CN100335622C - Synthesis of batroxobin gene and purification preparation of its expresson product - Google Patents
Synthesis of batroxobin gene and purification preparation of its expresson product Download PDFInfo
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- CN100335622C CN100335622C CNB031160549A CN03116054A CN100335622C CN 100335622 C CN100335622 C CN 100335622C CN B031160549 A CNB031160549 A CN B031160549A CN 03116054 A CN03116054 A CN 03116054A CN 100335622 C CN100335622 C CN 100335622C
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Abstract
The present invention relates to batroxobin prepared by using the gene recombination method, which has the key points of the synthesis and the expression of batroxobin genes, the purification of the expression product and the determination of the properties. The recombinant batroxobin not only can be used as a main component in a hemostatic, but also can be directly used as a medicine for reducing fibers.
Description
Technical field
The present invention relevant adopts recombinant DNA technology to produce a kind ofly to come from pallas pit viper and belong to synthetic, the expression of batroxobin (Batroxobin) albumen, particularly batroxobin gene in the poisonous snake venom and the purifying preparation and the character conclusive evidence of expression product.
Background technology
Austrian scholar Von Klobusitzky in 1963 separates from Brazilian fer-de-lanc (Bothrops atrox) venom and obtains a kind of serine proteinase enzyme, just so-called batroxobin.Since then, the investigator has obtained as many as 20 kinds of serine protease quasi-molecules from pallas pit viper belongs to the venom of different poisonous snakes, they can both cut mammiferous plasma fibrinogen by enzyme, make it to change into scleroproein, thereby influence hemorrhage-coagulation process of animal.In these proteolytic enzyme, especially to comparatively clear (Stocker K., Barlow G.H., Methods Enzymol.45:214-223,1976) of physics, chemical property and the clinical application research of batroxobin.
The specific effect substrate of batroxobin is a Fibrinogen, and different with zymoplasm is, the A chain of its cutting fibre proteinogen, and do not act on the B chain.Arg in its hydrolysis plasma fibrinogen A chain
16-Gly
17The position is during peptide bond, can discharge fibrinopeptide A, thereby apace the Fibrinogen in the blood is transformed into scleroproein, then these scleroproeins just can be gathered into loose being easy to and come wound closure by the thrombus of scleroproein enzymic hydrolysis, the effect of realization quick-acting haemostatic powder.But, using heavy dose of time marquis, batroxobin has fibrinogen concentration in the blood of reduction, improves the hydrodynamic characteristic of blood viscosity and blood, thereby plays the effect (US Pat.No:3849252,1974) of fiber eliminating enzyme.Based on more such biochemical characteristics, batroxobin successfully has been developed to hemostatic drug (trade(brand)name Reptilase or Hemocoagulase) and has been fallen fine medicine (trade(brand)name Defibrase).In Europe, batroxobin is substituting human thrombin as haemostatic medicament.
At home, consider that the function of its molecule has similar zymoplasm (Thrombin) part, existing middle translated name is called ' hemocoagulase '.Sayings such as ' haemocoagulase ', ' hemocoagulin enzyme ' are also arranged previously.This reaches by Shenyang match promise, Changchun, Beijing match is given birth to and ' reptilase ' imitation ' Ba Quting ' of Peng Lai drugmaker of Hua Taisi family joint research and development is also succeeded in developing August calendar year 2001, and this medicine (protection period is 2001.8.21-2007.8.21) also is to extract to be prepared from from snake venom.But need to prove, according to British Pharmacopoeia ([) (MARTINDALE The Extra Pharmacopoeia, Thirty-firstEdition, Edited by James E F Reynolds, Royal PharmaceuticalSociety, London, 1996, P756) and the Merck index (The Merck lndex, 1989, P1017) describe in, commodity are called the medicine of Reptilase (Chinese commodity are called " reptilase ") or Hemocoagulase, and its main component is a batroxobin, also contains factor X activator (Factor-X Activator in addition, for example, composition such as RVV-X albumen).
Though the investigator has had many understandings to the character of batroxobin, but, molecular biology research to this snake venom composition is made slow progress always, just finished cDNA and genomic dna examining order (Nobuyukiltoh etal J.Biol.Chem.262 (7): 3132～3135,1987 up to 1987,1988 to batroxobin gene by the investigator of Japan; J.Biol.Chem., 263:7628～7631,1998).
1991 Japanese rattan pool drugmaker (Fujisawa Pharmaceutical) the investigator adopt gene recombination method, in Ecoli, adopt the amalgamation and expression system, formal representation with inclusion body (lnclusion Body) goes out this composition, cut the method for fusion rotein open and it is reported that obtaining what is called has biologic activity batroxobin (Maeda M etal by preparing electrophoresis and zymoplasm again, J Biochem (Tokyo), 109 (4): 632-637,1991), but also applied for patent (Pat.No.:JP2124092,1990) for this reason.But, from after this, have only the batroxobin that from snake venom, extracts preparation be used for the treatment of multiple face indication (as, myocardial infarction, senile dementia, apoplexy, sudden deafness or the like) patent (US Pat.No:5595974,5869044,6106830,6399576,6416717; Eur.Pat.No:0984279,0826374,0750912,0719791) and research report, recombinant batroxobin is used for any correlative study report of animal experiment and clinical study work never again.At present, domestic research report for batroxobin pharmacodynamics aspect is a lot of, and Shang Weijian carries out recombinant expressed document or report to this composition.
Though batroxobin protein has only a peptide chain, because intramolecular disulfide bond is many, also have glycosylation modifiedly etc., so adopt genetic engineering means to produce this albumen very big technical difficulty is arranged.This also should be this one of the main reasons that does not always have recombinant product to come out.
The single chain protein that sophisticated batroxobin molecule is made up of 231 amino-acid residues, it is 25.5kD that Theoretical Calculation goes out its molecular weight, iso-electric point is 7.39, abroad the actual molecular weight of the batroxobin of biological extraction is 42kD from Bothrops atrox venom, and the deviation of this molecular weight is because glycosylation modified cause.Can find that from batroxobin protein matter primary structure its intramolecularly has two N-glycosylation site: Asn
146-Asn
147-Thr
148And Asn
225-Lys
226-Thr
228In addition, also can extract in other subspecies of Bothropsatrox and the venom of other kind poisonous snake and have the active albumen of batroxobin, but its molecular weight does not wait from 29.1kD to 42kD, and this may be owing to last difference of amino acid composition or glycosylated degree difference between the different subspecies are caused.12 halfcystines are arranged in the batroxobin molecule,, infer that these 12 halfcystines have Cys according to the result of study of known serine protease quasi-molecule
7-Cys
139, Cys
26-Cys
42, Cys
74-Cys
230, Cys
118-Cys
184, Cys
150-Cys
168And Cys
174-Cys
199The formation of these six kinds of intramolecular disulfide bonds.Consider production cost that extracts batroxobin from the venom of snake and the needs of further studying its structure, we adopt engineered method to produce recombinant batroxobin.，
Adopt the albumen that disulfide linkage is rich in engineered means production always to be a technical barrier, especially producing has many serine proteinase enzyme molecules to disulfide linkage.This is that what obtain in protokaryon almost is inclusion body entirely because disulfide linkage pairing error rate is very high, though can obtain a small amount of activated target protein by denaturation renaturation to inclusion body, its cost determination do not possess the actual production meaning.Can guarantee higher disulfide linkage pairing accuracy in the eukaryotic cell expression system (yeast, CHO and insect cell etc.), so this patent is to adopt the methanol yeast system.We are at methanol yeast (Pichia pastoris, Pichia methanolica) successfully given expression to the batroxobin protein that biologic activity is arranged in, output can reach every milliliter of fermented liquid 20 Ke Shi units (20KU/ml), and this output has tentatively possessed the production meaning.
Aminoacid sequence data according to disclosed batroxobin (X12747) among the U.S. Genebank, select the genetic codon of hobby of yeast or E.coli, synthetic batroxobin complete genome sequence, respectively this gene being inserted into the E.coli expression vector (as pET, pGEX class carrier, induces with IPTG; Or pLY, PBV200 carrier, use thermal induction; The pLEX carrier is induced with tryptophane) in merge or the research of non-fusion expression; Or be inserted into the secreted expression carrier pPIC9 of pichia pastoris, pPIC9K, expression vector in pPICZa class carrier or the pPIC3K class born of the same parents, or be inserted into the research of carrying out secreting, expressing in the secreted expression carrier pMETa class carrier of Pichia methanolica or the pMET class born of the same parents in the expression vector.In addition, also adopt the artificial complete synthesis batroxobin gene of codon of methanol yeast hobby fully, comparative studies these two batroxobin genes in the difference of expressing on the output.
Though in E.coli, express some albumen and be the basic means of comparatively conventional production pharmaceutical protein, but in practical study, find batroxobin protein, no matter (same GST, Trx or Nus merge) or non-fusion expression (the many methionine(Met) of N end) batroxobin protein is merged in employing, resulting all is inclusion body insoluble, non-activity, though expression amount can reach than higher level (account for bacterial protein 20～30%).Change among the host bacterium AD494 of the new many disulfide bond proteins of releasing of suitable expression of Novagen company and the Origami expression vector also of no avail.This inclusion body is after the denaturation renaturation process, and the soluble proteins that obtains does not detect any activity.
Three responsive site (Arg of possible yeast membrane proteolytic enzyme Kex2 are arranged in the batroxobin primary structure
44-Arg
45, Lys
77-Lys
78-Lys
79, Arg
203-Lys
204), generally speaking, this can may cause the excretory target protein to be degraded, and reduces the effective expression amount, finds to have the protein band of degraded the supernatant after inducing really.But by optimizing a series of conditions in the fermenting process, the proteic expression amount of final purpose still can satisfy (Fig. 3) of large-scale production needs.So we are chosen in efficiently expressing batroxobin among methanol yeast Pichia pastoris and the Pichia methanolica.
Summary of the invention
A kind of recombinant batroxobin, it is made by engineered method, and this method comprises following step:
(1), gene is synthetic: according to the aminoacid sequence of batroxobin in the Brazilian fer-de-lanc venom, select the used codon of batroxobin structure gene, the used codon of described batroxobin structure gene is the codon that yeast or intestinal bacteria are all compared preference, adopt the method for synthetic, obtain batroxobin structural gene sequence SEQ-2; Or adopt the method for same synthetic, obtain fully the molecular batroxobin structural gene sequence of password SEQ-3 by the methanol yeast hobby.
(2), the structure of expression vector: 5 of batroxobin gene '-end adds the point of contact of restriction enzyme Xho I, 3 '-two terminator codons of end interpolation TAA TGA and Not I point of contact, point of contact and the batroxobin protein N-at Xho I holds adding yeast KEX between first amino acid Val codon in addition
2The codon AAA AGA of protease recognition sequence Lys-Arg correspondence inserts this dna fragmentation in pPICZ α A or the pMET α A carrier, gets pPICZ α A-Bg or pMET α A-Bg expression vector, and the clone cultivates, and extracts plasmid;
(3), the expression of batroxobin: with expression vector pPICZ α A-Bg or pMET α A-Bg linearizing, prepare the competence of yeast host bacterium with DNA restriction restriction endonuclease, and carry out the electricity conversion; The clone, and express, screen, the high expression level bacterial strain obtained;
(4), the purifying of going up jar fermentation and expression product of batroxobin engineering bacteria.
1 batroxobin gene (Batroxobin gene, synthesizing Bg), order-checking and being configured to of expression vector make goal gene can insert pPICZaA or pMETaA carrier, at the point of contact that the 5`-of Bg end adds restriction enzyme XhoI, the 3`-end adds two termination codons of TAA TGA and NotI point of contact, hold the corresponding codon AAA AGA of adding KEX2 protease recognition sequence Lys-Arg between first amino acid Val codon at the XhoI point of contact with batroxobin protein N-in addition, the signal peptide sequence that this just guarantees can successfully excise when target protein is in being secreted into fermented supernatant fluid a-signal peptide (Fig. 1) or batroxobin self has with the same n terminal amino acid sequence of this albumen that extracts in the snake venom batroxobin of engineering bacterium expression.
The splicing of gene adopts recursion PCR (Recursive PCR) method, final PCR product post is reclaimed, through XhoI, the NotI double digestion, glue reclaims the dna fragmentation that meets design Bg length again, be inserted in pPICZaA or the pMETaA carrier (Fig. 2), select the mono-clonal on LZ (LB+25ug/ml Zeocin) flat board, the LZ medium liquid is cultivated, extract plasmid, XhoI-NotI double digestion screening contains 6 of the clones of Bg length, check order from the 5`-end and the 3`-end of Bg gene respectively with carrying out a-factor priming and 3`-AOX1 priming sequencing primer, finally obtain a clone who conforms with original design requirement fully, contain expression vector pPICZaA-Bg among this clone and can be used for transforming Pichia pastorisX-33 and GS115His
+The host bacterium.
The correct Bg gene of order-checking is cut out with the XhoI-NotI double digestion from the pPICZaA-Bg expression vector, change between the XhoI-NotI restriction enzyme sites of inserting the pMETaA carrier again, structure pMETaA-Bg expression vector, this expression vector is used for transforming Pichia methanolica PMAD11 host bacterium.The above-mentioned two kinds of expression plasmid carriers of a large amount of preparations of alkaline hydrolysis method are for the need of transformed yeast.
2. the determination of activity of the expression of batroxobin and expression product
1.1.pPICZaA-Bg transform Pichia pastorisX-33 after the linearizing
With DNA restriction enzyme SacI with the pPICZaA-Bg linearizing, prepare the competence of yeast host bacterium and carry out the electricity conversion according to the method (P21～23) among the P.methanolica Expression Kit Version B of Invitrogen company, cell after transforming is layered on YPD+500ug/ml Zeocin agarose plate (1%Yeast extract, 2%Polypeptone, 2%Glucose, 1.5%Agar, 500ug/ml Zeocin) goes up bed board, placed 3～4 days down, as seen have the yeast mono-clonal to grow for 30 ℃.Under 500ug/ml-600ug/ml Zeocin concentration conditions, on average each conversion has 200～400 clones not wait approximately.If the concentration of Zeocin is lowered, clone's number is multiplied the utmost point, when the electricity that adopts this method to carry out changes, with the Zeocin of 500～600ug/ml, also is fit to this expression vector and inserts other genes.The clone that choosing colony is big and full carries out expression screening.
2.2.pMETaA-Bg transform Pichia methanolicaPMAD11 after the linearizing
, adopt and last identical method the pMETaA-Bg linearizing with DNA restriction enzyme KpnI, transform PMAD11 (Pichia methanolica), and the yeast after will transforming is at MD agarose (1.34%YNB, 4X10
-5%Biotin, 2%Glucose, 1.5%Agar) the dull and stereotyped bed board of going up, placed 3～4 days down for 30 ℃, after waiting to grow mono-clonal, change 4 ℃-8 ℃ over to and placed 3-4 days down, as seen have a small amount of yeast mono-clonal to be with a redness slightly, this is that conversion is unsuccessful, only chooses the big and full clone who is creamy white and carries out expression screening.On average each transforms big York and obtains 500～1000 clones.
2.3. the screening of engineering bacteria
The clone that obtains in above-mentioned 2.1,2.2 is expressed, screens according to requiring in the Invitrogen company operational manual, obtain the bacterial strain of high expression level.
2.4. the determination of activity of expression product
Measuring method for activity with reference to the batroxobin that from snake venom, extracts, can measure the enzymic activity that gives expression in the fermented supernatant fluid with the people's standard blood plasma that has added Trisodium Citrate, this promptly can screen the clone, also can just test and assess to the output of engineering bacteria.Concrete measure active standard and be exactly: under 37 ℃, the fermented supernatant fluid of 100ul is joined 300ul contain in people's standard blood plasma of Trisodium Citrate, behind the mixing, observation is solidified the required time.
The vitality test in each stage is all with representing the used the number of minutes of the clotting of plasma in the expression output of batroxobin and the purifying in this patent, and its implication is people's standard clotting of plasma required time that the fermented liquid of 100ul or the solution that contains batroxobin make 300ul.
3. the purifying preparation of going up jar fermentation and expression product of batroxobin engineering bacteria
3.1 the jar of going up of engineering bacteria ferments,
(expression amount is 15～20mins), is inoculated into to shake in the bottle propagation as seed liquor, is forwarded to the 30L fermentor tank again, carries out the fermentation of pilot scale by the ordinary method of methanol yeast fermentation to be used in the engineering bacteria of the high expression level that filters out in the test tube.After inducing 50 hours, expression amount is 40secs～1min.
3.2 the purifying of fermented supernatant fluid preparation
With dilution of fermented supernatant fluid large volume or ultrafiltration and concentration desalination, make the PH of sample solution and the requirement that ionic strength (being specific conductivity) is fit to ion exchange chromatography.The first step is carried out cation-exchange chromatography, and we select the strong cation exchange medium, and the target protein that wash-out goes out is gone up sample again to the reinforcing yin essence Ion Exchange Medium, through a step molecular sieve, promptly can obtain the batroxobin (see figure 3) that purity meets the requirements at last.
Description of drawings
Fig. 1: batroxobin gene is with annexation between the signal peptide sequence
SEQ-2 is adapted at the batroxobin structure gene part expressed among yeast and the E.coli; SEQ-3 is the batroxobin structure gene part that is adapted at high expression level in the yeast; SEQ-5 is the pairing dna sequence dna of batroxobin natural signals peptide sequence.
The A:a-signal peptide connects with batroxobin structure gene; B: batroxobin natural signals peptide connects with its structure gene, and the KEX2 recognition sequence has been inserted in the centre.
Fig. 2: the structure of batroxobin gene expression vector (pPICZaA-Bg)
Fig. 3: the SDS-PAGE electrophoresis of batroxobin protein
1. the fermented supernatant fluid when not inducing; 2. induce the fermented supernatant fluid after 72 hours; 3. the batroxobin that is purified into; M: molecular weight standard in the albumen (LMW Marker Kit, Amersham PharmaciaBiotech)
Fig. 4: the sugar chain of batroxobin is removed in the enzyme excision
A. cut the batroxobin of processing without PNGase F enzyme; B.PNGase F enzyme is cut the batroxobin of handling after 1 hour, and wherein the B1 band is not excise sugar chain as yet, and the B2 band is the batroxobin that cuts the N-sugar chain: molecular weight standard in the M. albumen (LMW Marker Kit, Amersham PharmaciaBiotech)
Embodiment
Embodiment 1:
Recurrence PCR method obtains batroxobin gene (Bg-EP; Bg-P)
According to the aminoacid sequence of the batroxobin protein of announcing among the Genbank X12747, synthetic the full gene of batroxobin.In order to realize batroxobin efficiently expressing in different host bacterium (E.coli and yeast), two kinds of batroxobin gene (SEQ-2 have been synthesized respectively, SEQ-3): one is the batroxobin gene (Bg-EP) of taking into account E.coli and methanol yeast hobby codon, and one is to select the methanol yeast hobby molecular gene of password (Bg-P) fully.Utilize the Bg gene of DNA analysis software analysis total length, whole gene is divided into 22 bar segment, synthetic these fragments, complementary cohesive end length is not less than 15 base pairs between every adjacent primer, have XhoI point of contact and the pairing codon of Kex2 recognition sequence amino acid on 5 ' of batroxobin gene-end primer, have two terminator codons (TAA and TAG) and EcoRI point of contact on 3 '-end primer.By PCR method splicing Bg-EP and Bx-P gene.
Connect order between each primer fragment referring to Fig. 1.
Embodiment 2:
The expression of batroxobin gene Bg-EP, Bg-P
1. express batroxobin gene Bg-EP, Bg-P in the yeast
Bg-EP is inserted between the multiple clone site Xho-NotI of pPICZaA carrier, transforms Pichia pastoris host bacterium X-33.Carry out expression screening according to the method among the Pichia pastorisExpression Kit of Invitrogen company.Determination of activity is to utilize batroxobin can enzyme cut Fibrinogen, makes the human plasma that contains Trisodium Citrate solidify that this method carries out.Picking contains the yeast colony that the YPD flat board of 500ug/ml microbiotic Zeocin grows, and reality has been screened 80 clones altogether, and expression amount does not wait from 40mins to 15mins.With on the bacterial classification of 40min, the 30min, 25min and the 15min that sift out jar of fermentation, behind methanol induction 50hrs, the expression amount difference is not remarkable.Equally Bg-P is inserted among the pPICZaA, resulting engineering bacterium expression output does not have marked difference with Bg-EP.Single activity from expression product, the expression output of Bg gene (Bg-EP, Bg-P) in yeast has tangible stone wall phenomenon, the 40min output that filters out in the test tube and the bacterial classification of the 15min output activity expression amount difference with insignificance in fermentor tank.Though obviously thicken at the band that is equivalent to the batroxobin protein position, active not increase.
2.E.coli middle expression batroxobin gene Bg-EP
Synthesized following three the 5 '-end primer of Bg-EP gene respectively, they have BglII, BamH1 and EcoRI restriction enzyme site respectively:
Primer 1 (P1):
5′-CAT
AGATCTAGATGATGACGATAAAGTTATCGGTGGTGATGAATG-3’
Primer 2 (P2):
5′CAT
GGATCCGATGATGACGATAAAGTTATCGGTGGTGATGAATG-3’
Primer 3 (P3):
5′CAT
GAATTCATGGTTATCGGTGGTGATGAATG-3’
With pPICZa-Bg-EP is template, the primer BP22 that uses when using above-mentioned three 5 '-end primer and front gene splicing respectively, and passing through PCR just can be with the point of contact of transform-based because of two ends, and Bg-EP is inserted among the different expression vectors.The part of underscore is the DNA restriction endonuclease recognition sequence in above-mentioned three primers, bold-type letter partly is 5 ' of batroxobin gene Bg-EP-end complementary pairing sequence, it wherein between the restriction enzyme sites of primer-1 (P1), primer-2 (P2) and the Bg-EP enteropeptidase (Enterkinase) recognition sequence, the batroxobin that goes out with expressing fusion protein can be cut with enteropeptidase, discharge batroxobin protein.Between the restriction enzyme sites of primer-3 (P3) and Bg-EP is the methionine(Met) initiator codon, is inserted into the batroxobin protein that will produce the many methionine residues of N-end in the carrier.
2.1 utilize the PET series expression system of Novagen company
With P1, BP22 is primer, the pPICZaA-Bg-EP plasmid is a template, PCR goes out the gene fragment that length is about 730bp, be inserted into equally in two PET32a (+) carriers of cutting of these two kinds of enzymes after cutting through BgllI-NotI is two, the NovaBlue host bacterium of transformed competence colibacillus, the segmental clone of purpose is inserted in the double digestion screening, with plasmid extraction test kit extracting plasmid, transform BL21 (DE3) or Origami (DE3), bed board on the LA+Agar plate, choose positive colony and be inoculated into LA nutrient solution multiplication culture, IPTG just induces can give expression to the Trx-Batroxoboin fusion rotein.
2.2 utilize the pGEX series expression system of Amersham Pharmacia Biotech company
With P2, BP22 is primer, the pPICZaA-Bg-EP plasmid is a template, PCR goes out the gene fragment that length is about 730bp, after BamHI-NotI pair is cut, be inserted among the pGEX4T-1 that these two kinds of enzymes are cut equally, directly transform BL21 host bacterium, choose positive colony, be inoculated in the LA nutrient solution, induce with IPTG, the SDS-PAGE electrophoresis detection gives expression to the clone who conforms with theoretical big or small fusion rotein, promptly is the engineering bacteria that contains goal gene.Be further affirmation, but extracting plasmid double digestion again, the agarose gel electrophoresis checking.
2.3 utilize thermal induction type expression vector pLY expression system
The pLY expression vector is the varient of PBV serial carrier, and the characteristics of its maximum are to have cIt568, makes expression vector not have special requirement to the host bacterium.Use EcoRI, the two pLY carriers of cutting of NotI.With P3, BP22 is primer, the pPICZaA-Bg-EP plasmid is a template, PCR goes out the gene fragment that length is about 710bp, in the pLY carrier of handling above after EcoRI, NotI pair is cut, being inserted into, transform BL21, in the LA nutrient solution, breed, carry out thermal induction under 43 ℃-45 ℃ the condition, it is exactly the engineering bacteria that filters out, therefrom extracting that the SDS-PAGE electrophoresis gives expression to the clone that molecular weight is about the 26kD protein band.
Above-mentioned in E.coli expressed products all be inclusion body protein, almost do not have the target protein or the target protein fusion rotein of solubility.Fail to measure the batroxobin activity in the broken bacterium liquid.Contain His-Tag among the Trx-Batroxobin that the PET32a-Bg-EP expression vector is expressed, can adopt Ni2
+Chelating HP medium carries out purifying.According to Life Science News 8,2001Innovations Forum, the inclusion body purification-refolding method of Amersham Pharmacia Biotech issue, obtained soluble fusion protein, after cutting with the enteropeptidase enzyme, discharged target protein, but target protein there is not activity yet.Other two kinds of expression system expressed products are also all like this.In a word, fail in E.coli, to have given expression to active batroxobin protein.In view of the above, the resulting activated composition of investigator of inferring Japanese rattan pool drugmaker may be residual zymoplasm.
Embodiment 3:
The purifying of batroxobin preparation in the fermented supernatant fluid
After regulating fermented supernatant fluid and make PH be 4.0 with Glacial acetic acid, make specific conductivity be≤8mS/cm by the 1:20 dilution fermented liquid with distilled water again, this fermentation diluent is gone up sample earlier to be arrived through 25mM acetic acid-sodium acetate buffer, the SP-Sepharose FF that the PH4.0 pre-balance is crossed (Amersham PharmaciaBiotech) post, adopt gradient elution, obtain the target protein coarse-grain, be 1M glycine-sodium hydroxide solution of 10.0 again with distilled water and PH, to contain the SP-SepharoseFF elutriant dilution of batroxobin, be adjusted to 50mM glycine-sodium hydroxide buffering, PH10.0, the sample-loading buffer of specific conductivity≤3.5mS/cm, go up again through 50mM glycine-sodium hydroxide buffering, the Q-Sepharose FF of PH10.0 pre-equilibration (Amersham Pharmacia Biotech) post, adopt the mode of downward modulation PH, carry out gradient elution, make the acetic acid-sodium acetate buffer of target protein at 100mM, wash-out among the PH4.0 is collected by S200 (Pamersham Pharmacia Biotech) substep more at last and just can be obtained purity and be not less than 97% pure product of batroxobin (scheming .4).The PH gradient elution adopts the buffered soln of following volumetric molar concentration and PH respectively:
25mMNa2HP04-NaH2P04，PH8.8
50mMNa2HP04-NaH2P04，PH7.0
100mMHAc-AcNa，PH5.0
The mode that adopts the damping fluid volumetric molar concentration to increase progressively can guarantee that the conversion of PH gradient is obvious, reduces the conditions of streaking that may occur in the elution process, and the buffering of high density has also been brought into play the eluting effect of salt (ionic strength) simultaneously.
Embodiment 4:
The renaturation of folding wrong batroxobin protein in the fermented supernatant fluid
Find when being described to the yeast expression batroxobin among the embodiment 2 that along with induction time prolongs, the protein band in batroxobin protein molecular size corresponding position obviously thickens, but active not increase.Infer that thus these albumen seemingly fold wrong batroxobin in the secretion process.For confirming the correctness of this supposition, the following reagent of special employing carries out the renaturation test to containing this proteic solution.
Concrete renaturation manipulation: before the renaturation, its batroxobin vigor is 30hrs (1800mins), when in the solution that contains the inactive protein band, adding following reagent, make final concentration as the concentration shown in the back is parenthetic, under 15 ℃ of conditions, secluding air, place about 12hrs after, measuring its batroxobin vigor is 30mins.
Na2HP04-NaH2P04，PH8.0(100mM)
Histidine (Histidine) (10mM)
GSH (reduced glutathion) (0.5mM)
GSSG (Sleep-promoting factor B) (0.3mM)
The aqueous solution that contains mentioned component is not measured activity, and this shows that activity is by the generation after the inactive protein renaturation, rather than the illusion that produces of the composition that is added.
Embodiment 5: the glycosylation structure determines in the batroxobin protein molecule
Calculating this proteic molecular weight according to batroxobin aminoacid sequence data is 25.5kD, and the apparent molecular weight that the SDS-PAGE electrophoresis draws is between 30～32Kd.With the batroxobin that we are purified into, mass spectroscopy measures accurately that molecular weight is 31.8kD.
With reference to the methanol yeast expression system when the foreign protein of expressing, as long as having in the protein molecular primary structure that N-is glycosylated may the site, generally all can N-type glycosylation phenomenon.Commercially available protein enzyme PNGase F (enzyme of N-sugar chain in the cutting glycoprotein) with New EnglandBioLabs company, operate according to the method in the working instructions, find behind the batroxobin protein SDS-PAGE electrophoresis before and after will handling: the batroxobin molecular weight after PNGaseF handles has reduced, and size is with the product suitable (Fig. 4) of intestinal bacteria non-fusion expression.This batroxobin intramolecularly that shows that yeast expression goes out has modifications such as N-glycosylation.
Protein and dna sequence dna are described
The data of SEQ-1 sequence:
(1). sequence signature:
A. length: 231 amino acid
B. type: amino acid
C. topology: the unknown
(2). molecule type: protein
(3). sequence description: SEQ-1
1?VIGGDECDIN?EHPFLAFMYY?SPRYFCGMTL INQEWVLTAA?HCNRRFMRIH
51?LGKHAGSVAN?YDEVVRYPKE?KFICPNKKKN VITDKDIMLI?RLDRPVKNSE
101?HIAPLSLPSN?PPSVGSVCRI?MGWGAITTSE DTYPDVPHCA?NINLF
NNTVC
151?REAYNGLPAK?TLCAGVLQGG?IDTCGGDSGG PLICNGQFQG?ILSWGSDPCA
201?EPRKPAFYTK?VFDYLPWIQS?IIAG
NKTATC?P
The data of SEQ-2 sequence
(1). sequence signature:
A. length: 693bp
B. type: nucleic acid
C. topology: linearity
(2). molecule type: the DNA of synthetic
(3). sequence description: SEQ-2
1?GTTATCGGTG?GTGATGAATG?TGATATTAAC?GAACATCCAT?TCTTGGCCTT
51?TATGTATTAT?TCTCCACGTT?ATTTCTGTGG?TATGACCTTG?ATTAACCAAG
101?AGTGGGTTTT?GACCGCCGCC?CATTGTAACC?GTCGTTTTAT?GCGTATTCAT
151?TTGGGTAAAC?ATGCCGGTTC?TGTTGCCAAC?TATGATGAAG?TTGTTCGTTA
201?TCCAAAAGAA?AAGTTTATTT?GTCCAAACAA?GAAAAAGAAC?GTTATTACCG
251?ATAAAGATAT?CATGTTGATT?CGTTTGGATC?GTCCAGTTAA?AAACTCTGAA
301?CATATTGCCC?CATTGTCTTT?GCCATCTAAC?CCACCATCTG?TTGGTTCTGT
351?TTGTCGTATT?ATGGGTTGGG?GTGCCATTAC?CACCTCTGAA?GATACCTATC
401?CAGATGTTCC?ACATTGTGCC?AACATTAACT?TGTTTAACAA?CACCGTTTGT
451?CGTGAAGCCT?ATAACGGTTT?GCCAGCCAAA?ACCTTGTGTG?CCGGTGTTTT
501?GCAGGGTGGT?ATCGATACCT?GTGGTGGTGA?TTCTGGTGGT?CCATTGATTT
551?GTAACGGTCA?GTTCCAGGGT?ATTTTGTCTT?GGGGTTCTGA?TCCATGTGCC
601?GAACCACGTA?AACCAGCCTT?TTATACCAAA?GTTTTTGATT?ATTTGCCTTG
651?GATTCAGTCT?ATTATTGCCG?GTAACAAAAC?CGCCACCTGT?CCA
The data of SEQ-3
(1). sequence signature:
A. length: 693bp
B. type: nucleic acid
C. topology: linearity
(2). molecule type: the DNA of synthetic
(3). sequence description: SEQ-3
1?GTTATTGGTG?GTGATGAATG?TGATATTAAC?GAACATCCAT?TTTTGGCTTT
51?TATGTACTAC?TCTCCAAGAT?ACTTTTGTGG?TATGACTTTG?ATTAACCAAG
101?AATGGGTTTT?GACTGCTGCT?CATTGTAACA?GAAGATTTAT?GAGAATTCAT
151?TTGGGTAAGC?ATGCTGGTTC?TGTTGCTAAC?TACGATGAAG?TTGTTAGATA
201?CCCAAAGGAA?AAGTTTATTT?GTCCAAACAA?GAAGAAGAAC?GTTATTACTG
251?ATAAGGATAT?TATGTTGATT?AGATTGGATA?GACCAGTTAA?GAACTCTGAA
301?CATATTGCTC?CATTGTCTTT?GCCATCTAAC?CCACCATCTG?TTGGTTCTGT
351?TTGTAGAATT?ATGGGTTGGG?GTGCTATTAC?TACTTCTGAA?GATACTTACC
401?CAGATGTTCC?ACATTGTGCT?AACATTAACT?TGTTTAACAA?CACTGTTTGT
451?AGAGAAGCTT?ACAACGGTTT?GCCAGCTAAG?ACTTTGTGTG?CTGGTGTTTT
501?GCAAGGTGGT?ATTGATACTT?GTGGTGGTGA?TTCTGGTGGT?CCATTGATTT
551?GTAACGGTCA?ATTTCAAGGT?ATTTTGTCTT?GGGGTTCTGA?TCCATGTGCT
601?GAACCAAGAA?AGCCAGCTTT?TTACACTAAG?GTTTTTGATT?ACTTGCCATG
651?GATTCAATCT?ATTATTGCTG?GTAACAAGAC?TGCTACTTGT?CCA
The data of SEQ-4 sequence
(1). sequence signature:
A. length: 26 amino acid
B. type: amino acid
C. topology: linearity
(2). molecule type: signal peptide
(3). sequence description: SEQ-4
1?MVLIRVIANL?LILQVSYAQK?SSELKR
The data of SEQ-5 sequence
(1). sequence signature:
A. length: 78bp
B. type: nucleic acid
C. topology: linearity
(2). molecule type: the DNA of synthetic
(3). sequence description: SEQ-5
1?ATGGTTTTGA?TTAGAGTTAT?TGCTAACTTG?TTGATTTTGC?AAGTTTCTTA
51?CGCTCAAAAG?TCTTCTGAAT?TGAAGAGA
Claims (8)
1, a kind of recombinant batroxobin is characterized in that: it is made by engineered method, and this method comprises following step:
(1), gene is synthetic: according to the aminoacid sequence of batroxobin in the Brazilian fer-de-lanc venom, select the used codon of batroxobin structure gene, the used codon of described batroxobin structure gene is the codon that yeast or intestinal bacteria are all compared preference, adopt the method for synthetic, obtain batroxobin structural gene sequence SEQ-2; Or adopt the method for same synthetic, obtain fully the molecular batroxobin structural gene sequence of password SEQ-3 by the methanol yeast hobby.
(2), the structure of expression vector: 5 of batroxobin gene '-end adds the point of contact of restriction enzyme Xho I, 3 '-two terminator codons of end interpolation TAA TGA and Not I point of contact, point of contact and the batroxobin protein N-at Xho I holds adding yeast KEX between first amino acid Val codon in addition
2The codon AAA AGA of protease recognition sequence Lys-Arg correspondence inserts this dna fragmentation in pPICZ α A or the pMET α A carrier, gets pPICZ α A-Bg or pMET α A-Bg expression vector, and the clone cultivates, and extracts plasmid;
(3), the expression of batroxobin: with expression vector pPICZ α A-Bg or pMET α A-Bg linearizing, prepare the competence of yeast host bacterium with DNA restriction restriction endonuclease, and carry out the electricity conversion; The clone, and express, screen, the high expression level bacterial strain obtained;
(4), the purifying of going up jar fermentation and expression product of batroxobin engineering bacteria.
2, a kind of preparation method of recombinant batroxobin is characterized in that: it is made by engineered method, and this method comprises following step:
(1), gene is synthetic: according to the aminoacid sequence of batroxobin in the Brazilian fer-de-lanc venom, select the used codon of batroxobin structure gene, the used codon of described batroxobin structure gene is the codon that yeast or intestinal bacteria are all compared preference, adopt the method for synthetic, obtain batroxobin structural gene sequence SEQ-2; Or adopt the method for same synthetic, obtain fully the molecular batroxobin structural gene sequence of password SEQ-3 by the methanol yeast hobby.
(2), the structure of expression vector: 5 of batroxobin gene '-end adds the point of contact of restriction enzyme Xho I, 3 '-end adds two terminator codons of TAA TGA and Not I point of contact, and point of contact and the batroxobin protein N-at Xho I holds adding yeast KEX between first amino acid Val codon in addition
2The codon AAA AGA of protease recognition sequence Lys-Arg correspondence inserts this dna fragmentation in pPICZ α A or the pMET α A carrier, gets pPICZ α A-Bg or pMET α A-Bg expression vector, and the clone cultivates, and extracts plasmid enzyme restriction and obtains the clone;
(3), the expression of batroxobin: limit restriction endonuclease with expression vector pPICZ α A-Bg or pMET α A-Bg linearizing with DNA; The competence of preparation yeast host bacterium, and carry out the electricity conversion; The clone, and carry out expression screening, obtain the high expression level bacterial strain;
(4), the purifying of going up jar fermentation and expression product of batroxobin engineering bacteria.
3, the preparation method of a kind of recombinant batroxobin according to claim 2, it is characterized in that: described batroxobin engineering bacteria is methanol yeast engineering bacteria (pichia pastoris) CCTCC M203006, with a large amount of batroxobins that biologic activity is arranged that produce of the mode of secreting, expressing.
4, the preparation method of a kind of recombinant batroxobin according to claim 3, it is characterized in that: in yeast during secreting, expressing, utilize zymic α-signal peptide, or the pairing polynucleotide sequence SEQ-5 of the natural signals peptide that utilizes Brazilian fer-de-lanc batroxobin, wherein be by yeast KEX between signal peptide sequence and the batroxobin protein
2Protease recognition sequence is connected.
5, the preparation method of a kind of recombinant batroxobin according to claim 4 is characterized in that: the pairing polynucleotide sequence SEQ-5 of the natural signals peptide of described batroxobin is the codon synthetic that adopts pasteur Bi Shi methanol yeast to be liked.
6, the preparation method of a kind of recombinant batroxobin according to claim 2, it is characterized in that: fermented supernatant fluid is diluted or ultrafiltration desalination pre-treatment after, through cation exchange medium, pass through anionic exchange medium more earlier, prepare the pure product of batroxobin by gel permeation chromatography at last.
7, the degraded plasma fibrin medicine made of recombinant batroxobin according to claim 1.
8, the application of recombinant batroxobin according to claim 1 in preparation hemostatic drug.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101705240B (en) * | 2009-07-23 | 2012-07-04 | 扬子江药业集团北京海燕药业有限公司 | Synthesis of batroxobin gene and preparation method of expression product thereof |
| CN101952423B (en) * | 2007-12-28 | 2014-03-12 | Biobud有限公司 | Mutated nucleotide sequences of batroxobin, mutated alpha factor secretion signal sequence and processes for preparing batroxobin using same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100424172C (en) * | 2005-09-30 | 2008-10-08 | 沈阳守正生物技术有限公司 | Oriented mutant gene engineering barr kinase and its use |
| CN100494366C (en) | 2006-10-19 | 2009-06-03 | 康辰医药股份有限公司 | Thrombin |
| CN100564532C (en) * | 2006-12-18 | 2009-12-02 | 中国人民解放军军事医学科学院生物工程研究所 | A kind of batroxobin and preparation method thereof and own coding gene |
| CN101274096B (en) * | 2007-03-29 | 2011-04-27 | 上海万兴生物制药有限公司 | Stable Batroxobin medicament composition |
| AU2007350619B2 (en) | 2007-03-30 | 2013-12-19 | Shanghai Tenry Pharmaceutical Co., Ltd. | A purified recombinant batroxobin with high specific activity |
| CN101319207B (en) * | 2007-06-06 | 2010-12-29 | 沈阳守正生物技术有限公司 | Site-directed mutagenesis genetic engineering batroxobin and uses thereof |
| CN112062863B (en) * | 2020-09-18 | 2023-10-20 | 重庆医科大学 | Fusion protein of activated blood coagulation factor XIIIA and batroxobin BTX as well as construction method and application thereof |
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| US3849252A (en) * | 1971-01-18 | 1974-11-19 | Pentapharm Ag | Enzyme composition and process for the manufacture thereof |
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| US3849252A (en) * | 1971-01-18 | 1974-11-19 | Pentapharm Ag | Enzyme composition and process for the manufacture thereof |
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| Experssion of cdna for batroxobin ,a thrombin-like snakevenom enxyme Msahiro Maeda et al,J.Bilchem,Vol.109 1991 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101952423B (en) * | 2007-12-28 | 2014-03-12 | Biobud有限公司 | Mutated nucleotide sequences of batroxobin, mutated alpha factor secretion signal sequence and processes for preparing batroxobin using same |
| CN101705240B (en) * | 2009-07-23 | 2012-07-04 | 扬子江药业集团北京海燕药业有限公司 | Synthesis of batroxobin gene and preparation method of expression product thereof |
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