of C end from N; (2) with nickel ion chelating affinity chromatography the fusion rotein that step (1) obtains is carried out purifying; And (3) cut the fusion rotein that step (2) purifying obtains with the enteropeptidase enzyme, so that Rat parathyroid hormone 1-34 1-34 peptide is discharged from described fusion rotein.Utilize method of the present invention, can be fast, purifying hPTH (1-34) simply, efficiently, improved the rate of recovery greatly.
In order to obtain the pure product of hPTH (1-34) polypeptide, after utilizing enteropeptidase that hPTH (1-34) is discharged from described fusion rotein as mentioned above, method of the present invention comprises the steps: further preferably that also (4) cut the mixture that obtains to step (3) enzyme and carry out chromatography with anion-exchange column, collects to penetrate protein solution; (5) make step (4) collect the protein solution that penetrates that obtains and cross the reversed phase chromatography post, collect elution peak; And (6) cross cationic exchange coloum with the protein solution that step (5) obtains.
In a specific embodiment of the present invention, enteropeptidase recognition site in the described fusion rotein-Rat parathyroid hormone 1-34 1-34 peptide is encoded by following nucleotide sequences:
GACGACGACGACAAGTCCGTTTCCGAAATCCAGCTGATGCACAA
CCTGGGTAAACACCTGAACTCCATGGAACGTGTTGAATGGCTGC
GTAAAAAACTGCAGGACGTTCACAACTTC。
In described step (1), can use the coli expression carrier system, also can use yeast expression system.If use coli expression system, the expression vector of structure energy expressed fusion protein earlier.In a specific embodiment, the present invention has utilized can be from the commercially available expression vector pET32a (+) that buys, the nucleotide sequence of people PTH (1-34) of will encoding is inserted into the downstream of the contained Trx of this commercially available carrier (Trx) gene, and the gained expression vector is named as pET-PTH (1-34).Preferred described step (1) is finished under the following conditions: the e. coli bl21 (DE3) that will contain expression vector pET-PTH (1-34) ferments in the substratum of at least a LB of being selected from, TB and M9CA; Leavening temperature is 30-40 ℃, and fermented liquid pH is 6.5-7.5 and fermentation dissolved oxygen DO 〉=30%.When being cultured to OD 600=4.0 o'clock, adding final concentration in substratum was the IPTG of 0.3-1.0mM, the beginning abduction delivering, and the fermentation inducement time is 3-5 hour.
In described step (2), in the ratio adding enteropeptidase of 1mg enteropeptidase cutting 5mg fusion rotein.Preferred this enzyme is cut step and is finished under following condition: 1mg/mL fusion rotein, 50mM Tris-HCl (pH8.0), 1mM CaCl 2, about 25 ℃, enzyme was cut about 20 hours.
Description of drawings
Fig. 1 cuts the evaluation collection of illustrative plates for recombinant expression plasmid pET-PTH (1-34) enzyme.Among Fig. 1, the content of each swimming lane representative is: M, dna molecular amount standard (λ DNA/Hand III, the molecular weight size is marked in a figure left side); 1, recombinant plasmid pET-PTH (1-34); 2, recombinant plasmid pET-PTH (1-34) cuts through EcoR I enzyme; 3, pET32a (+) plasmid DNA is cut through Pst I enzyme; 4, recombinant plasmid pET-PTH (1-34) cuts through Pst I enzyme.
Fig. 2 is recombinant plasmid pET-PTH (1-34) forward sequencing result.
Fig. 3 is recombinant plasmid pET-PTH (1-34) backward sequencing result.
Fig. 4 is the SDS-PAGE electrophorogram of the expression screening of recon.Among Fig. 4, M, for protein molecular weight standard (the molecular weight size is marked in a figure left side); What swimming lane 1 showed is to induce preceding sampling result; That swimming lane 2-9 shows respectively is 1-8 recon abduction delivering result.
SDS-PAGE electrophoretic analysis after Fig. 5 engineering bacterium fermentation is expressed.Among Fig. 5, M, be protein molecular weight standard, each band molecular weight size (kDa) is marked in a figure left side; What swimming lane 1 showed is the fermentation thalline of collecting; What swimming lane 2 showed is that IPTG induces preceding thalline.
Fig. 6 rhPTH (1-34) respectively goes on foot the SDS-PAGE electrophoretic analysis of purification of samples.Among Fig. 6,1, the fermentation thalline; 2, centrifugal supernatant behind the broken bacterium; 3, nickel ion chelating affinity chromatography sample; 4, the enteropeptidase enzyme of fusion rotein is cut sample; 5, Q Sepharose High Performance column chromatography sample; 6, reversed phase column chromatography sample; 7, SP Sepharose Fast Flow column chromatography sample; M, be protein molecular weight standard, each band molecular weight size (kDa) is marked in the figure right side.
Fig. 7 is that the RP-HPLC of rhPTH (1-34) analyzes collection of illustrative plates.
Fig. 8 is the mass spectroscopy collection of illustrative plates of rhPTH (1-34).
Fig. 9 shows commercially available pET32a (+) plasmid map.
Figure 10 is the dna sequence dna that contains rhPTH (1-34) encoding sequence of synthetic.Wherein, 5 '-CTG and AAG-3 ' cut the protection base for enzyme, GGTACCBe Kpn I restriction enzyme site, GACGACGACGACAAG is an enteropeptidase restriction enzyme site encoding sequence, TCCGTTTCCGAAATCCAGCTGATGCACAACCTGGGTAAACACCTGAACTCCATGGA ACGTGTTGAATGGCTGCGTAAAAAACTGCAGGACGTTCACAACTTC is the sequence of coding PTH, TAA is a terminator codon GTCGACBe Sal I restriction enzyme site.
Embodiment
In order to obtain fusion rotein of the present invention, need first structure can express this Expression of Fusion Protein carrier.This expression vector can be inserted into the downstream that is subjected to the Trx gene under the promotor control by the dna sequence dna of the hPTH (1-34) that will encode and make up and finish.As described in the background section, Trx is a kind of yeast, bacterium, animal, intravital protein of plant of being prevalent in, and this protein also is for example yeast or colibacillary intrinsic protein of present hPTH (1-34) expressive host commonly used.This albumen can be regulated the balance of Protein Folding and accumulation process in cell, Trx can interact with many albumen, strengthens the solvability of fusion rotein, thereby has reduced the formation (Thomas of inclusion body, J.G. etc., Appl Biochem Biotechnol.66 (3): 197-238).Can use complete Trx in the present invention, also can use a part or its mutant of Trx, selected part or mutant have and same or analogous space structure of Trx and function.
Expression vector of the present invention can be the zymic expression vector, also can be colibacillary expression vector.Described hPTH (1-34) can be the new dna sequence dna of complete synthetic, also can be the dna sequence dna of disclosed coding hPTH (1-34).For the downstream that the dna sequence dna of the hPTH (1-34) that will encode inserts the Trx gene, preferred use has contained the cloning vector of described Trx gene or its partial sequence.This carrier also can obtain by purchase.For example, pET32a (+) a kind of carrier that comes to this.PET32a (+) can efficiently express the polypeptide that contains 109 amino acid whose Trx-Tag, and after foreign gene inserted its multiple clone site, the fusion rotein of generation contained Trx-Tag and the S-Tag sequence that can shear, is convenient to detect and purifying.
In order in subsequent step, hPTH (1-34) to be discharged from fusion rotein, preferably introduce proteins encoded lytic enzyme recognition site nucleotide sequence at 5 ' end of the dna sequence dna of coding hPTH (1-34).Described proteolytic ferment can be zymoplasm, Kex2-600, proline(Pro) endopeptidase, enteropeptidase.Enteropeptidase is preferred, because this endonuclease capable is at the C-terminal hydrolysis fusion rotein of recognition site.Therefore, the more preferably direct and then encoding sequence of hPTH (1-34) in the back of the nucleotide sequence of coding enteropeptidase recognition site, like this enteropeptidase can finally required hPTH (1-34) is complete, discharge exactly.If adopt the dna sequence dna of synthetic coding hPTH (1-34),, when the described dna sequence dna of synthetic, also need introduce suitable restriction enzyme site at the 5 ' end and the 3 ' end of this sequence for the ease of the clone.For the ease of purifying in the future, can on the appropriate location of fusion rotein, insert the sequence of being convenient to purifying, for example preferably insert His-Tag at the N of used Trx end or C end.The gene that not only has Trx among the commercially available carrier pET32a (+), and also have His-Tag in the upstream of this gene.
The present invention also provide a kind of on a large scale, hPTH (1-34) expression method efficiently.For a large amount of preparation hPTH (1-34), the recombinant expression vector that makes up need be transformed suitable host's competent cell, for example yeast cell or Bacillus coli cells, and in suitable substratum, ferment, with the results fusion rotein.The host cell that adopts in a specific embodiment of the present invention is the E.coli BL21 (DE3) that can commercially availablely obtain.In the born of the same parents of this cell and the equal inactivation of intercellular periplasm protein enzyme, when carrying out the solubility expression of foreign protein, be not easy like this by the protease hydrolysis of host bacterium, but thereby stable existence.
In order during fermentation effectively to control, but the expression vector of suggestion induction type usually.The pET serial carrier paracolon expression vector that comes to this.This carrier is the E.coli expression vector that utilizes T7 phage rna polymerase/promoter systems to make up, and has promptly integrated the T7 rna polymerase gene on the karyomit(e) of E.coliBL21 (DE3) or JM109 (DE3), and regulated and control by the lac operon.So, when inducing, cause the synthetic of t7 rna polymerase, thereby induced the expression of goal gene on the pET carrier with IPTG.T7 phage rna/promotor has very strong startup activity, and in carrier multiple clone site sequence upstream strong ribosome binding sequence (rbs) is arranged, so the pET carrier can be expressed foreign protein efficiently.
Described fermention medium is according to host's difference and different.Selecting intestinal bacteria for use is the host, is under the situation of expression vector with the pET serial carrier, and fermention medium can be preferably the TB substratum for LB, TB, M9CA etc.Leavening temperature is 30-40 ℃, is preferably 37 ℃; PH6.5-7.5 is preferably pH 7.0; Dissolved oxygen DO 〉=30%, inducing the IPTG final concentration of usefulness is 0.3-1.0mM, is preferably 0.5mM; Induction time is 3-5h, is preferably 3.5-4h.Under above-mentioned suitable condition, can make fermented liquid concentration reach that 30g bacterium weight in wet base/more than the L fermented liquid, the purpose fusion protein expression is more than 25%.
In order to obtain the pure product of rhPTH (1-34) polypeptide, at first be dissolved in the lysate with the fusion rotein of the broken bacterium method of high-pressure homogenization with emiocytosis; Carry out preliminary purification with nickel ion chelating affinity chromatography then, sample behind the preliminary purification is cut with the enteropeptidase enzyme, sample after enzyme is cut carries out chromatography with anion-exchange column, collection penetrates protein solution, to penetrate protein solution and cross the reversed phase chromatography post, and at last sample be crossed cationic exchange coloum and removed organic solvent and obtain rhPTH (1-34) albumen stoste.Utilize this method, 70 liters of fermented liquids can about 2000g weight in wet base thalline, can obtain about 3.5g rhPTH (1-34) polypeptide stoste by purifying.
Pharmacodynamics test, toxicology test studies show that before clinical: rhPTH (1-34) subcutaneous injection of the present invention's preparation has the effect of remarkable promotion scleroblast bone forming, and its safe dose is 15 μ g/kg.Therefore, to be used for human body therapy be safely and effectively to the rhPTH (1-34) of the present invention preparation.
One section sequence of hPTH (1-34) and hydrophilic Trx is merged in the present invention, and this fusion rotein is expressed with soluble form in born of the same parents, has avoided the lower renaturing inclusion bodies step of complex process and yield.Fusion rotein N end contains His-Tag, can pass through Ni 2+Huge legendary turtle and affinity chromatography fast, purifying simply, efficiently, improved the rate of recovery greatly.There is the enteropeptidase cleavage site between Trx and hPTH (1-34), guarantees that the fusion rotein of purifying can discharge complete hPTH (1-34) through the enteropeptidase cutting.Adopt enteropeptidase with the fusion rotein enzymolysis that gives expression to, and utilize a series of column chromatography that target protein hPTH (1-34) purifying is come out, purity can reach more than 99%, even reaches 100%.
Below will be with the example of intestinal bacteria as the host, the mode by specific embodiment is illustrated the present invention, but is to be understood that these embodiment do not limit this in any form
Invention scope.
Embodiment 1 expresses the design and the synthetic of the dna sequence dna of hPTH (1-34)
According to hPTH (1-34) aminoacid sequence (seeing Table 1), according to the e. coli codon preferences, the dna sequence dna of the suitable escherichia coli expression that synthetic is optimized.When synthesizing hPTH (1-34) gene, introduce Kpn I restriction enzyme site GGTACC at 5 of this gene ' end, introduce termination codon TAA and Sal I restriction enzyme site GTCGAC at 3 ' end, and 5 ' Kpn I restriction enzyme site back that end is introduced adds that the enteropeptidase enzyme cuts the encoding sequence GACGACGACGACAAG of recognition site, obtains sequence 1 (see figure 10).For ease of later subclone, synthetic gene is cloned on the pUC18, be used to preserve this synthetic dna sequence dna.Plasmid pUC18 contains Kpn I identical with plasmid pET32a (+) and Sal I restriction enzyme site.
Table 1, hPTH (1-34) codon use table
Sequence number Amino acid Selected codon Optional codon Not optional codon
1 Ser TCC TCT，TCA，TCG，AGT，AGC
2 Val GTT GTC，GTA，GTG
3 Ser TCC TCT，TCA，TCG，AGT，AGC
4 Glu GAA GAG
5 Ile ATC ATT ATA
6 Gln CAG CAA
7 Leu CTG TTA，TTG，CTT，CTC CTA
8 MET ATG
9 His CAC CAT
10 Asn AAC AAT
11 Leu CTG TTA，TTG，CTT，CTC CTA
12 Gly GGT GGC，GGG GGA
13 Lys AAA AAG
14 His CAC CAT
15 Leu CTG TTA，TTG，CTT，CTC CTA
16 Asn AAC AAT
17 Ser TCC TCT，TCA，TCG，AGT，AGC
18 Met ATG
19 Glu GAA GAG
20 Arg CGT CGC CGA， CGG， AGA， AGG
21 Val GTT GTC，GTA，GTG
22 Glu GAA GAG
23 Trp TGG
24 Leu CTG TTA，TTG，CTT，CTC CTA
25 Arg CGT CGC CGA， CGG， AGA， AGG
26 Lys AAA AAG
27 Lys AAA AAG
28 Leu CTG TTA，TTG，CTT，CTC CTA
29 Gln CAG CAA
30 Asp GAC GAT
31 Val GTT GTC，GTA，GTG
32 His CAC CAT
33 Asn AAC AAT
34 Phe TTC TTT
Annotate: aminoacid sequence is from the N end beginning label of hPTH polypeptide.
Embodiment 2 construction of recombinant plasmid processes
Following molecule clone technology working method, if no special instructions, equal reference literature: molecular cloning experiment guide (Huang Peitang etc. translate, work such as [U.S.] Sa nurse Brooker, Science Press, 2002).Used DNA extraction test kit (UNIQ-10), DNA glue reclaims test kit (UNIQ-10) and connection test kit etc. available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd in the DNA operation.Cloning vector pUC18, restriction enzyme are available from Fermentas LifeScience company.Expression vector pET32a (+), intestinal bacteria TOP10 and BL21 (DE3) are all available from Novagen company.The clone is TOP10 with escherichia coli host, and expressing with escherichia coli host is BL21 (DE3).BL21 (DE3) genotype is: hsdS gal (λ cIts857ind1 Sam7 nin5 lacUV5-T7 geneI).BL21 (DE3) has T7 RNA pol gene, and T7 RNA polymerase produces in a large number under the inducing of IPTG, thereby has opened expression of exogenous gene, can make exogenous gene high-efficient expressed.
1. preparation target gene fragment
Extract the pUC18 plasmid DNA that contains hPTH (1-34) encoding sequence with DNA extraction agent box, with Kpn I/Sal I double digestion, agarose gel electrophoresis 1% separates small segment, cutting-out contains the segmental gel in the 130bp left and right sides, reclaim test kit with gel DNA and reclaim 130bp left and right sides fragment, electrophoresis checking back is standby.
2. prepare the expression vector fragment
Extract pET32a (+) plasmid DNA with DNA extraction agent box, with Kpn I/Sal I double digestion, 1% agarose gel electrophoresis separates big fragment, downcuts to contain big segmental gel, reclaims test kit with gel DNA and reclaims big fragment, and electrophoresis checking back is standby.
3. construction recombination plasmid pET-PTH (1-34)
1,2 ready dna fragmentations are mixed by different volume ratio (2/8,3/7,4/6,5/5), connect 30min with the T4DNA ligase enzyme at 16 ℃, connect product transformed into escherichia coli TOP10 competent cell, coat LB agarose plate (1% peptone that contains 100 μ g/ml Amp, 0.5% yeast extract paste, 1%NaCl, 2% agar)), 37 ℃ of overnight incubation.
4. recombinant screen
10 bacterium colonies of picking contain 37 ℃ of overnight incubation in the LB substratum of 100 μ g/ml Amp at 5ml, extract plasmid DNA with DNA extraction agent box.With EcoR, PstI the DNA enzyme that is extracted is cut respectively, carried out 1% agarose gel electrophoresis evaluation recon then.Because EcoR I is single restriction enzyme site in pET32a (+), removed by Kpn I/Sal I double digestion when making up recon, so recon can not be cut by EcoR I; And the single site of Pst I among the pET32a (+) is not or not the polyclone district, and contain a Pst I site in rhPTH (1-34) gene, so when cutting pET32a (+) vector plasmid with Pst I enzyme, obtaining molecular weight is the unique DNA fragment of 5.9kb, and recombinant plasmid is cut two dna fragmentations (seeing also Fig. 1) that occur about 1.2kb and 4.7kb with Pst I enzyme.Restriction analysis is the result show, 10 bacterium colonies are correct recon, correct recombinant plasmid called after pET-PTH (1-34).
5. the sequencing of plasmid pET-PTH (1-34):
Recombinant plasmid pET-PTH (1-34) is carried out sequence verification, and the forward sequencing result is seen Fig. 2, and backward sequencing the results are shown in Figure 3.Wherein encoding fusion protein Trx-contains (His) 6As follows with the nucleotide sequence of the connection peptides-Rat parathyroid hormone 1-34 1-34 peptide of enteropeptidase recognition site:
ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACG
GATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGC
AGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATG
AAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTG
AACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCG
TGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGC
AACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCC
TCGACGCTAACCTGGCCggttctggttctggccatatg caccatcatcatcatcattcttctg
gtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaattcgaacgccagcacatggacag
cccagatctgggtacc
Figure A20061007421600131
TCCGTTTCCGAAATCCAGCTGAT
GCACAACCTGGGTAAACACCTGAACTCCATGGAACGTGTTGA
ATGGCTGCGTAAAAAACTGCAGGACGTTCACAACTTCTAA。Wherein, the sequence that the non-black-body capitalization is formed is the Trx encoding sequence, and the sequence that lowercase is formed is to contain Trx-to contain (His) 6With the connection peptides encoding sequence of proteolytic enzyme recognition site coding region, the italics that has underscore partly is (His) 6Coding region, two line partly are enteropeptidase recognition site coding regions, and the sequence that bold race capital is formed is hPTH (1-34) dna sequence dna, and the TAA of italic is a terminator codon.HPTH (1-34) dna sequence dna and Design Theory in the engineering bacteria that the order-checking proof makes up are in full accord.In addition, need to prove, in the encoding sequence of above-mentioned fusion rotein, in the connection peptides encoding sequence that lowercase is formed (His) 6-Tag and enteropeptidase coding region are essential sequences for obtaining pure hPTH (1-34), so other sequence in the lowercase is dispensable, and also replaceable have the sequence of function for other.
The abduction delivering of embodiment 3 engineering bacterias
With recombinant plasmid pET-PTH (1-34) DNA transformed into escherichia coli BL21 (DE3), the resulting genetic engineering bacterium that is used to express rhPTH (1-34) fusion rotein that is.8 single bacterium colonies of picking, 5ml contain 100 μ g/ml Amp the LB substratum (1% peptone, 0.5% yeast extract paste, 1%NaCl) in 37 ℃ of overnight incubation, transferring with 1/100 volume contains 37 ℃ of cultivations in the LB substratum of 100 μ g/ml Amp in 50ml, and residue bacterium liquid is frozen with 15% glycerine packing.OD 600Reach at 0.5 o'clock, add IPTG and carry out abduction delivering to final concentration 0.5mM, the SDS-PAGE electrophoresis is carried out in sampling after 4 hours.Compare with the contrast of inductive not, the recon after inducing all has the expression band about expection molecular weight 20kDa, and expression amount is more than 25% of whole bacterial protein.Get the highest bacterium of output No. 6 as engineering bacteria, called after BL21 (DE3)-PTH (1-34) puts in the glycerine and preserves.Protein expression screening electrophorogram is seen accompanying drawing 4.
The fermentation of embodiment 4 engineering bacteria BL21 (DE3)-PTH (1-34)
Get 1 of the glycerine bacterial classification (1mL) of engineering bacteria BL21 (the DE3)-PTH (1-34) that expresses Trx-hPTH, be inoculated into 400mL LB substratum (1% peptone, 0.5% yeast extract paste, 1%NaCl) in, 37 ℃, 200rpm shake-flask culture 15h, activated seed.Get activated seed and be connected to (1.2% peptone, 2.4% yeast extract paste, 0.4% glycerine, 17mM KH in the 3.5L TB substratum by 10% inoculum size 2PO 4, 72mM K 2HPO 4) (5L B.Braun fermentor tank), cultivate 4h for 37 ℃.Then this nutrient solution is connected to by 5% inoculum size that (100L B.Braun fermentor tank) ferments in the 70L TB substratum, temperature is 37 ℃, pH7.0, DO 〉=30%, is cultured to OD 600=4.0 o'clock begin abduction delivering, and the IPTG final concentration of inducing usefulness is 0.5mM, and induction time is 4h.With this understanding, cell density is 30g bacterium weight in wet base/L fermented liquid, purpose fusion protein expression 25%.The result sees also Fig. 5.
The purifying of embodiment 5rhPTH (1-34)
Adopt to connect speed scheming (the CEPA Z41 that wanders about as a refugee, B.Braun company, Germany) the fermentation thalline among the collection embodiment 4, with buffer A (10mM PBS (phosphate buffered saline buffer), 500mM NaCl, 30mM imidazoles, pH8.0) suspend, use APV-1000 high pressure homogenizer (APV Co. Denmark) to break bacterium then, the fusion rotein of emiocytosis is dissolved in the buffer A the centrifugal 30min of 9000rpm.Getting supernatant liquor adopts following method that rhPTH (1-34) is carried out purifying successively:
1. nickel ion chelating affinity chromatography
High-pressure homogenization is broken on the bacterium supernatant liquor in using buffer A equilibrated nickel ion chelating affinity chromatography (Chelating Sepharose Fast Flow, GE Healthcare) post, behind the buffer A thorough washing, with buffer B (10mM PB, 500mM NaCl, 200mM imidazoles, pH8.0) wash-out, collect elution peak, obtain fusion protein sample.
2. the enzyme of fusion rotein is cut
Preparation endonuclease reaction liquid, it is composed as follows: 1mg/mL fusion rotein, 50mMTris-HCl (pH8.0), 1mM CaCl 2, enteropeptidase (ratio in 1mg enteropeptidase cutting 5mg fusion rotein adds enteropeptidase).25 ℃ of enzymes were cut 20 hours.
3. anion exchange chromatography
Enzyme is cut the back protein solution be splined on (50mM Tris-HCl, pH8.0) equilibrated Q Sepharose High Performance (GE Healthcare) chromatography column with damping fluid C.The theoretical iso-electric point of rhPTH (1-34) is 8.29, and is positively charged when pH8.0, do not combine with anion-exchange column, and foreign protein then combines with anion-exchange column because of electronegative.Collection penetrates liquid, can obtain purity and be rhPTH (1-34) protein sample more than 95%.
4. reversed phase column chromatography
Select for use reversed-phase column Source 15RPC (GE Healthcare) to carrying out polishing purification by the rhPTH that obtains through the ion-exchange chromatography purifying mentioned above (1-34) protein sample, make gradient elution with 24-64% ethanol, occur rhPTH (1-34) albumen elution peak when 40-60% ethanol, purity reaches more than 98%.
5. cation exchange column chromatography
With the sample of reversed-phase column wash-out damping fluid D (10mM PB, pH7.0) after the dilution, go up in through damping fluid D equilibrated SP Sepharose Fast Flow (GE Healthcare) chromatography column, behind the damping fluid D thorough washing, with the damping fluid D wash-out that contains 400mM NaCl, obtain purity greater than 99% rhPTH (1-34) albumen.
Respectively go on foot the purification effect (see figure 6) with the SDS-PAGE electrophoretic analysis.
The calibrating of test example 1 rhPTH (1-34)
1, SDS-PAGE purity check
Adopt the Tris-Tricine SDS-PAGE (Guo Yaojun of system, protein electrophorese experimental technique, Science Press, 1999) carry out non-reduced type electrophoresis, with Bio-Rad Gel Doc 2000 gel imaging system sweep measurings, rhPTH (1-34) purity is 100% (seeing the 7th road among Fig. 6).
2, RP-HPLC purity check
With the purity of HPLC method mensuration protein and peptide class, accuracy height and its retention time also can be used as index qualitatively.Chromatography column is Delta-Pak C18 5 μ m 3.9 * 150 (Waters Co.), and (0.1% trifluoroacetic acid (TFA) is at 95%dH for buffer A 2In O and 5% acetonitrile) (0.1%TFA is 95% and 5%dH to buffer B 2Among the O) linear gradient elution 70min, flow velocity 1ml/min, 220nm ultraviolet detection.Analytical results shows that the HPLC collection of illustrative plates of the rhPTH of above-mentioned prepared (1-34) is a simple spike, and purity is 100%.The RP-HPLC analytical results is seen Fig. 7.
3, N, C-terminal amino acid sequence analysis
Employing Edman edman degradation Edman mensuration obtains terminal 15 aminoacid sequences of rhPTH (1-34) N-according to embodiment 5 purifying:
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu; Adopt terminal 3 aminoacid sequences of carboxypeptidase y method mensuration rhPTH (1-34) C-to be: His-Asn-Phe.N, C-terminal amino acid sequence analysis result and theoretical sequence are identical, illustrate that rhPTH (1-34) primary structure that we prepare is correct.
4, the mass spectroscopy of rhPTH (1-34)
The LCQ-Classic mass spectrograph of employing Finnigan company carries out mass spectroscopy to the rhPTH (1-34) of purifying, and the molecular weight that records rhPTH (1-34) is the 4117.5Da (see figure 9).Consistent with the theoretical value (4118.8Da) of rhPTH (1-34).
5, intracellular toxin and pyrogen test
According to the regulation of " biological products bacterial endotoxin test rules " in " Chinese biological goods rules " (version in 2000), the endotoxin content that detects prepared rhPTH (1-34) sample with limulus reagent test is not higher than 10EU/20 μ g rhPTH (1-34).According to the regulation of " Chinese biological goods rules " (version in 2000) " biological products pyrogen test rules ", it is negative to adopt the rabbit method to measure its pyrogen.
The 2 PTH determinations of activity of test example
Inoculation UMR-106-01 cell (available from ATCC) in 96 porocyte culture plates, inoculum size is 1～2 * 10 5Individual cell/mL, 100 μ L/ holes, 37 ℃, 5%CO 2Overnight incubation.Wash cell once with serum free medium, add substratum and (contain 20mM Hepes, 0.1% bovine serum albumin, and 0.2mM IBMX (3-isobutyl-1-methylxanthine, Sigma), pH7.4) 180 μ L, add 20 μ L again and be diluted to the hPTH (1-34) and the standard substance (available from WHO biological product standards product laboratories (NIBSC)) thereof of different concns, establish the contrast that contains and do not contain IBMX simultaneously, all do two multiple holes with this substratum, 37 ℃, 5%CO 2Hatch 45min.Remove substratum, every hole adds 200 μ L 0.1N HCl, incubation 30min, and fully lysing cell is got supernatant and is adopted cAMP (low pH) KIT (R﹠amp; D company, Cat No.DE0355) mensuration cAMP value.Data utilize computer software to handle, and press following formula formula calculation result:
Figure A20061007421600171
The result shows that we rhPTH (1-34) of preparation has identical biological activity with the WHO reference substance, and its specific activity is greater than 1.0 * 10 5U/mg rhPTH (1-34).
Test example 3 Pharmacodynamic test of active extract
Use rat ovary and extract (ovanceclomiwd, OVX) method is set up simulation primary osteoporosis model, gives to observe bone amount, bone biomechanical, the relevant blood with bone metabolism of bone norphometry, its result of treatment of urine biochemical indicator comprehensive evaluation after 8 weeks of rhPTH (1-34) treatment.Test-results shows:
1) ovariectomy 12 week the group rats the uterus weight in wet base obviously vacation pluck the ovum group and reduce, bone amount (femur and lumbar spine bmd) and bone biomechanical property (femur three-point bending load, lumbar vertebrae compressive load) all vacation pluck the ovum group and obviously reduce, show that the osteoporosis rat model is set up due to the estrogen deficiency;
2) rhPTH (1-34) has obvious result of treatment to OVX induced osteoporosis rat.RhPTH (1-34) treated for 8 weeks, low dosage (10 μ g/Kg) group promptly shows obvious raising effect to femur, lumbar vertebra amount (dry weight, heavy, the bone density of ash) biomechanical property (femur three-point bending ultimate load, lumbar vertebrae compression ultimate load) and the lumbar vertebra girder area of model osteoporosis rat, and improves with the therapeutic dose increase.
Blood calcium phosphorus has the fluctuation change of increasing and descend when 3) observing rhPTH (1-34) injection 4h in this experiment, and is normal behind the 24h.
Therefore, rhPTH (1-34) has the effect of remarkable promotion scleroblast bone forming, and osteoporosis rat is had obvious result of treatment.
Sequence table
＜110〉Shenzhen Hua Shengyuan genetically engineered Development Co., Ltd
＜120〉prepare the method for human parathyroid hormone 1-34
<130>FI-060213-59
<160>2
<170>PatentIn version 3.2
<210>1
<211>117
<212>DNA
<213>Artificial
<220>
＜223〉encoding sequence of enteropeptidase recognition site-human parathyroid hormone 1-34 peptide
<400>1
gacgacgacg acaagtccgt ttccgaaatc cagctgatgc acaacctggg taaacacctg 60
aactccatgg aacgtgttga atggctgcgt aaaaaactgc aggacgttca caacttc 117
<210>2
<211>576
<212>DNA
<213>Artificial
<220>
＜223〉coding contains the nucleotide sequence of the fusion rotein of Trx and human parathyroid hormone 1-34
<400>2
atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60
gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120
ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180
atcgatcaaa accctggcac 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 caagtccgtt 480
tccgaaatcc agctgatgca caacctgggt aaacacctga actccatgga acgtgttgaa 540
tggctgcgta aaaaactgca ggacgttcac aacttc 576

Claims (10)

1. method for preparing hPTH (1-34), this method comprises the steps:
(1) expression vector that enables expressed fusion protein is expressed, and it is Trx-(His) that described fusion rotein is held the sequence of C end from N 6-enteropeptidase recognition site-Rat parathyroid hormone 1-34 1-34 peptide;
(2) with nickel ion chelating affinity chromatography the fusion rotein that step (1) obtains is carried out purifying; And
(3) fusion rotein that step (2) purifying is obtained is cut with the enteropeptidase enzyme, so that Rat parathyroid hormone 1-34 1-34 peptide is discharged from described fusion rotein.
2. the described preparation method of claim 1 also further comprises:
(4) step (3) enzyme is cut the mixture that obtains and carry out chromatography, collect and penetrate protein solution with anion-exchange column; And
(5) make step (4) collect the protein solution that penetrates that obtains and cross the reversed phase chromatography post.
3. claim 1 or 2 described preparation methods, the enteropeptidase recognition site in the wherein said fusion rotein-Rat parathyroid hormone 1-34 1-34 peptide is encoded by following nucleotide sequences: GACGACGACGACAAGTCCGTTTCCGAAATCCAGCTGATGCACAACCTGGGTAAACA CCTGAACTCCATGGAACGTGTTGAATGGCTGCGTAAAAAACTGCAGGACGTTCACA ACTTC.
4. the expression vector that uses among the described preparation method of claim 3, wherein said step (1) is coli expression carrier pET-PTH (1-34), and expression is e. coli bl21 (DE3) with the host.
5. preparation method according to claim 4, described step (1) is finished under the following conditions:
The e. coli bl21 (DE3) that will contain expression vector pET-PTH (1-34) ferments in the substratum of at least a LB of being selected from, TB and M9CA; Leavening temperature is 30-40 ℃, and fermented liquid pH is 6.5-7.5 and fermentation dissolved oxygen DO 〉=30%.
6. according to the method for claim 5, wherein ought be cultured to OD 600=4.0 o'clock, adding final concentration in substratum was the IPTG of 0.3-1.0mM, the beginning abduction delivering.
7. preparation method according to claim 6, the wherein said fermentation inducement time is 3-5 hour.
8. according to each described preparation method among the claim 5-7: wherein substratum is TB, and leavening temperature is about 37 ℃, and fermented liquid pH is about 7.0, and the final concentration of IPTG is about 0.5mM, and inducing fermentation time is about 4 hours.
9. preparation method according to claim 1: wherein in step (2), in the ratio adding enteropeptidase of 1mg enteropeptidase cutting 5mg fusion rotein.
10. method according to claim 9, described enzyme is cut step and is finished under following condition: 1mg/mL fusion rotein, 50mM Tris-HCl (pH8.0), 1mM CaCl 2, about 25 ℃, enzyme was cut about 20 hours.
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