CN1111602C - A kind of method of using molecule mate to promote protein secretion - Google Patents
A kind of method of using molecule mate to promote protein secretion Download PDFInfo
- Publication number
- CN1111602C CN1111602C CN98110840A CN98110840A CN1111602C CN 1111602 C CN1111602 C CN 1111602C CN 98110840 A CN98110840 A CN 98110840A CN 98110840 A CN98110840 A CN 98110840A CN 1111602 C CN1111602 C CN 1111602C
- Authority
- CN
- China
- Prior art keywords
- gene
- stimulating factor
- colony stimulating
- macrophage colony
- expression vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention relates to utilize molecular chaperones and desired protein at the prokaryotic cell prokaryocyte coexpression in the escherichia coli host particularly, to promote the method for desired protein secreting, expressing.Specifically, the present invention relates to be used for the recombinant expression vector at prokaryotic cell prokaryocyte secreting, expressing desired protein, said expression vector contains the site after the coding molecule companion's that is operably connected gene order to the first promotor.There is the coding desired protein gene order that is operably connected in coding molecule chaperone sequence downstream to the signal peptide dna sequence dna that contains second promotor.
Description
The present invention generally relates to and utilizes molecular chaperones and the coexpression of desired protein in the prokaryotic organism body, with the method for the desired protein of production solubility.Specifically, the present invention relates to use can be in the prokaryotic organism body the new recombinant expression vector of secreting, expressing desired protein, carry the bacterial cell of said new recombinant expression vector, and the coexpression that utilizes molecular chaperones and target protein matter, with the method for secreting, expressing form production solubility desired protein.
In recent years, deep research has been carried out in the effect of molecular chaperones, and found that it participates in the not many total biological function of organism of the same race, for example form and keep proteinic higher structure, the proteinic film of striding transports, regulates cell Growth and Differentiation and growth cycle to regulate (Ellis such as functions of immune system, R.J.et al, Molecular Chaperons, Annu.Rev.Biochem., 60:321-347,1991).
Most of secretory proteins of organism, comprise the protein in inner membrane protein, outer membrane protein and the periplasmic space, and be secreted into the ad hoc structure that proteinic N end in the extracellular environment and C end have all had the secretion signal effect, for example the N of secretory protein holds and has about 20 to 30 the amino acid signal peptide sequences of length.The aminoterminal of signal peptide sequence is generally positively charged amino acid, and the participation precursor molecule combines with plasma membrane.Thereafter for highly hydrophobic, (particularly under the condition near stain remover or the lipid molecule) nonpolar amino acid that highly forms α spiral trend arranged.Some constitutional featuress of these and other of signal peptide sequence, the film of striding that not only directly influences precursor molecule is secreted and its correct cutting, and has the suitable conformation that helps mature protein.On the other hand, the structure of sophisticated protein molecule to exocytosis thereafter also have material impact (positive forever male, water chestnut natural pond literary composition man. protein nucleic acid ferment, 1992; 37 (3): 223).These protein are secreted into the extracellular, must keep its specific film excretory conformation that is suitable for by the effect of certain factor and/or enzyme.
Studies show that in recent years, the closely-related excreted factor of proteinic secretion (Schatz, P.J.et al., Annu.Rev.Genet.24:215-248,1990 in some and the thalline in the intestinal bacteria; Hajime Tokuda 1993, protein nucleic acid ferment, 38:947-956), these excreted factor and signal peptidase and some other known and unknown factor interactions are participated in the film of wearing of intracellular protein and are transported and the course of processing.In fixed at least seven kinds of Sec genes, Sec B is as a member of its family, plays a significant role in proteinic transmembrane transport system.
Sec B is present in the endochylema with tetramer form, by the protein that 155 amino acid are formed, is a kind of molecular chaperones (Kumamoto C.A.et al., Mol.Microbiol., 5:19-22,1991).It can combine with great majority synthetic secretary protein on rrna, makes it to keep its loose activity conformation folding, that be suitable for transmembrane transport.Current body protein is that Sec A combines with another family member with ATP enzymatic structure feature promptly be transported to inner membrance under Sec B helps after, and under the help of Sec A and some other correlation factors, finish molecule stride the film secretion process.
In the member of Sec family, Sec B is present in the interior unique solubility of intestinal bacteria endochylema, and has excreted factor (Kumamoto C.A.et al., Proc.Natl.Acad.Sci.USA, 86:5320-5324,1989 of many negative charges; Watanabe M.et al., Cell, 58:695-705,1989).Sec B in the peptide chain folding process of secretor type precursor protein matter by the β laminated structure between molecular skeleton interact with it (Breukink E.et al., Eur.J.Biochem., 208:419-425,1992).Combining site that it is generally acknowledged Sec B molecule and protein precursor molecule exists a β lamella core, and both sides then are αLuo Xuanjiegou (Spurlino, J.C., et al., J.Biol.Chem., 266:5202-5219,1991) separately.
Because bacterial cell is easy to growth and operation, so in the genetically engineered field,, usually use bacterial cell as the host in order to produce desired protein with in enormous quantities and high-recovery.Yet,
In bacterial cell, most of exogenous protein all are with the inclusion body formal representation of insoluble and non-activity.In addition, under the situation of using the exogenous promoter in bacterial cell, bring into play function, generally all be insoluble and inactive form by expressed protein.Therefore, people are attempting to seek the synthetic protein transmembrane transport of improvement always, produce the approach of desired protein with soluble form.Utilizing target protein matter and molecular chaperones such as the proteic coexpression of Sec B is one of possible approach that realizes this purpose.Find in the former research work of the inventor, owing to be subjected to the restriction of factors such as the proteic content of the intrinsic Sec B of bacterial cell, a large amount of synthetic exogenous protein usually are accumulated in the cell on rrna, the random folding of precursor molecule takes place, cause intermolecular gathering, cause these precursor molecules can not with various associated molecule companions Sec B protein binding particularly, and forfeiture is striden film and is passed on ability.The inventor finds in its long term studies practice and confirms that further there is higher affinity in the proteic binding site of Sec B and the many clean positive charge of excretory precursor protein matter band zones for the treatment of, therefore infer the fusion gene that might design and make up the structural gene sequence that comprises the secretion signal peptide-coding sequence and wait to express, attempt to improve the secernment efficiency of exogenous protein by the synchronous expression of control gained fusion gene and Sec B gene.The result is surprisingly found out that, using suitable synthetic promotor, for example under the situation of intestinal bacteria alkaline phosphatase promotor, utilize coexpression system of the present invention, successfully realize the purpose of high secernment efficiency expression desired protein, thereby finished the present invention.
Therefore, an object of the present invention is to provide the recombinant expression vector that is used for expressing at prokaryotic cell prokaryocyte desired protein, wherein said expression vector comprises the site after the coding molecule companion's that is operably connected gene to the first promotor.
According to expression vector of the present invention, the gene order of the coding desired protein that is operably connected is wherein arranged in the downstream of coding molecule companion's dna sequence dna to the signal peptide dna sequence dna that contains second promotor.
According to " desired protein " of the present invention, be meant human granulocyte, macrophage colony stimulating factor.
According to expression vector of the present invention, wherein said coding molecule companion's gene is an intestinal bacteria Sec B gene.
According to expression vector of the present invention, wherein said first and/or second promotor is an intestinal bacteria alkaline phosphatase promotor, and first and second promotors are identical or different.
According to expression vector of the present invention, the gene of wherein said coding desired protein is through the mutator gene transformed or the gene of chemosynthesis after the wild type gene that obtains from natural origin screening, the screening.
Another object of the present invention provides the prokaryotic cell prokaryocyte that is used to express required exogenous protein that transforms with above-mentioned expression vector.
According to prokaryotic cell prokaryocyte of the present invention, said prokaryotic cell prokaryocyte is a Bacillus coli cells.A further object of the present invention provides the method for prokaryotic cell prokaryocyte host with soluble form secreting, expressing desired protein of utilizing, and this method comprises:
(1) obtain the encoding dna sequence dna of desired protein;
(2) dna sequence dna with the coding desired protein of step (1) is operably connected on the dna sequence dna of the suitable mate molecule of coding, obtains being used for the recombinant expression vector of coexpression desired protein and molecular chaperone protein matter under suitable promotor control;
(3) recombinant expression vector with step (2) transforms suitable bacterial host cell;
(4) host cell of culturing step (3) under being suitable for the condition of soluble secreted form expression desired protein;
(5) from culture, isolate insoluble part and from soluble fractions, reclaim desired protein.
This purpose preferred embodiment according to the present invention may further comprise the steps:
(1) obtain the encoding cDNA sequence of human granulocyte-macrophage colony stimulating factor;
(2) will the encode dna sequence dna of intestinal bacteria SecB gene is operably connected on the plasmid of the dna sequence dna that contains coding human granulocyte-macrophage colony stimulating factor, obtains being used for coexpression human granulocyte-macrophage colony stimulating factor and the proteic recombinant expression vector of intestinal bacteria SecB under suitable promotor control; On this recombinant expression vector, be arranged in order first promotor and intestinal bacteria Sec B gene, second promotor, signal peptide and human granulocyte-macrophage colony stimulating factor gene in the direction of the clock;
(3) recombinant expression vector with step (2) transforms suitable prokaryotic host cell;
(4) host cell of culturing step (3) under being suitable for the condition of soluble secreted form expressing human granulocyte-macrophage colony stimulating factor;
(5) from culture, isolate insoluble part and from soluble fractions, reclaim human granulocyte-macrophage colony stimulating factor.
A further object of the present invention provides the method for prokaryotic cell prokaryocyte with soluble form secreting, expressing desired protein of utilizing, and this method comprises:
(1) obtain the encoding dna sequence dna of desired protein, and it is operably connected to is suitable in prokaryotic hosts, expressing on first expression vector of desired protein;
(2) obtain the dna sequence dna of coding molecule chaperone, and it is operably connected to is suitable in prokaryotic hosts, expressing on second expression vector of desired protein;
(3) the suitable prokaryotic hosts of the first and second recombinant expression vector cotransformations of usefulness step (1) and (2);
(4) under being suitable for, cultivate the host cell that goes on foot (3) with the condition of soluble form secreting, expressing desired protein;
(5) from culture, isolate insoluble part and from soluble fractions, reclaim desired protein.
According to the preferred embodiment of the inventive method, wherein said desired protein is selected from protein and the polypeptide of all animals, plant and the microorganism that comprise the people.
Fig. 1 shows the dna encoding sequence of the Sec B that the clone obtains from intestinal bacteria.
Fig. 2 shows the human granulocyte that the clone obtains from the human embryonic lung cell, the dna encoding sequence of macrophage colony stimulating factor.
Fig. 3 shows the structure of the recombinant vectors M13mp19-Sec B be used to clone Sec B gene.
Fig. 4 shows the structure of the recombinant vectors pAGMT-8 that is used for expressing human granulocyte, macrophage colony stimulating factor.
Fig. 5 shows and to carry the structure that is used at the recombinant vectors pAGMST-8 of intestinal bacteria coexpression secretion signal protein s ec B and human granulocyte, macrophage colony stimulating factor.
Fig. 6 a～6c shows respectively with behind plasmid pAGMST-8 and the control plasmid pAGMT-8 transformed into escherichia coli host cell, the SDS-PAGE collection of illustrative plates of different components.
Fig. 7 shows that fermentation culture carries Sec B gene, but this upstream region of gene the engineering strain EGMST-8 of band signal SPphoA sequence and the lysate that does not contain the EGMT-8 of Sec B gene are not made the Northern engram analysis.
The invention provides the formal representation that is suitable in prokaryotic hosts with the solubility secretory protein The recombinant expression carrier of desired protein, wherein said recombinant expression carrier comprises the required egg of expression First transcript unit of white matter and second transcript unit of expressing molecular chaperone protein matter, wherein said First transcript unit and second transcript unit can be present in same restructuring table with series system Reach carrier, also can be present in respectively two different restructuring tables for the same prokaryotic hosts of cotransformation Reach in the carrier.
As previously mentioned, although some prokaryotic, particularly Escherichia coli usually are used to recombinate Dna technique is expressed manyly to has outside various BA and biological function different Source protein matter, but since these prokaryotes bodies particularly colibacillary inherent function characteristic determine Fixed, most of protein of being expressed all mainly form inclusion body in cell, and with inclusion body Form is present in the cell, thus give continue after the step of separation and the required expression product of purifying bring very Big difficulty is difficult to obtain the protein of high bioactivity. The former recombinant technique branch of using of the inventor Secrete in the practice of expressing granulocyte, macrophage colony stimulatory factor (GM-CSF) and find, The GM-CSF protein of high expressed in escherichia coli host, about being accumulated in more than 80% carefully In the born of the same parents, and be present in expression product only 10%～20% in the culture supernatant. In order to solve this One problem has carried out grinding extensively and profoundly to the possibility of the secernment efficiency that improves destination protein matter Study carefully, and made up recombinant vector and the corresponding expression system that is used for secreting, expressing. It is frightened that the result makes us Find that very the synthetic E. Coli Alkaline Phosphatase promoter control that utilizes us to make up is lower The recombinant expression system of the required destination protein matter of coexpression and molecular chaperones Sec B albumen is at shaking table Under the condition of culture, can make the secreting, expressing efficient of desired protein GM-CSF improve about 5～6 Doubly, further optimize fermentation conditions and be expected further to improve secreting, expressing efficient.
Recombinant expression carrier of the present invention comprises the coding that is operably connected on first promoter The dna sequence dna of molecular chaperones and the insertion site that can insert second transcript unit. Utilize Said insertion site can be inserted basically by volume in said recombinant expression carrier at an easy rate The dna sequence dna of code desired protein forms with second promoter that is operatively coupled on its upstream Second transcript unit. At second promoter of second transcript unit and coding desired protein Add an other targeting sequencing (signal peptide sequence) between the dna sequence dna, and melt at gained The downstream of closing gene adds terminator.
As promoter sequence, can use any desirable proteins that is suitable in prokaryotes, expressing The promoter of matter and/or molecular chaperones for example can be used bacterium promoter and phage promoter. These promoters for example comprise but be not only limited to tac promoter, lac promoter, T7 promoter, Gal promoter and alkaline phosphatase (phoA) promoter, but the present invention phoA preferably Promoter.
As with the molecular chaperone of dna sequence dna coexpression of coding desired protein, this The gene that preferably has the molecular chaperones Sec B that helps secreting function in bright. Sec B is secretion Unique soluble protein that is present in the born of the same parents in the factor S ec family. Other EF Sec A, Sec D, Sec E, Sec F, Sec Y etc. are positioned on the film. Sec B can Be combined with most protein, it is special loose folding that it is kept, and is suitable for the space structure that passes on Resemble. Sec B in connection with protein be transported on the film, be combined with Sec A, other the secretion because of Under the mutual assistance of son, finish the membrane process of striding of protein. The binding site of Sec B need not special Amino acid sequence, it is incorporated into zone positively charged and that have the β-pleated sheet conformation, thereby can with greatly Most protein bound. This also is preferred molecular chaperones Sec B conduct and required egg during we invent The reason of white matter coexpression.
As the initial vector that is used for making up recombinant expression carrier of the present invention, can use any energy Enough carriers that in bacterial cell, stably keeps and copy. Such initial vector comprises but not only Be limited to pTZ18R, pBR322, pKK223-3, pUC18, pUC19, pUC119 Deng, but this patent multicopy plasmid pTZ18R preferably.
As the host, can use any being suitable for therein with high efficiency secreting, expressing desirable proteins The bacterial cell of matter and molecular chaperones, particularly Bacillus coli cells. The preferred host of the present invention is thin Born of the same parents are Escherichia coli YK537 of alkaline phosphate ester enzyme gene deletion.
When making up recombinant expression carrier of the present invention, can be at first according to well known by persons skilled in the art Method (for example referring to J.Sambrook, E.F.Fritsck﹠T.Manicetis, Molecular Cloning, A Laboratory Mannual, Znd ed., Cold Spring Harbour Press, NY, 1989) promoter is imported in the initial plasmid. Then, with coding molecule companion's dna sequence dna Be connected to the downstream of first promoter. Using the E. Coli Alkaline Phosphatase deficient strain In the situation of YK537, preferably use alkaline phosphatase promoter (phoA) to open as first Mover. Can suitably adjust the distance between first promoter sequence and the molecular chaperone, to obtain Get the optimum expression activity. Can from known Escherichia coli, separate and clone the branch of known its sequence Sub-companion's sequence. The present invention designs pair of primers according to Sec B gene order (465bp), Using polymerase chain reaction (PCR) restrains from the chromosomal DNA of Escherichia coli YK537 Swell Escherichia coli Sec B gene.
In order to improve expression efficiency, can add a terminator order in the downstream of molecular chaperones in case of necessity Row, but also can when design and cloning molecular companion sequence, add suitable termination signal. Can Finish in accordance with known methods being connected between molecular chaperones sequence and terminator sequence. To divide among the present invention Sub-chaperone is inserted in plasmid for example plasmid pAE-8 or the pAET-8 (bacterial strain of this Plasmid Transformation Regulation by budapest treaty is deposited in the Chinese microorganism strain guarantor before present patent application Hide administration committee common micro-organisms center, preservation registration number is CGMCC NO.0287) Between promoter sequence and the terminator sequence, contain promoter sequence, the branch that is one another in series with structure The recombinant vector of sub-chaperone and terminator sequence. If expressing, the molecular chaperones that so obtains carries Have suitable connection desired protein gene cloning site in the body, this carrier namely can be used as this Bright expression vector. If constructed carrier does not have suitable cloning site, then can suitably modify The end of sequence also adds cloning site. Can use and have connecing of different restriction enzyme sites more Head is as cloning site.
Can be with the dna sequence dna of the coding desired protein of second promoter that is operably connected Import in the cloning site of above-mentioned expression vector. Be imported into the coding desired protein DNA Second promoter sequence that sequence is operably connected, can be identical with above-mentioned first promoter or Different. First promoter and second promoter are the phoA promoter among the present invention. Must Can between first transcript unit and second transcript unit, add connecing of a suitable length when wanting Head, and can suitably adjust the length of joint, in order to more effectively express required protein.
Use known method, transform Host Strains with expression vector, for example CaCl2Facture will be expressed Carrier is transformed into bacterial cell. Cultivate and proliferative cell after, select to be converted according to the resistance to the action of a drug Cell, take obtain as the object of the invention required, with the weight of high yield secreting, expressing desired protein The group body. In the situation of using escherichia coli host, preferably utilize drug resistance gene as selection Marker gene for example can be used ampicillin resistance gene, chloramphenicol resistance gene, be blocked that is mould Plain resistant gene or tetracycline resistance gene.
Be purpose of the present invention, the transformant for preparing as stated above not necessarily comprises carries branch simultaneously The same plasmid vector of sub-chaperone and desirable proteins plasmagene. As a kind of alternative side Method, can be respectively with first recombinant vector that carries first promoter and molecular chaperone with take With second promoter and the coding desired protein exogenous DNA array second recombinant expressed year The bacterial host that the body cotransformation is suitable is to obtain recombinant of the present invention. Relatively, be used for corotation Two recombinant vectors changing are preferably respectively with its different drug resistance selection marker base separately Cause.
Can by methods known in the art, in appropriate culture medium, cultivate above-mentioned transformant. Cultivate After finishing, for example can separate the culture supernatant by simple centrifugal method, and from the gained culture Separate and the required protein of purifying in the supernatant. The method bag that is used for purifying and recovery desired protein Draw together but be not only limited to ammonium sulfate precipitation method, ultrafiltration, ion exchange chromatography, hydrophobic chromatography, The combination of gel filtration, affinity chromatography or these methods.
Available method of the present invention comprises with the protein of soluble form secreting, expressing but is not only limited to Microprotein, phytoprotein or animal protein, or their antibody or various forms of Fused protein, or natural or artificial variation's body of these protein.
The following example is intended to further describe for example the present invention, rather than limits the present invention in any form.Though be to describe the present invention for example as specific examples in the following example with the secretor type coexpression of molecular chaperones Sec B albumen and target protein matter GM-CSF, but it will be appreciated by those skilled in the art that, except that utilizing molecular chaperones Sec B gene and GM-CSF coexpression, also can be according to the spirit and principles in the present invention, use other molecular chaperoneses that same or similar function is arranged, coexpression and GM-CSF have the protein of same or similar constitutional features.Therefore, under the prerequisite of essence spirit of the present invention and principle, to of the present invention any be equal to alternative, change or improve and all will fall into the present invention and await the reply in the claim scope.Embodiment 1: the preparation of molecular chaperones Sec B gene
Fig. 1 has shown the nucleotide sequence of Sec B gene.In order to obtain being used for intestinal bacteria molecular chaperones Sec B gene of the present invention, intestinal bacteria YK537 (providing by MAKARI professor YAMASAKI of Tokyo Univ Japan) at first are provided in the LB substratum that contains penbritin (100 μ g/ml) (every liter contains 10g peptone, 5g yeast extract and 10g sodium-chlor), and press currently known methods and separate chromosomal DNA.Show aligning primer (1) and primer (2) under design and use dna synthesizer are synthetic: primer (1): 5 '-G
GAATTCATGTCAGAACAAAACAACAC-3,
EcoR I primer (2): 5 '-AA
CTGCAGTTATCAGGCATCCTGATGTTC-3,
Pst?I
With the total DNA of the karyomit(e) of YK537 as template, under the effect of Taq polysaccharase, utilize primer (1) and primer (2), obtain the dna fragmentation that two ends have about 480 bp of length of EcoR I and Pst I restriction enzyme site respectively with the amplification of polymerase chain reaction (PCR) method.The reaction conditions of PCR is: 94 ℃ of reactions 30 seconds, 50 ℃ of reactions 60 seconds were extended 60 seconds down at 72 ℃ at last, repeat 35 circulations altogether after, in 72 ℃ down about 600 seconds of reaction to finish pcr amplification.
Electrophoretic separation (1% low melting point glue) PCR reaction mixture is equivalent to the electrophoretic band of 480bp and reclaims it from the gel cutting-out.Use SmaI, calf intestinal alkaline phosphatase (CIP) is handled M13mp19, is connected (see figure 3) with the fragment of above-mentioned 480 bp under the effect of T4 dna ligase after reclaiming big fragment, screens forward and reverse two kinds and connects products.Extracting single stranded DNA from the culture supernatant of phage is pressed the nucleotide sequence of currently known methods with universal primer mensuration forward and reverse junction fragment, proves that products therefrom is 155 proteic nucleotide sequences of amino acid whose SecB of coding.Embodiment 2: the preparation of the cDNA of coding human granulocyte, macrophage colony stimulating factor
Present embodiment is described the preparation of the cDNA of coding human granulocyte, macrophage colony stimulating factor (hGM-CSF).
Human embryonic lung cell HFL cell strain (available from Shanghai cell biological institute of the Chinese Academy of Sciences) is cultivated in the RPMI-1640 substratum that contains 20% foetal calf serum (FBS), after treating that cell grows to 70% individual layer, change the fresh culture that contains TNF-α (final concentration 1000 U/ml) into.Inducing culture is harvested cell after 36 hours, and extracts total RNA with guanidine thiocyanate method (Chomczynskiet al).
Design and show for synthetic time the primer (3) and the primer (4) of sequence: primer (3): 5 '-GCACCCGCCCGCTCGCCCAG-3 ' primer (4): 5 '-A with dna synthesizer
AAGCTTATCACTCCTGGACTGGCTCCC-3 '
Hind?III
Get 2 μ g through the total RNA of TNF-c inductive human embryonic lung cell as template, use above-mentioned primer (4) as the reverse transcription initial primers, add 1 μ lSuper Script (R) ThermoScript II after 15 minutes in 65 ℃ of denaturing treatment.37 ℃ of reactions were warming up to 95 ℃ and keep 5 minutes with termination reaction after 40 minutes, and the cDNA that obtains with reverse transcription is a template then, use primer (3) and primer (4) carry out pcr amplification (94 ℃ 30 seconds, 50 ℃ 60 seconds, 72 ℃ 60 seconds, after repeating 35 circulations, in about 600 seconds of 72 ℃ of reactions).
After the PCR reaction mixture carried out electrophoretic separation, obtain the amplified production of about 390 bp of length, clone this fragment, hold two-way mensuration sequence from the 5 ' end and 3 ' of sequence by known double-stranded sequencing by embodiment 1 described method.(Fig. 2) result as seen, the cDNA sequence of the hGM-CSF of people (Science 228:310,1985) such as measured sequence and Wong report is in full accord.Embodiment 3: the structure of recombinant vectors pAGMT-8
The concise and to the point structure of describing the recombinant expression vector that carries external source GM-CSF encoding sequence and secreting, expressing signal of present embodiment.
Use to make up before us, the recombinant plasmid pAET-8 of dna sequence dna that carries encoding human epidermal growth factor (hEGF) is as the parent material of present embodiment.In pAET-8, be connected with intestinal bacteria alkaline phosphatase promoter sequence (phoA) and signal peptide sequence (SPphoA) synthetic in advance and that assembling is come in succession in 5 ' upstream of EGF coding region.Therefore, after importing goal gene in the downstream of these controlling elements and changing plasmid over to suitable host, even under the situation that does not contain other intestinal bacteria secretion signals, it is the required protein of secreting, expressing effectively also.
For construction recombination plasmid pAGMT-8, at first cut above-mentioned plasmid pAET-8 with EcoR I enzyme, handle to form tack with mung-bean nuclease (MungBean Nuclease) after from the endonuclease reaction mixture, reclaiming big fragment, with Hind III the EGF gene is downcut again after reclaiming big fragment, in the presence of the T4 dna ligase, make it to be connected, promptly obtain about 4.1 Kb of length and name the recombinant vectors that carries the GM-CSF encoding sequence (Fig. 4) for pAGMT-8 with the dna fragmentation that contains the GM-CSF encoding sequence with Hind III digestion in advance by embodiment 2 described methods preparations.The difference of this plasmid (pAGMT-8) and pAET-8 only is to have been replaced by the encoding sequence of GM-CSF the sequence of the coding EGF on the plasmid pAET-8.Embodiment 4: the structure of recombinant vectors pAGMST-8
Present embodiment is described the structure be used at the recombinant vectors of intestinal bacteria coexpression molecular chaperone protein and human granulocyte, macrophage colony stimulating factor.
As previously mentioned, although have signal element phoA and the SPphoA that is used to drive foreign gene such as EGF genetic expression in the pAGMT-8 plasmid that we make up, but we find have a large amount of expression products to pile up after the fermentation culture when this expression system is used for expression of GM-CSF in host cell, as and if GM-CSF product accumulating amount is inversely proportional to secretory volume, therefore, for solving the expression product secretion problem of GM-CSF, we are further on the basis of existing expression vector pAGMT-8, made up and carried the recombinant vectors that is operably connected to coding intestinal bacteria Sec B gene after the phoA promotor, this gene with the phoA promotor as transcription unit independently, it can be connected in series in the upstream or the downstream of the transcription unit of coding GM-CSF, perhaps, also can be present in two discrete carriers with the transcription unit of coding G M-CSF or other target protein matter.
Use M13mp19-Sec B and pAGMT-8 to make up the plasmid pAGMST-8 that has Sec B gene as shown in Figure 5.
At first obtain containing the dna fragmentation of Sec B gene with EcoRI and PstI digestion M13mp19-Sec B, on the other hand, synthetic have primer (5) and a primer (6) that shows sequence down: primer (5): 5 '-AGCGGATAACAATTTCACACAGGA-3 ' primer (6): 5 '-G
GAATTCTTTATTTTCTCCATGTAC-3 '
EcoRI
With pAGMT-8 is template, uses primer (5) and primer (6) to carry out PCR and reacts, and the gained fragment finally obtains containing the dna fragmentation of intestinal bacteria alkaline phosphatase promoter sequence after with Pst I and EcoR I double digestion.Handle plasmid pAGMT-8 with PstI and calf intestinal alkaline phosphatase (CIP), reclaim after the big fragment dna fragmentation with the above-mentioned Sec of containing B gene. be connected in the presence of the T4 dna ligase with the dna fragmentation that contains intestinal bacteria alkaline phosphatase promoter sequence, recovery obtains cutting the screening forward with Kpn I with EcoR I enzyme after the big fragment of about 4.7Kb and is connected product, promptly gets the pAGMST-8 recombinant plasmid.This plasmid has another phoA promotor that is connected on the control intestinal bacteria secretion signal Sec B genetic expression of the phoA promotor upstream of controlling GM-CSF genetic expression.The difference of two transcription units that are connected in series is, is in the upstream and instructs entrained signal peptide sequence SPphoA in second transcription unit that instructs GM-CSF genetic expression that is not in the downstream in the transcription unit of Sec B genetic expression.(bacterial strain that this plasmid transforms is deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center by the regulation of budapest treaty before in present patent application, and preservation registration number is CGMCC NO.0288) embodiment 5: the conversion of recombinant vectors pAGMST-8 and expression
Present embodiment is described the recombinant plasmid transformed e. coli host cell that makes up with as stated above, and has the active protein of human GM-CSF with the expression of solubility secreted form in this host.
To be derived from e. coli k12 strain series, the fresh bacterium that spends the night of alkaline phosphatase defective escherichia coli bacterial strain YK537 is forwarded in the LB substratum with 1% volume, in 37 ℃ of shaking culture to OD
600Be about 0.4 (approximately cultivating 3 hours).10,000rpm collected thalline (all needing aseptic technique) in centrifugal 1 minute, added calcium solution (the 50mM CaCl of 100 μ l precoolings by every 1.5ml bacterium liquid
2, 10mM Tris.Cl pH8.0,15 pounds of sterilizations 15 minutes, 4 ℃ of preservations) and jog suspension thalline, ice bath 10 minutes, 4, centrifugal 10 minutes of 4 ℃ of 000rpm, the supernatant that inclines adds equal-volume and preserves liquid (100mM CaCl
2, 15% glycerine was sterilized 4 ℃ of preservations 15 minutes for 15 pounds), by 200 μ l packing, can deposit half a year for-80 ℃.
1～10ng plasmid pAGMST-8 adds in the 100 μ l competence bacteria liquid, mixing, and ice bath 30 minutes, 42 ℃ of heat-shockeds 90 seconds add 5 times of volume LB and recover to cultivate 1 hour in 37 ℃, get proper volume afterwards and are coated with flat board, are inverted in 37 ℃ and cultivate 12 hours.
The screening mono-clonal, cultivation and enzyme are cut evaluation and are finally obtained the corresponding engineering bacterium.
The engineering bacteria that will spend the night inserts in the 200ml LB substratum (content of penbritin is 100 μ g/ml), cultivates 18 hours to induce the DNA transcript and expression of the GM-CSF under the phoA promoters driven in 37 ℃.Induce the centrifugal culture in back and collect supernatant liquor.Group is also used the recombinant plasmid pAGMT-8 transformed into escherichia coli YK537 that does not have Sec B gene in contrast, and cultivates transformant cell and separation and Culture thing supernatant by above-mentioned with quadrat method, separates and purifying expression product hGM-CSF from the gained supernatant liquor then.Embodiment 6: to the analysis of transformant strain tunning
Present embodiment is described and is used intestinal bacteria molecular chaperones Sec B and target protein matter GM-CSF coexpression, to transforming the various analytical resultss of bacterial strain fermentation culture product.
With the control plasmid pAGMT-8 transformed into escherichia coli YK537 that carries the plasmid pAGMST-8 of intestinal bacteria secretion signal Sec B gene and do not carry Sec B gene of the present invention, obtain GM-CSF respectively and produce bacterium recombinant chou bacterial strain EGMST-8 and EGMT-8 respectively.Each the transformant bacterial strain that so obtains is induced the expression of goal gene in low-phosphorous substratum.With bacterial strain after inducing and supernatant, use the SDS lysate, 100 ℃, after the heating in 5 minutes, carry out SDS-PAGE and analyze, dye with Coomassie brilliant blue (Coomassie Brilliant Blue) behind the electrophoresis.
Fig. 6 a shows the result who the culture different piece of these transformant is carried out the SDS-PAGE analysis.As can be seen from the figure, contain hGM-CSF amount lower (swimming lane 3 and 4) in the culture supernatant of the control strain EG MT-8 that transforms with plasmid pAGMT-8, and hGM-CSF is mainly seen in ( swimming lane 1,2,5,6) in the insoluble part.Swimming lane 8 is a molecular weight standard.In the fermenting culture supernatant liquor of the EGMST-8 engineering strain that plasmid pAGMST-8 transforms as seen a large amount of ripe hGM-CSF (swimming lane 2 and 3 among Fig. 6 b, swimming lane 1 is a molecular weight standard) are arranged then.
Fig. 6 c show EGMST-8 and EGMT-8 through fermentation inducement to peak expression, the centrifugal supernatant of removing is used the SDS electrophoresis liquid, 100 ℃ of 5 minutes cracking thalline, the electrophorogram after centrifugal.The EGMST-8 bacterial strain with abduction delivering after visible strong Sec B band in the tropina, and in the bacterium pre-GM-CSF amount seldom, only visible sophisticated GM-CSF band (swimming lane 1); The EGMT-8 bacterial strain behind abduction delivering in the tropina Sec B band very shallow, but have strong pre-GM-CSF and sophisticated GM-CSF band (swimming lane 2).Swimming lane 3 is a molecular weight standard.
Above-mentioned analytical results shows, under the situation of GM-CSF and molecular chaperones coexpression, the target protein matter of expressing in the host is promptly striden under the help of film secretory protein at the molecular chaperones of coexpression with it, amount with remarkable increase is secreted into outside the host cell, thereby has increased the efficient that emiocytosis is expressed widely.Therefore, expect that secreting, expressing of the present invention system might be widely used in the secreting, expressing of many biologically active proteins matter.
In addition, we also use enzyme linked immunosorbent assay analysis method (ELISA) that the expression product in the culture each component of the engineering strain EGMT-8 of fermentor cultivation and EGMST-8 has been carried out semi-quantitative analysis.The ELISA Kit analytical results of using Amersham company shows, in the lysate (comprising cell pericentral siphon, cytoplasm and inclusion body) of EGMT-8 fermenting culture, have great expression product GM-CSF, and the GM-CSF content in fermented liquid supernatant only accounts for 1/6 of insoluble part.On the contrary, the GM-CSF content in the EGMST-8 fermented liquid supernatant is higher 5～6 times than the former approximately.
Fig. 7 shows that fermentation culture carries Sec B gene, but this upstream region of gene the engineering strain EGMST-8 of band signal SPphoA sequence and the lysate that does not contain the EGMT-8 of Sec B gene are not made the Northen engram analysis.The result as seen, this project bacterium is under the shake-flask culture condition, have dense Sec B band (Fig. 6 c) in its cell, and in further Northen engram analysis, use known array Sec B gene to make probe, use random primer labelling behind the prehybridization, and in 42 ℃ of hybridization 20 hours, carry out radioautograph after the washing, as seen the result has stronger Sec B mRNA hybridization band.On the contrary, the EGMT-8 bacterial strain that does not carry Sec B gene sees that in identical fermentation and next of testing conditions faint hybridization band is arranged.Swimming lane 2,4 is EGMST-8; Swimming lane 1,3 is EGMT-8.Embodiment 7: the amplification fermentation culture of engineering strain EGMST-8, the separation and purification of product and bioactive mensuration
The EGMST-8 glycerol stock that the contains 15% glycerine 20 μ l that are taken at-70 ℃ of preservations are inoculated in (penbritin content is 100 μ g/ml) in the 3mlLB substratum, 37 ℃ of overnight incubation, (penbritin concentration is 100 μ g/ml to get the suitable dilution of a little bacterium liquid do back coating LB flat board in second day, contain 15g agar among every liter of LB), in 37 ℃ of constant incubators, be inverted and cultivate picking list bacterium colony (penbritin concentration is 100 μ g/ml) in the test tube that contains 3ml LB after 16 hours, put into 37 ℃ of shaking table overnight incubation (rotating speed 200rpm), inserted 1 liter of containing 200ml LB substratum and shake (penbritin concentration 100 μ g/ml) in the bottle with the 1% ratio bacterium that will spend the night in second day, at 37 ℃, cultivated in the shaking table of 200rpm 10-12 hour seed.
The adding of 200ml seed culture fluid is contained 10 liters of fermention mediums, and (every liter contains peptone 20g, yeast extract 10g, glucose 30g, sal epsom 1g) fermentor tank (New BrunswickScientific company, model: Bio Flo IV), cultivated about 18～20 hours in 37 ℃ of aeration-agitations.As culture OD
600Value is about at 30～36 o'clock, and the productive rate of GM-CSF reaches the about 100～120mg/L of peak value.Utilize identical fermentation condition fermentation culture colibacillus engineering strain EGMT-8, the maximum output of GM-CSF has only about 20mg/L.
After fermentation is finished, centrifugal separating cell throw out and nutrient solution supernatant, according to ordinary method, be that methods such as 10,000 daltonian ultra-filtration membranes (Millipore Co.) ultrafiltration, hydrophobic chromatography, anionresin and gel-filtration are come purifying expression product hGM-CSF with interception successively for example.
Can use the TF-1 cell as target cell (being incubated in the RPMI-1640 substratum that contains 10%FCS) according to the standard method of setting up, the OD of working sample behind cultivation and the lysing cell in microtiter plate
570Light absorption ratio is with the activity unit of calculation sample after the regression analysis.The result proves, the hGM-CSF that uses secreting, expressing of the present invention system to produce has higher biologic activity than standard model.
Claims (7)
1. be used for recombinant expression vector at prokaryotic cell prokaryocyte secreting, expressing human granulocyte-macrophage colony stimulating factor, described expression vector comprises the intestinal bacteria See B gene of operationally will encoding and is inserted into the site after first promotor and is positioned at signal peptide dna sequence dna downstream, described site, after second promotor, and described signal peptide dna sequence dna can be connected with the dna sequence dna of the coding human granulocyte-macrophage colony stimulating factor in downstream.
2. according to the expression vector of claim 1, wherein said first and/or second promotor is an intestinal bacteria alkaline phosphatase promotor, and first and second promotors are identical or different.
3. according to claim 1 expression vector, the gene of wherein said coding human granulocyte-macrophage colony stimulating factor is through the mutator gene transformed or the gene of chemosynthesis after the wild type gene that obtains from the natural origin screening, the screening.
4. prokaryotic cell prokaryocyte that is used for the expressing human granulocyte-macrophage colony stimulating factor is characterized in that it being with any one expression vector cell transformed in the claim 1 to 3.
5. according to the prokaryotic cell prokaryocyte of claim 4, described prokaryotic cell prokaryocyte is a Bacillus coli cells.
6. the secretory expression method of a human granulocyte-macrophage colony stimulating factor is characterized in that may further comprise the steps:
(1) obtain the encoding cDNA sequence of human granulocyte-macrophage colony stimulating factor;
(2) will the encode dna sequence dna of intestinal bacteria SecB gene is operably connected on the plasmid of the dna sequence dna that contains coding human granulocyte-macrophage colony stimulating factor, obtains being used for coexpression human granulocyte-macrophage colony stimulating factor and the proteic recombinant expression vector of intestinal bacteria SecB under suitable promotor control; On this recombinant expression vector, be arranged in order first promotor and intestinal bacteria SecB gene, second promotor, signal peptide and human granulocyte-macrophage colony stimulating factor gene in the direction of the clock;
(3) recombinant expression vector with step (2) transforms suitable prokaryotic host cell;
(4) host cell of culturing step (3) under being suitable for the condition of soluble secreted form expressing human granulocyte-macrophage colony stimulating factor;
(5) from culture, isolate insoluble part and from soluble fractions, reclaim human granulocyte-macrophage colony stimulating factor.
7. according to the method for claim 6, wherein said bacterial host cell is a Bacillus coli cells.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98110840A CN1111602C (en) | 1998-05-15 | 1998-05-15 | A kind of method of using molecule mate to promote protein secretion |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98110840A CN1111602C (en) | 1998-05-15 | 1998-05-15 | A kind of method of using molecule mate to promote protein secretion |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1236011A CN1236011A (en) | 1999-11-24 |
| CN1111602C true CN1111602C (en) | 2003-06-18 |
Family
ID=5220857
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98110840A Expired - Fee Related CN1111602C (en) | 1998-05-15 | 1998-05-15 | A kind of method of using molecule mate to promote protein secretion |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1111602C (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0329681D0 (en) * | 2003-12-23 | 2004-01-28 | Delta Biotechnology Ltd | Gene expression technique |
| CN100334216C (en) * | 2004-11-16 | 2007-08-29 | 南京大学 | Method and use of producing soluble recombinant protein in colibacillus |
| CN100432231C (en) * | 2006-08-22 | 2008-11-12 | 中山大学 | Prokaryotic secretion expression carrier and application thereof |
| WO2012089164A1 (en) * | 2010-12-31 | 2012-07-05 | 中国科学院上海生命科学研究院 | Functional construction of secb-mediated post-translational targeting pathway and use thereof |
| CN107271680B (en) * | 2017-05-22 | 2019-04-23 | 中山大学附属第三医院 | The detection method of human myelin oligodendroglia glycoprotein autoimmune antibody |
| CN107723290A (en) * | 2017-08-14 | 2018-02-23 | 蔡祥胜 | One kind restructuring human oocyte's oolemma ZP3 albumen and preparation method thereof |
| CN108977455B (en) * | 2018-08-01 | 2021-07-20 | 武汉康复得生物科技股份有限公司 | Recombinant plasmid for producing oxalate decarboxylase, escherichia coli expression system, method and application |
| CN112940955B (en) * | 2021-04-18 | 2022-10-18 | 江南大学 | Pichia pastoris for efficiently synthesizing lactoferrin and construction method and application thereof |
| CN117327681B (en) * | 2022-12-31 | 2024-06-18 | 义翘神州(泰州)科技有限公司 | Chaperone plasmid for promoting correct folding of mammalian cell expression protein and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0774512A2 (en) * | 1995-09-14 | 1997-05-21 | Tadayuki Imanaka | A method for production of protein using molecular chaperon |
| JPH1084970A (en) * | 1996-07-23 | 1998-04-07 | H S P Kenkyusho:Kk | New transcription control factor |
-
1998
- 1998-05-15 CN CN98110840A patent/CN1111602C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0774512A2 (en) * | 1995-09-14 | 1997-05-21 | Tadayuki Imanaka | A method for production of protein using molecular chaperon |
| JPH1084970A (en) * | 1996-07-23 | 1998-04-07 | H S P Kenkyusho:Kk | New transcription control factor |
Non-Patent Citations (1)
| Title |
|---|
| EMBOJ 17(4) 1998-02-16 DELEPELAIRE P WANDERSMAN C THE SECB CHAPERONE IS NVOLVED IN THE SECRETION OF THE SERRATIA MARCESCENS * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1236011A (en) | 1999-11-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN1111602C (en) | A kind of method of using molecule mate to promote protein secretion | |
| KR101778436B1 (en) | Acetobacter sp. SLV-7 as a novel strain with high acetic acid producing ability and use thereof | |
| CN103509729A (en) | Construction method of engineering bacterium for producing coenzyme Q10, engineering bacterium and application of engineering bacterium | |
| CN109266675A (en) | A kind of Bacillus subtilis genes engineering bacteria producing lipopeptid and its construction method and application | |
| CN101974587A (en) | Efficient expression method of human serum albumin | |
| CN102796720A (en) | (+)gamma-lactamase with racemate gamma-lactam resolution activity and its application | |
| CN103509816A (en) | Coenzyme-Q10-production engineered bacteria construction method, engineered bacteria, and application thereof | |
| CN110468143A (en) | The preparation method and application of antibacterial peptide NZX | |
| CN112225785A (en) | GntR family transcription inhibitor mutant, mutant gene and application thereof in preparation of vitamin B2In (1) | |
| CN107287197A (en) | Histidine attenuator mutant and histidine operon and their application for solving feedback repression | |
| CN108368512A (en) | A kind of preparation method of recombinant human granulocyte colony stimulating factor | |
| CN101475944B (en) | Promoter replacement method for improving Bacillus amyloliquefaciens yield | |
| CN102485890A (en) | Expression of recombined human protein disulphide isomerase (hPDI491) with Pichia pastoris in secretion manner | |
| CN114480177A (en) | Levan Levan-producing microbacterium capable of producing Levan with high yield and application of Levan | |
| CN100591758C (en) | Method for producing target protein by deleting or amplifying ibpA and/or ibpB gene encoding inclusion body-related protein | |
| CN110331121B (en) | Recombinant bacterium for high-yield lipopeptide and application thereof | |
| RU2790669C1 (en) | New leuconostoc mesenteroides dextran producer strain | |
| CN102747057A (en) | Cordyceps sinensis hirsutella sinensis purine anabolism enzyme, gene thereof, and application thereof | |
| CN102690797A (en) | Enzyme for synthesizing and metabolizing adenine of Cordyceps sinensis(Berk.)Sacc. Hirsutella sinensis, and gene and application of enzyme | |
| CN103233004B (en) | Artificial DNA (deoxyribonucleic acid) molecule and method for detecting expression of target gene | |
| KR101091150B1 (en) | Novel Bacillus sp．HY―２０ strain isolated from Apis melifera and xylanase produced from it | |
| CN1946844B (en) | Generation of recombinant genes in prokaryotic cells by using two extrachromosomal elements | |
| CN106834161B (en) | Bacillus subtilis Z12 and application thereof | |
| CN112501088A (en) | Bacillus, hyaluronidase produced by bacillus and production method of hyaluronidase | |
| CN108251447A (en) | A kind of plasmid, its construction method and its application of energy high efficient expression lipase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C06 | Publication | ||
| PB01 | Publication | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C56 | Change in the name or address of the patentee |
Owner name: SHANGHAI INST. OF LIFE SCIENCE, CAS Free format text: FORMER NAME OR ADDRESS: SHANGHAI INST. OF BIOCHEMISTRY, CHINESE ACADEMY OF SCIENCES |
|
| CP03 | Change of name, title or address |
Address after: 200031 No. 320, Yueyang Road, Shanghai Patentee after: Shanghai Institute of life Sciences, Chinese Academy of Sciences Address before: 200031 Technical Development Office of Shanghai Institute of Biochemistry, Chinese Academy of Sciences, Yueyang Road, Shanghai, Qiu Jin, 320 Patentee before: Shanghai Research Institute of Biochemistry Chinese Academy of Sciences |
|
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20030618 Termination date: 20100515 |