CN101591660A - A kind of in intestinal bacteria the method for excreting and expressing recombinant human granulocyte-colony factor - Google Patents
A kind of in intestinal bacteria the method for excreting and expressing recombinant human granulocyte-colony factor Download PDFInfo
- Publication number
- CN101591660A CN101591660A CNA2008101131387A CN200810113138A CN101591660A CN 101591660 A CN101591660 A CN 101591660A CN A2008101131387 A CNA2008101131387 A CN A2008101131387A CN 200810113138 A CN200810113138 A CN 200810113138A CN 101591660 A CN101591660 A CN 101591660A
- Authority
- CN
- China
- Prior art keywords
- gcsf
- signal peptide
- expression
- protein
- recombinant human
- 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.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 21
- 241000894006 Bacteria Species 0.000 title claims abstract description 10
- 230000000968 intestinal effect Effects 0.000 title abstract description 5
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 48
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 35
- 230000014509 gene expression Effects 0.000 claims abstract description 22
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 14
- 210000004027 cell Anatomy 0.000 claims abstract description 8
- 239000000284 extract Substances 0.000 claims description 3
- 230000006698 induction Effects 0.000 claims description 3
- 210000003714 granulocyte Anatomy 0.000 claims 1
- 230000004936 stimulating effect Effects 0.000 claims 1
- 230000028327 secretion Effects 0.000 abstract description 7
- 210000001322 periplasm Anatomy 0.000 abstract description 5
- 230000003248 secreting effect Effects 0.000 abstract description 4
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 abstract 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract 1
- 101000746367 Homo sapiens Granulocyte colony-stimulating factor Proteins 0.000 description 30
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 28
- 235000018102 proteins Nutrition 0.000 description 28
- 239000013612 plasmid Substances 0.000 description 13
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 9
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 9
- 239000013604 expression vector Substances 0.000 description 8
- 241000588724 Escherichia coli Species 0.000 description 6
- 238000004153 renaturation Methods 0.000 description 6
- 238000013461 design Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 4
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 210000003000 inclusion body Anatomy 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000004587 chromatography analysis Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 230000009465 prokaryotic expression Effects 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 229910052742 iron Inorganic materials 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 201000010000 Agranulocytosis Diseases 0.000 description 1
- YCRAFFCYWOUEOF-DLOVCJGASA-N Ala-Phe-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 YCRAFFCYWOUEOF-DLOVCJGASA-N 0.000 description 1
- DCVYRWFAMZFSDA-ZLUOBGJFSA-N Ala-Ser-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O DCVYRWFAMZFSDA-ZLUOBGJFSA-N 0.000 description 1
- 208000032467 Aplastic anaemia Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 235000007926 Craterellus fallax Nutrition 0.000 description 1
- 101710095468 Cyclase Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 240000007175 Datura inoxia Species 0.000 description 1
- 206010018687 Granulocytopenia Diseases 0.000 description 1
- RTSQPLLOYSGMKM-DSYPUSFNSA-N Ile-Trp-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(C)C)C(=O)O)N RTSQPLLOYSGMKM-DSYPUSFNSA-N 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- HAQLBBVZAGMESV-IHRRRGAJSA-N Met-Lys-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(O)=O HAQLBBVZAGMESV-IHRRRGAJSA-N 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 239000012506 Sephacryl® Substances 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 239000004098 Tetracycline Substances 0.000 description 1
- XOCUXOWLYLLJLV-UHFFFAOYSA-N [O].[S] Chemical compound [O].[S] XOCUXOWLYLLJLV-UHFFFAOYSA-N 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000010322 bone marrow transplantation Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000000247 postprecipitation Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011112 process operation Methods 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000013094 purity test Methods 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 229960002180 tetracycline Drugs 0.000 description 1
- 229930101283 tetracycline Natural products 0.000 description 1
- 235000019364 tetracycline Nutrition 0.000 description 1
- 150000003522 tetracyclines Chemical class 0.000 description 1
- 108010087967 type I signal peptidase Proteins 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
Images
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind ofly at intestinal bacteria secretion type expression recombinant human granulocyte-colony factor (rHG-CSF), this carrier is by adding DsbA signal peptide (SEQ ID NO:1) at the N of foreign protein end, and uses λ P
LPromotor can make up a kind of carrier of pericentral siphon solinocrine type express recombinant protein, and transformed into escherichia coli DH5 α competent cell is expressed in the colibacillus periplasm chamber by temperature-induced realization foreign protein.This method expression amount height, secernment efficiency height are applicable to secreting, expressing various exogenous genes efficiently and stably.
Description
Technical field
The present invention relates to a kind of colibacillus periplasm solinocrine type express recombinant human granulocyte-colony factor (G-CSF) method, more specifically, relate to a kind of the end and add DsbA signal peptide (MKKIWLALAGLVLAFSASA (SEQID NO:1)) by N at G-CSF, and the new expression vector that foreign protein is expressed in the colibacillus periplasm chamber; The explanation of this expression vector and application.
Technical background
Genetically engineered recombinant methionyl human G-CSF (rhG-CSF), be mainly used in the granulocytopenia after cancer patients's chemotherapy. be used for the oligoleukocythemia of leukemia, acquired immune deficiency syndrome (AIDS) of autologous bone marrow transplantation, some type and aplastic anemia etc. in addition, all show to have significant curative effect.Mostly by escherichia coli expression, though expression amount is high, form inclusion body at present, need just can obtain activated protein through the sex change renaturation, yield is very low, and N end methionine(Met) can not be removed.
Adopt prokaryotic expression system (mainly being coli expression system) to have the advantage of many uniquenesses., cell clear such as genetic background growth rapidly, fermentation period is short, be easy to screening and gene recombination operation, expression system is than horn of plenty etc.But also there are some shortcomings in prokaryotic expression system: can't glycosylation and be easy to form inclusion body etc. as foreign protein.The inclusion body that foreign protein forms must pass through sex change, fold renaturation again, just can obtain active protein product.The said process operation is loaded down with trivial details, annealing efficiency is low, need be optimized respectively for different foreign proteins.Therefore,, in application in practice, will increase work efficiency greatly so, save production cost if can adopt prokaryotic expression system to realize soluble-expression or the expression of pericentral siphon solinocrine in the born of the same parents of foreign protein.
People taked multiple mode to promote the soluble-expression of foreign protein.With gsh (GST) or sulphur oxygen cyclase protein amalgamation and expression, can play short solvable effect to the part foreign protein; Utilize protein disulfide to form the folding assisted effect of associated protein, cis-trans propyl isomerism enzyme and other molecular chaperoneses, design expression vector, can obviously promote the interior soluble-expression of born of the same parents of foreign protein.But, the soluble-expression mode also has apparent in view shortcoming in the born of the same parents: belong to the reductibility environment in the born of the same parents, be unfavorable for that foreign protein forms correct disulfide linkage bridge, and then influence the formation of correct structure picture, and need ultrasonication or pressure breaking cell walls could discharge soluble-expression component in the born of the same parents, be unfavorable for large scale culturing and purifying.Comparatively speaking, because the pericentral siphon chamber belongs to the oxidized form environment, help forming correct disulfide linkage bridge, and extract and do not need fracturing cell walls when component is expressed in the pericentral siphon chamber, can reduce the purifying cost significantly, be a kind of ideal phraseology so pericentral siphon solinocrine type is expressed.Usually foreign protein and appropriate signals peptide are merged, foreign protein is secreted in the pericentral siphon chamber by the guiding function of signal peptide.The copy number of control expression vector is selected suitable promotor and substratum, adopts suitable abduction delivering mode and culture condition (culture temperature, induce opportunity and induction time), can improve the efficient of pericentral siphon solinocrine type expression.
Experiment showed, the G-CSF that is connected to behind the signal peptide, realize that not only pericentral siphon solinocrine type expresses, the G-CSF of expression need not renaturation obtain activated product, and correct through the order-checking protein structure, N-holds no unnecessary amino acid.The principle of secreting, expressing is: purpose G-CSF encoding sequence is fused to after the signal coding sequence, expression product is when secreting to born of the same parents' pericentral siphon (periPlasmic space) by the Bacillus coli cells inner membrance, signal peptide can be by correct identification of signal peptidase and excision.Foreign protein after the secretion does not contain N end methionine(Met), and space folding (comprise and form disulfide linkage) one-tenth has had the activated protein of native conformation, need not follow-up renaturation process, thereby simplified production technique. in addition, the foreign protein that is accumulated in born of the same parents' pericentral siphon has been avoided the degraded of some proteolytic enzyme in the cell, thereby more stable.
Summary of the invention
Purpose of the present invention just provides the method that a kind of prokaryotic secretion is expressed G-CSF, and the versatility of this method is good, expression amount is high, secernment efficiency is high, is applicable to secreting, expressing various exogenous genes efficiently and stably, as G-CSF, IL-2 etc.
The present invention relates to recombinant DNA technology, relate more specifically to the construction process that a kind of new prokaryotic secretion is expressed the G-CSF plasmid.The invention still further relates to prokaryotic secretion and express expression and the extracting method of G-CSF.
Description of drawings
The structure iron of Fig. 1 pDsbA-GCSF.
The structure iron of Fig. 2 pBV-DsbA-GCSF.
Fig. 3 is the chamber protein electrophoresis between week
When expressing GCSF, gene is inserted into the downstream of DsbA sequence, this is so that during expressing gene, expressed peptide chain is by DsbA Signal peptide is directed in the pericentral siphon chamber and is folded to form activated protein. Selected in the method the strong terminator sequence of rrnB, and also had steady Tailor-made using. As for elements such as origin of replication ori and marker gene (such as resistant gene), without any particular limitation. Such as, use Can select tetracycline resistance gene, neomycin resistance gene or ammonia joint penicillin resistance gene etc. in the resistant gene of screening.
In another aspect of the present invention, provide a kind of host of containing general pericentral siphon solinocrine type expression vector pDsbA thin Born of the same parents, commonly used is bacillus coli DH 5 alpha, is coli strain W3110 best.
Carrier provided by the present invention has following characteristics:
1. the short folded signal peptide DsbA of coexpression effectively promotes correct fold of GCSF in the pericentral siphon chamber, improves the activated protein expression level;
2. when adopting this carrier to carry out the expression of pericentral siphon solinocrine type, do not need broken bacteria cell wall, directly extract pericentral siphon chamber component and promptly can gather in the crops target protein.
3. expressed proteins all is the activated protein that function is arranged by this way, does not need complex steps and extremely low sex change and the renaturation process of efficient, only needs comparatively simple purification step just can obtain a large amount of foreign proteins;
4. this expression vector is expressive host with intestinal bacteria, but bulk fermentation production, production technique is simple, and operation is with low cost easily.
Embodiment
In an example of the present invention, in order to obtain to make up several members of secreted expression carrier, at first according to known λ P
LThe sequence of promotor, the synthetic PCR primer of use, increasing from escherichia coli vector pBLV carrier with the method for PCR obtains λ P
LPromotor.Sequences Design according to DsbA is extended primer, with existing GCSF is template, with the N end of pcr amplification to GCSF, DsbA-GCSF fragment after pBR322 fragment after two ends design restriction enzyme site is cut suitable enzyme then, promoter sequence and suitable enzyme are cut links together, and has just formed a kind of new GCSF secretion expression carrier pDsbA-GCSF.Also DsbA-GCSF is building up to simultaneously the pBv220 carrier and forms pBV-DsbA.Two kinds of plasmids are transformed DH5 α respectively.
For the transformed host cells that filters out, can under the usual culture condition that uses in this area, cultivate to express GCSF, wherein very important to the control of the OD value before inducing, suitable induced concentration can make the GCSF expression rate higher.Cultivate the OD value when the 0.8-1.0 when 30 ℃, elevated temperature to 42 ℃ is induced and was collected thalline in 6 hours rapidly, and GCSF has preferably and expresses.
Embodiment 1
The sequence of λ PL promotor is synthesized and is used the PCR primer, clones from escherichia coli vector pBLV with the method for PCR to obtain, and be that template is carried out pcr amplification with it.Used upstream primer is: 5 ' GTGAATTCAGATCTCTCACCTACCAAAC3 ' (containing EcoRI), downstream primer is: 5 ' GTCATATGACTAGTCCTCCTTAATTTTTAACCAATG 3 ' (containing the Spel site).The PCR product is behind the EcoRI/Spel double digestion, and low melting point glue reclaims standby.
Embodiment 2
People GCSF gene is that my company is existing, design primer PCR from former preservation plasmid obtains, primer is extended in two of upstream designs, primer 1:5 '-GGT TTA GTT TTA GCG TTT AGC GCA TCG GCGACACCATTAGGCCCTGCCAGC-3 ', primer 2: 5 '-GAGGAATTCATG AAA AAG ATT TGG CTGGCG CTG GCT GGT TTA GTT TTA GCG TTT AGC-3 ' downstream primer, 5 '-GCGGGATCCTCACGGCTGGGCAAG-3 ', the fragment that amplifies is about 600bp.Second GCA of 5 ' end links to each other with last amino acid whose coding DNA of DsbA signal peptide, thereby first amino acid of coding people GCSF maturation protein is (Thr), therefore signal peptide excision back excretory GCSF N-terminal does not contain Met, and consistent with natural GCSF.The aminoacid sequence of signal peptide is: Met Lys Lys Ile Trp Leu Ala Leu Ala Gly Leu Val Leu Ala Phe Ser Ala SerAla
Embodiment 3
The structure of pBV-DsbA-GCSF plasmid
In this embodiment, make up the plasmid that contains DsbA-GCSF sequence and rmB terminator sequence. that the rmB sequence is provided is plasmid pBv220, and this is a kind of known expression plasmid.The heterologous protein sequence that will express in this plasmid is placed in the downstream of PRPL promotor, and this promotor can make and be positioned at its downstream albumen sequence table and reach when thermal induction.Cut among the plasmid pBV220 with BamHI/SaLI is two, form plasmid pBV-DsbA-GCSF.The structure of PBV-DsbA-GCSF plasmid as shown in Figure 2, what be close to DsbA-GCSF sequence downstream is exactly the rrnB terminator sequence.
Embodiment 4
The structure of expression vector pDsbA with EcoRI/SpeI double digestion plasmid pBR322, is isolated big fragment as shown in Figure 4 then.Plasmid pBV-DsbA-GCSF with obtaining among the EcoRI/SpeI double digestion embodiment 3 separates small segment then.Above-mentioned 2 kinds of isolating fragments are mixed by a certain percentage, connect with ligase enzyme and spend the night, thereby be built into plasmid pDsbA-GCSF.Then this recombinant expression vector is changeed people intestinal bacteria W3110, set up the engineering bacteria PDsbA-hGCSF/W3110 of people GCSF secretion type expression.
For the transformed host cells that filters out, can under the usual culture condition that uses in this area, cultivate to express GCSF, wherein very important to the control of the OD value before inducing, suitable induced concentration can make the GCSF expression rate higher.Cultivate the OD value when the 0.8-1.0 when 30 ℃, elevated temperature to 42 ℃ is induced and was collected thalline in 6 hours rapidly, and GCSF has preferably and express (account between week chamber proteic more than 40%) as Fig. 3.
The extraction of GCSF and purifying
GCSF slightly carries: slightly carrying damping fluid is 10mMTris-HCL (pH7.4) (20% sucrose), and every mL adds 33ul0.5mMEDTA.Thick extracting method: take out thalline from-70 ℃, add the above-mentioned damping fluid of people 10ml by the wet bacterium of every gram, the suspension bacterium, be placed on 4 ℃ of vibration 1h, suspension is through 4 ℃, 12000g, 20min is centrifugal, and supernatant is collected in the back, and supernatant is behind the positive press filtration of 5um filter membrane, be the GCSF extracting solution. cross the cation-exchange chromatography post, pillar with sample on the 10ml/min flow velocity, is used the GCSF crude extract instead the 20ml/min flow velocity after the abundant balance of buffer A (20mMNaAC-HAC pH5.0), with 20% buffer B (IMNaCI, 20mMNaAC-HACpH5.0) wash post to the 280nm uv-absorbing to baseline, wash post with 30% buffer B again, collect the eluted protein peak.
Hydrophobic chromatography: filler is the Phemyl-sepharose HP of Pharmacia company.Post is through level pad A (1.5M (NH4) 2S04,20mM NaAC-HACpH5.0) abundant balance. add people's solid (NH4) 2S04 in the component that ion exchange chromatography obtains, making its final concentration is 1.5M, the centrifugal post precipitation that goes is with sample on the 5ml/min flow velocity, use the 10ml/min flow velocity instead, wash post to the 280nm uv-absorbing to baseline with 40% buffer B (20mM NaAC-HAC pH5.0), remake the 40-60%B linear gradient elution, collect GCSF activated protein peak.
Ultrafiltration and concentration:, on the Minitan ultra-fine filter, use PTGC filter membrane (MW10000) ultrafiltration and concentration to 90-100ml the GCSF active constituent that hydrophobic chromatography obtains.Gel-filtration: filler is the Sephacryl S-100 of Pharmacia company, and column volume is 5cm * 115cm.After the earlier abundant balance of pillar, with sample chromatography on the ultrafiltration and concentration sample, flow velocity is 60ml/h, collects GCSF activated protein peak.The evaluation of GCSF behind the purifying: to the GCSF behind the aforesaid method purifying, carry out the HPLC purity testing with ordinary method, the result records its purity greater than 98%.
In this embodiment, GCSF all is secreted into the Bacillus coli cells pericentral siphon more than 95% when the optimum condition bottom fermentation finishes, expression amount accounts for 40% of born of the same parents' periplasm protein total amount, the output of GCSF is 350-500mg in the 1L fermented liquid, and this is than so far for to exceeding nearly 5 times with the GCSF productive rate of inclusion body formal representation.In addition needn't renaturation, so technology is easy, and the activity of the GCSF that obtains is very high.
Claims (3)
1. a pericentral siphon solinocrine type is expressed the method for this stimulating factor of express recombinant human granulocyte colony, and its structure as shown in Figure 1.The N end that it is characterized in that protein gene contains signal peptide.
2. the expression method of claim 1, wherein said signal peptide is DsbA signal peptide SEQ ID NO:1, uses λ P
LPromotor utilizes the temperature adjusting induction exogenous gene to express.
3. the purposes of claim 1,2 described methods is characterized in that expressing in the cell pericentral siphon by signal peptide guiding recombinant human granulocyte-colony factor rHG-CSF, helps forming being dissolved with activated protein, need not break bacterium, directly extracts albumen from the pericentral siphon chamber.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101131387A CN101591660A (en) | 2008-05-28 | 2008-05-28 | A kind of in intestinal bacteria the method for excreting and expressing recombinant human granulocyte-colony factor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2008101131387A CN101591660A (en) | 2008-05-28 | 2008-05-28 | A kind of in intestinal bacteria the method for excreting and expressing recombinant human granulocyte-colony factor |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101591660A true CN101591660A (en) | 2009-12-02 |
Family
ID=41406540
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2008101131387A Pending CN101591660A (en) | 2008-05-28 | 2008-05-28 | A kind of in intestinal bacteria the method for excreting and expressing recombinant human granulocyte-colony factor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101591660A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154189A (en) * | 2010-12-31 | 2011-08-17 | 山东新时代药业有限公司 | Fermentation culture method of rhG-CSF (recombinant human granulocyte colony-stimulating factor) recombination engineering bacteria |
| CN102517260A (en) * | 2011-12-29 | 2012-06-27 | 中国人民解放军军事医学科学院野战输血研究所 | Fusion protein carrying NS3 preponderant area of non-structural protein of bovine viral diarrhea virus as well as recombinant expression method and application thereof |
| CN103044531A (en) * | 2012-09-29 | 2013-04-17 | 重庆原伦生物科技有限公司 | Purification method of methicillin-resistant staphylococcus aureus (MREA)-resistant vaccine recombinant protein antigen HI2 |
| CN103797122A (en) * | 2011-04-08 | 2014-05-14 | 安瑟生物科技私人有限公司 | Novel expression and secretion vector systems for heterologous protein production in escherichia coli |
-
2008
- 2008-05-28 CN CNA2008101131387A patent/CN101591660A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102154189A (en) * | 2010-12-31 | 2011-08-17 | 山东新时代药业有限公司 | Fermentation culture method of rhG-CSF (recombinant human granulocyte colony-stimulating factor) recombination engineering bacteria |
| CN102154189B (en) * | 2010-12-31 | 2015-11-11 | 鲁南制药集团股份有限公司 | A kind of fermentation culture method of rhG-CSF recombinant bacterial strain |
| CN103797122A (en) * | 2011-04-08 | 2014-05-14 | 安瑟生物科技私人有限公司 | Novel expression and secretion vector systems for heterologous protein production in escherichia coli |
| CN102517260A (en) * | 2011-12-29 | 2012-06-27 | 中国人民解放军军事医学科学院野战输血研究所 | Fusion protein carrying NS3 preponderant area of non-structural protein of bovine viral diarrhea virus as well as recombinant expression method and application thereof |
| CN103044531A (en) * | 2012-09-29 | 2013-04-17 | 重庆原伦生物科技有限公司 | Purification method of methicillin-resistant staphylococcus aureus (MREA)-resistant vaccine recombinant protein antigen HI2 |
| CN103044531B (en) * | 2012-09-29 | 2014-07-30 | 重庆原伦生物科技有限公司 | Purification method of methicillin-resistant staphylococcus aureus (MREA)-resistant vaccine recombinant protein antigen HI2 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR100195632B1 (en) | Peptide and DNA Arrays | |
| US5665863A (en) | Polypeptides having granulocyte colony stimulating activity, their preparation and pharmaceutical compositions containing them | |
| JP3085973B2 (en) | Application of a novel DNA fragment as a sequence encoding a signal peptide related to secretion of mature protein by recombinant yeast, expression cassette, transformed yeast and method for producing corresponding protein | |
| JPH04502861A (en) | Production of heterologous proteins in plants and plant cells | |
| JP5868999B2 (en) | Cost-effective method for expressing and purifying recombinant proteins in plants | |
| US20020194643A1 (en) | Method for producing hemin proteins using plant cells, resulting proteins and products containing same | |
| JPS6236183A (en) | Ugenoyobibunpi | |
| CN101591660A (en) | A kind of in intestinal bacteria the method for excreting and expressing recombinant human granulocyte-colony factor | |
| Ayed et al. | High level production and purification of human interferon α2b in high cell density culture of Pichia pastoris | |
| JP3433807B2 (en) | Method for producing a useful substance using Bacillus brevis incorporating an expression vector carrying a novel amino acid sequence and a DNA encoding the amino acid sequence | |
| EP3027645A2 (en) | Process for production of insulin and insulin analogues | |
| JP2001500388A (en) | Method for producing recombined protein from Saccharomyces cerevisiae using a highly efficient expression vector | |
| CN101280019A (en) | Fusion protein of human serum albumin and human granulocyte colony-stimulating factor mutant and its preparation | |
| CN111320702A (en) | Method for efficient secretion fusion expression and recombinant preparation of bacillus prodigiosus nuclease in methanol yeast | |
| CN101063124A (en) | Preparation method for fused protein of human interleukin-11 and human serum albumins and its product | |
| CN101538318B (en) | A kind of signal peptide and its coding gene and application | |
| EA005586B1 (en) | Fused protein and process for producing recombinant insulin | |
| CN103966253A (en) | Method for efficiently preparing recombinant human interluekin-33 protein | |
| CN102277371A (en) | Method for preparing BNP (brain natriuretic peptide) | |
| Lasnik et al. | Human granulocyte colony stimulating factor (hG-CSF) expressed by methylotrophic yeast Pichia pastoris | |
| AU621051B2 (en) | Method for purifying granulocyte-macrophage colony stimulating factor | |
| CN102485890B (en) | With Pichia sp. excreting and expressing recombinant human protein disulfide isomerase | |
| CN112646044B (en) | TFF2-Fc fusion protein and high-efficiency expression production method thereof | |
| EP0171000A2 (en) | Process for producing heterologous matured protein or peptide | |
| US20230002468A1 (en) | N-terminal extension sequence for expression of recombinant therapeutic peptides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20091202 |