CN1261918A - Protein producing cell containing multiple copies of a desired gene and a screenable marker but no selection marker - Google Patents

Protein producing cell containing multiple copies of a desired gene and a screenable marker but no selection marker Download PDF

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CN1261918A
CN1261918A CN 98806873 CN98806873A CN1261918A CN 1261918 A CN1261918 A CN 1261918A CN 98806873 CN98806873 CN 98806873 CN 98806873 A CN98806873 A CN 98806873A CN 1261918 A CN1261918 A CN 1261918A
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cell
bacillus
protein
gene
microorganism
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斯蒂恩·T·乔根森
基姆·B·佩德森
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Novo Nordisk AS
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Abstract

A method of constructing a microbial multicopy protein production cell which does not comprise an inserted selection marker gene; a microbial multicopy protein production cell obtainable by said method; and a method of producing a protein of interest using such a microbial multicopy protein production cell.

Description

But have multiple copied goal gene and selection markers and do not have the white matter cell of laying eggs of selected marker
Invention field
The present invention relates to a kind of structure and do not contain the lay eggs method of white matter cell of the microorganism multiple copied that inserts the selected marker; By the obtainable microorganism multiple copied of this method white matter cell of laying eggs; And use the lay eggs method of white matter cells produce target protein matter of this microorganism multiple copied.
Background of invention
The method that a large amount of preparations can be expressed the microorganism strains of high yield target protein matter has been described in this area.
Normally based on the structure of multi-copy strains, promptly this bacterial strain contains the gene more than the coding target protein matter of a copy to these methods.
These multi-copy strains are usually by following construction of strategy:
1) one section sequence is imported in the karyomit(e) of microorganism cells, make this cell chromosome contain dna structure A-P-M-A thus, wherein
A represents homologous DNA sequence,
The dna sequence dna of P presentation code target protein matter,
The dna sequence dna (as antibiotics resistance) of M presentation code selected marker,
2) the described cell of propagation under the selective pressure that selected marker is increased;
3) identify the cell that has obtained the resistance of increase by the homologous recombination of homologous sequence A.
The multi-copy strains that makes up contains structure---A-P-M-A-P-M-A-P-M-A---then
Patent US 4959316, and US 5695976, and US 5733753 and WO 94/14968 have described these and made up the method for multi-copy strains, and the reference of Yin Ruing provides more details in addition.
Summary of the invention
Based on the consideration of environment, in the protein industrial production selected marker especially the antibiotics resistance mark be used for cell, be not preferred in the whole bag of tricks.
Therefore, the problem to be solved in the present invention provides a kind of multiple copied cell construction novel method of not using any selected marker in actual multiple copied sepn process, provide thus to obtain not contain and inserted the choosing property released marker gene, especially do not inserted the possibility of the multiple copied cell of antibiotics resistance marker gene.
The solution of problem confirms based on the inventor, can use can screen protein (screenable protein) and serve as a mark and substitute use selectivity protein as known in the art (selectable protein) and serve as a mark and make up multi-copy strains.
Therefore, first part of the present invention relates to a kind of structure and does not contain the lay eggs method of white matter cell of the microorganism multiple copied that inserts the selected marker, comprises
A) one section sequence is imported in the karyomit(e) of microorganism cells, make this cell chromosome contain dna structure A-P-S-A thus, wherein
A represents homologous DNA sequence,
The dna sequence dna of P presentation code target protein matter,
The S presentation code can screen the protein DNA sequence
B) breed described cell;
C) screening produces the proteinic cell of selectivity of increasing amount; And
C) cell of being identified recovery c); And randomly
D) cell that reclaims in the use step d) is as parent material repeating step b-d.
Term " can screen protein ", and a kind of protein of expression is not that the cell growth is necessary, if promptly it is removed from cell, so under similar growth conditions, to compare can screen protein the time with comprising in the described cell, this cell still can be with substantially the same growth velocity growth.Suitable " can screen protein " can be a fluorescigenic protein under suitable the exposure, such as egfp (GFP) or its variant (more details as follows).
This term is corresponding with the protein that is defined as " selectivity protein " herein; " selected marker protein " or " selective marker protein " expression is a kind of to be the cell necessary protein of growing, if promptly it is removed from cell, so under similar growth conditions, compare with comprising " selectivity protein " in the described cell, this cell can not grown with substantially the same growth velocity.
This " selected marker protein " can be a kind of antibiotics resistance protein usually.Therefore, when a cell grows in when containing in the corresponding antibiotic substratum, the proteinic amount of antibiotics resistance can influence the actual growth rate of cell basically in the cell.
Cell of term " do not contain the multiple copied that inserts the selected marker lay eggs white matter cell " expression does not contain the selected marker who is inserted into cell in the preparation multiple copied is laid eggs the white matter cell processes.Preparation contains the lay eggs discussion of currently known methods of white matter cell of this insertion selected marker multiple copied and sees above-mentioned " background " part in this area.
The dna sequence dna of the polypeptide of term " gene " presentation code tool specific activity, the i.e. dna sequence dna of the active polypeptide of term " selected marker " presentation code tool selected marker.
The multiple copied cell that the method according to this invention is identified has obtained to increase screened protein " S " gene of number in karyomit(e) by the spontaneous homologous recombination of homologous sequence " A ".
The incident that spontaneous homologous recombination is normally quite rare.
Yet, screen the cell of suitable quantity (preferably more than 10 by the method according to this invention 5Individual cell), the inventor confirms to identify this cell.
According to the present invention the final multiple copied cell that makes up, contain---A-P-S-A-P-S-A-P-S-A---structure, contain the target protein matter " P " of multiple copied thus.
A benefit of prepared according to the methods of the invention multiple copied cell is, it does not contain the selected marker " M " (" background " part is seen in the description of currently known methods in this area, and wherein the feature of the final multiple copied cell that makes up is the selected marker of containing this insertion) of insertion.
These genes are the existence of antibiotics resistance gene particularly, gets permission angle from environmental and product and considers it is undesirable.
Second section of the present invention relates to by the obtainable microorganism multiple copied of any method of the present invention white matter cell of laying eggs, and it is characterized in that gene (" P ") and expression that this cell contains the target protein matter that multiple copied expresses can screen proteinic gene (" S ").
The described cell of term " multiple copied is expressed the gene (" P ") of target protein matter " expression contains the gene of this target protein matter of expression of at least 2 copies; More preferably described cell contains the gene that at least 4 copies are expressed this target protein matter; Most preferably described cell contains the gene that at least 7 copies are expressed this target protein matter.
The described cell of term " multiple copied express can screen proteinic gene (" S ") " expression contain at least 2 copies expression this can screen proteinic gene; More preferably described cell contains at least 4 copy expression, and this can screen proteinic gene; Most preferably described cell contains at least 7 copy expression, and this can screen proteinic gene.
Third part of the present invention relates to the method that produces at least one target protein plasmagene in microorganism cells, and method comprises:
1) under permission produces the condition of target protein matter, cultivate according to claim 9) the microorganism multiple copied white matter cell of laying eggs; And
2) from laying eggs the white matter cell, the nutrient solution that obtains or microorganism multiple copied reclaim this target protein matter.
Embodiment of the present invention only are described below by embodiment.
Accompanying drawing
These figure have shown according to the present invention, in work embodiment 1 and work embodiment 2, by making up the lay eggs method of white matter cell of microorganism multiple copied, the plasmid that the preparation multiple copied uses when laying eggs the white matter cell.
More descriptions to this plasmid are provided in embodiment 1 and embodiment 2 subsequently.
Detailed Description Of The Invention
Sequence is imported in the karyomit(e) of microorganism cells so that cell chromosome contains dna structure A-P-S-A:
Sequence is imported in the karyomit(e) of microorganism cells so that cell chromosome contains dna structure A-P-S-A (step a)) can be undertaken by the similar strategy that the dna structure that will contain A-P-M-A (" M " is the selected marker) structure as known in the art is building up in the microorganism cells karyomit(e).
This dna structure is building up to can carries out as followsly in the microorganism cells karyomit(e), will contain structure A-P-S-A, or more optimize the carrier that contains structure A-P-S, import microorganism cells, this structure is integrated in the karyomit(e) of this microorganism cells subsequently.Or can contain the direct importing of dna fragmentation that can screen protein " S " with one with similar method and contained in the karyomit(e) of A-P-A structure, on karyomit(e), to produce dna structure A-P-S-A.
Concerning those of skill in the art, selecting in the karyomit(e) of a specially suitable strategy with A-P-S-A structure importing microorganism cells is routine work.
WO 94/23073, and US 5695976, and US 5733753 and US 4959316 have very at large described these strategies, and these documents are introduced as this dna structure is imported the chromosomal detailed description of microorganism cells herein.
Moreover work embodiment has herein described the example (seeing below) of suitable strategy.
In addition, term " imports sequence in the karyomit(e) of microorganism cells so that cell chromosome contains dna structure A-P-S-A " and can be defined as " the DNA structure that will contain structure A-P-S-A imports in the karyomit(e) of microorganism cells ".
Screening produces the proteinic cell that screens of increasing amount:
In order to identify the multiple copied cell, preferably screen a large amount of cells according to the present invention.Preferably (in step c)) screens 10 at least 5Individual cell, more preferably screening at least 10 6Individual cell, and more preferably screen 10 7At least individual cell.
In one embodiment of the invention, the screened protein in step c) be a kind of under suitable exposure fluorescigenic protein, this fluorescence protein is egfp (GFP) or its mutation particularly.
Egfp (GFP) or the existing in the art description of its mutation have been described in this area, and more details are seen Crameri etc., Nature Biotechnology 14:315-319 (1996); Cormack etc., GENE 173:33-38 (1996); WO 97/11094.GFP fluoresces under proper exposure.
Alternative suitable the screened protein of another example is beta-galactosidase enzymes, can detect it with the suitable enzyme substrates that fluoresces according to means known in the art.
Screening can use any standard screening system that measures the fluorescin quality to carry out.
High screening capacity and commercially available instrument based on this method of enforcement, preferably make the flow cytometry of apparatus cell sorting ability carry out step C by known flow cytometry technology) screening (Cormack etc., " FACS-optimize egfp (GFP) mutant " GENE173:33-38 (1996)).An example of this flow cytometry is the FACSCalibur flow cytometry that Becton Dickinson and company produces.
Therefore, in the further embodiment of the present invention, the flow cytometry completing steps c that the cell sorting ability is arranged by use) screening, fluorescing of screening tool increasing amount can be screened proteinic cell, particularly egfp (GFP) or its mutation of this fluorescing proteins herein, in screening process, GFP or its mutation are sent fluorescence under suitable exposure.
In the present invention further in the embodiment, microorganism cells of the present invention is a bacterial cell, this bacterial cell is the cell of bacillus (Bacillus) herein especially, subtilis (Bacillus subtilis) particularly, slow genus bacillus (Bacillus lentus), bacillus brevis (Bacillus brevis), bacillus acidocldarius (Bacillus stearothermophus), Alkaliphilic bacillus (Bacillus alkalophlus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus coagulans (Bacillus coagulans), Bacillus circulans (Bacillus circulans), bacillus lautus (Bacillus lautus), bacillus thuringiensis (Bacillus thuringiensis) is put under house arrest the cell of genus bacillus (Bacillus clausii) or Bacillus licheniformis (Bacilluslichenifomis).
Further in the embodiment, target protein matter (" P " in the step a)) is a kind of enzyme, particularly the protein enzyme in the present invention, lipase, amylase, tilactase, Starch debranching enzyme (pullanase), cellulase, glucose isomerase, protein disulfide isomerase, cyclodextrin glucanotransferase (CGTase, cyclodextrin gluconotransferase), phytase, glucose oxidase, Transglucosylase, laccase, or zytase.
The obtainable microorganism multiple copied of any method according to the present invention white matter cell of laying eggs:
As mentioned above, the lay eggs characteristics of white matter cell of this multiple copied are that gene and a plurality of copy that contains the expression target protein matter (P) of a plurality of copies expressed the gene that can screen protein (" S ").
As above further show that this multiple copied further feature of white matter cell of laying eggs is it does not contain the selected marker who inserts cell in the preparation multiple copied is laid eggs the white matter cell processes.
In microorganism cells, produce the method for at least one target protein matter:
The lay eggs substratum of white matter cell of culturing micro-organisms multiple copied can be any traditional substratum that is suitable for the cell growth studied.The target protein matter of expressing can be secreted in the substratum routinely, therefrom reclaim by well-known method then, comprise by centrifugal or filtration isolated cell from substratum, by salt precipitating proteins materials such as ammonium sulfate, then pass through such as ion exchange chromatography chromatography method isolated proteins such as affinity chromatography.
The present invention is described further by following non-limiting example.
Moreover, this application require right of priority Danish Patent Application DK 0792/97 content and follow the summary of this application to be incorporated herein by reference herein.
Material and method
Common molecular biology method:
Unless otherwise mentioned, DNA operation and conversion are carried out (Sambrook etc., (1989) molecular cloning---laboratory manual, cold spring harbor laboratory, cold spring port NY according to the molecular biology method of standard; Ausubel, F.M. etc. (eds), " current protocols in Molecular Biology " .John Wileyand Sons, 1995; Harwood, C.R., and Cutting, S.M. (eds), the molecular biosciences method .John Wiley and Sons 1990 of genus bacillus).
Unless otherwise mentioned, PCR manipulates standard method and the PCR response data carries out, can be referring to for example textbook (PCR practical approach IRL press, (1991)).
DNA operates the uses such as explanation of used enzyme according to supplier.
The enzyme that the DNA operation is used
Unless otherwise mentioned, all enzymes, restriction enzyme for example, ligase enzymes etc. are all available from New England Biolabs company.
Flow cytometry
The FACSCalibur flow cytometry that flow cytometry uses Becton Dickinson company to produce carries out.
On the FACSCalibur flow cytometry according to supplier's guidance, the FACSCalibur in August, 1996 for example TMFlow cytometry is carried out in the description that system user instructs.The flow cytometry terminology is according to this FACSCalibur TMSystem user instructs and uses.
Substratum
TY, BPX and LB agar describe in EP 0 506 780, and LBPSG agar is to have added phosphoric acid salt (0.01M K 3PO 4), the LB agar of glucose (0.4%) and starch (0.5%).
Embodiment 1
The structure that contains the bacterial isolates of the box that can increase, but this box includes the classical antibiotics resistance marker gene and the screening-gene of encoding green fluorescent protein.
The structure of plasmid
A)pSJ2773
United States Patent (USP) 5 7,333 753 has been described plasmid pSJ2059 and its instrument as the gene amplification that produces the amyL site of inserting Bacillus licheniformis (Bacillus lichenifomis).For plasmid can be shifted by joint, the oriT district of plasmid pUB110 is inserted into pSJ2059 to produce plasmid pSJ2773 (Fig. 1).Described as WO 96/23073, the transfer of pUB110 depends on and is positioned at 5 ' cis acting district oriT (Selinger, L.B., McGroger to (orf β), N.F., Khachatourians, G.G., and Hynes, M.F. (1990) etc., the transfer of closely-related plasmid pUB110 of general genus bacillus plasmid pXO503 and pBC16 needs trans-acting open reading frame β .J.Bacteriol., and 172,3290-3297).Use primer LWN5232 and LWN5233 to carry out in the pcr amplification pUB110 sequence pos.1020 to the fragment (McKenzie of the 550bp of pos.1575, T., Hoshino, T., Tanaka, T., Sueoka, N. the nucleotide sequence of (1986) .pUB110: some outstanding with duplicate and it regulates features relevant .Plasmid, 15,93-103).
LWN5232：
5’-GTCGGAGCTCATTATTAATCTGTTCAGCAATCGGGC-3’
LWN5233：
5，-GTCGGAGCTCTGCCTTTTAGTCCAGCTGATTTCAC-3’
The SacI site of escherichia coli plasmid (pUC19 derivative) is cloned in the fragment SacI enzymic digestion of amplification earlier.And downcut this fragment with the SacI enzyme, be cloned into pUC19 derivative pDN3000 SacI site (Diderchsen, B., Wedsted, U., Hedegaard, L., Jensen, B.R., Sjoholm, C. (1990). the extracellular enzyme that comes from bacillus brevis (Bacillus brevis) of encoding, the clone J.Bacteriol. of the aldB of acetolactate decarboxylase, 172,4315-4321), obtain pSJ2742 (Fig. 2).The OriT fragment is gone up with the BamHI-BglII fragment of 0.5kb from plasmid pSJ2742 and is downcut then, be connected on the pSJ2059 plasmid of BamHI enzymic digestion, connect mixture and be transformed in the competent cell of subtilis (Bacillus subtilis) DN1885, in the resistance of 30 ℃ of screenings erythromycin (5 μ g/ml) and kantlex (10 μ g/ml).A transformant that obtains is the SJ2773 that contains pSJ2773.
B)pSJ4574
As acquisition coding " F64L-S65T-GFP " as described in the WO97/11094, the gene of a mutant green fluorescent protein kind.This gene is to use primer #109563 and #128360 through pcr amplification.
#109563：
5’-GACTGCATGCGGGGAGGAGAATCATGAGTAAAGGAGAAGAAC-3’
#128360：
5’-CGTGAATTCGAGCTCTGCAGATCCCTTTAGTGTCAATTGG-3’
Fragment with EcoRI and BspHI enzymic digestion pcr amplification.
Use from the promotor of bacillus amyloliquefaciens (Bacillus amyloliquefaciens) alpha-amylase gene amyQ and ribosome bind site so that the encoding gene of " F64L-S65T-GFP " expressed, use primer LWN8741 and LWN8742 from bacillus amyloliquefaciens (Bacillus amyloliquefaciens) chromosomal DNA by pcr amplification amyQ promotor.
LWN8741：
5’-GTCACTGATCAATCGATTGTTTGAGAAAAGAAG-3’
LWN8742：
5’-GTCACTCATGAGTTTCCTCTCCCTCTC-3’
The PCR fragment that obtains with BclI and BspHI enzymic digestion, with this fragment cloning to pUB110 deutero-genus bacillus (Bacillus) carrier.Check order to inserting fragment, find this sequence and the amyQ promoter sequence of delivering consistent (Palva, I., Pettersson, R.F., Kalkkinen, N., Lehtovaara, P., Sarvas, M., Soderlund, H., Takkinen, K., Kaariainen, L. (1981) bacillus amyloliquefaciens (Bacillus amyloliquefaciens) alpha-amylase gene promotor and NH 2-the nucleotide sequence in signal peptide district of end not.Gene?15，43-51)。Then again promotor is downcut from this carrier with the ClaI-BspHI fragment now.
With AccI and EcoRI enzymic digestion cloning vector pUC19, carrier segments is connected with ClaI-BspHI fragment that contains the amyQ promotor and the BspHI-EcoRI fragment that contains " F64L-S65T-GFP " encoding gene in three fragments connect.Connect the very strong green fluorescence of transformant tool that mixture transformed into escherichia coli SJ2 (Diderichsen etc., 1990) obtains with this.One of them transformant is the SJ4574 that contains pSJ4574.
C)pSJ4621
To be inserted into from " F64L-S65T-GFP " encoding gene of amyQ promoter expression the amplification vector plasmid pSJ2773 through the following steps:
(i) with HindIII and AflIII enzymic digestion pSJ2773 plasmid, the fragment of purifying 3.95bp.
(ii) use AflIII and EcoRI enzymic digestion pSJ2773 plasmid, the fragment of purifying 4.25bp.
(iii) use EcoRI and HindIII enzymic digestion pSJ4574 plasmid, the fragment of purifying 1.0bp.
Connect the fragment of three purifying, mixture is transformed among subtilis (B.subtilis) DN1885, in the resistance of 30 ℃ of selections to kantlex (10 μ g/ml) and erythromycin (5 μ g/ml).Two fluorescigenic transformant of preserving are the SJ4622 bacterial strains that contains the SJ4621 bacterial strain (Fig. 4) of pSJ4621 and contain pSJ4622.
Engage the structure of F+strain:
To make competence as WO 96/23073 embodiment 4 described subtilis (B.subtilis) bacterial strain PP289-5 also transforms with plasmid pSJ4621 (obtaining bacterial strain SJ4623 and SJ4624) and pSJ4622 (obtaining bacterial strain SJ4625 and SJ4626).On the flat board that contains D-L-Ala (100 μ g/ml) in the resistance of 30 ℃ of selections to erythromycin (5 μ g/ml) and tsiklomitsin (5 μ g/ml).
Plasmid is to the transfer of Bacillus licheniformis (B lichenifomis):
The F+strain that bacterial strain SJ4623 and SJ4625 are used as engaging process changes in Bacillus licheniformis (Bacillus lichenifomis) F-strain with the plasmid vector that will increase, and engaging process is undertaken by WO96/23073 is described basically.These F-strains contain the alpha-amylase gene amyL of a chromosome copies.
The conversion zygote that obtains has the resistance to erythromycin (5 μ g/ml) and kantlex (10 μ g/ml), and the green fluorescence that transforms the zygote bacterium colony demonstrates has expressed the F64L-S65T-GFP gene.
The chromosomal integration of amplification box:
To transform the zygote bacterium colony and on the LBPSG flat board that contains erythromycin (10 μ g/ml), rule, and, grow the amplification vector plasmid integration and go into chromosomal bacterium colony 50 ℃ of cultivations.Then with the single colony inoculation on these 50 ℃ of flat boards to not containing in the antibiotic TY substratum (10ml), 30 ℃ of grow overnight, so that plasmid replication recovers, the final excision that the result is a plasmid from the karyomit(e), lacking under the situation of selective pressure, the plasmid of cutting-out will be lost from cell.Repeat the once growth in not containing antibiotic TY substratum, cell is coated on the flat board that contains kantlex (10 μ g/ml).These cells are xeroxed then and are coated on the flat board that contains erythromycin (5 μ g/ml), separate to provide kalamycin resistance and to the bacterium colony of erythromycin-sensitive.These bacterium colonies show tangible green fluorescence after some days.
Separation with bacterial strain of several gene copies
From the bacterial strain of above-mentioned structure, separate and contain a plurality of copy coding α-Dian Fenmei, the Bacillus licheniformis of " F64L-S65T-GFP " and kalamycin resistance gene (Bacillus lichenifomis) bacterial strain.
When these bacterial strains were the kantlex concentration increase that exists in growth medium, isolating by previously described enrichment procedure, the survival strains of this method screening had a plurality of gene copies (seeing US5,733,753).Therefore, screening be can be at 10 μ g/ml, 20 μ g/ml, 30 μ g/ml, 40 μ g/ml, 50 μ g/ml, the bacterial strain of growing when 100 μ g/ml and 200 μ g/ml.
Measure the amylase production of the bacterial strain that obtains in shake-flask culture.
Measure the proteinic cell content of these culture cells " F64L-S65T-GFP " simultaneously, set up kalamycin resistance, the dependency of α-Dian Fenmei output and cell fluorescence.
In experimental arrangement of the present invention, the method for separating the bacterial strain with a plurality of gene copies does not rely on based on the direct survival of kalamycin resistance gene to be selected, and depends on the physical sepn that general cell is demonstrated the cell of higher cell fluorescence.If the copy number of " F64L-S65T-GFP " expressing gene that cell contains increases, this cell will have higher cell fluorescence, thereby this method will make the cell with a plurality of gene copies be separated.
If do not have the antibiotics resistance marker gene in the cell as polygene copy cellular segregation starting point, the cell of Huo Deing will contain the goal gene (as amylase gene) of multiple copied so, and " F64L-S65T-GFP " encoding gene, but do not contain antibiotics resistance gene.More details are seen this civilian embodiment 2 back parts (seeing below).
According to experimental arrangement as described above, the method that makes up this cell is to use a kind of amplification vector plasmid, and kalamycin resistance gene wherein is in site-specific nature recombinase recognition site, as the flank in the res site of plasmid pAMbetal.In case cell obtains the amplification box (by kalamycin resistance, the disappearance of the expression of F64L-S65T-GFP and erythromycin resistance shows) of integration, just removes kalamycin resistance gene by the proteic carrier transfered cell of the pAMbetal resolvase of will encoding.WO 96/23073 describes the technology of the antibiotics resistance marker gene removal of using res site and resolvase mediation integration in detail.
Make up among another method embodiment 2 below of this cell and describe.
Embodiment 2
Have the screening-gene of encoding green fluorescent protein but include the amplification box, and do not have a structure of bacterial isolates of amplification box of antibiotics resistance marker gene
Strategy among this embodiment is to make up an amplification vector plasmid, wherein promoterless GFP gene follows the downstream that is inserted in bacillus amyloliquefaciens (Bacillus amyloliquefaciens) amyL gene coded sequence closely, then is the amyL promoter region of a copy again.
Plasmid construction:
A) pSJ4284 and pSJ4285
Use primer #109561 and #109562 by PCR from bacillus amyloliquefaciens (Bacillusamyloliquefaciens) the chromosomal DNA amplification coding AmyL C-fragment of the amyL gene of about 400 base pairs of end parts not.
#109561：
5’-GACTGAATTCCAAACATGGTTTAAGCC-3’
#109562：
5’-GACTAAGCTTGGATCCGCATGCCTATCTTTGAACATAAATTG-3’
The fragment of amplification is connected on the plasmid pUC19 of EcoRI and HindIII enzymic digestion with EcoRI and HindIII enzymic digestion, produces plasmid pSJ4284 and pSJ4285 (Fig. 5).
B) pSJ4286 and pSJ4285
Use primer #109564 and #109565 by the fragment of PCR from bacillus amyloliquefaciens (Bacillusamyloliquefaciens) chromosomal DNA amplification initial about 200 base pairs from amyL gene coding region downstream and then.
#109564：
5’-GACTGAATTCGGATCCGCAGAGAGGACGGATTTCC-3’
#109565：
5’-GACTAAGCTTCGATACCGTCATTTTCG-3’
The fragment of amplification is connected on the plasmid pUC19 of EcoRI and HindIII enzymic digestion with EcoRI and HindIII enzymic digestion, produces pSJ4286 and pSJ4287 (Fig. 6).
C) pSJ4294 and pSJ4295
The plasmid pSJ4285 that these plasmids (Fig. 6) insert BamHI+HindIII digestion by the BamHI-HindIII fragment of the 0.2kb that will downcut from pSJ4286 creates.
C) pSJ4313 and pSJ4314
The gene of coding " F64L-S65T-GFP ".
Use primer #109563 and #18842 by this gene of pcr amplification.
#109563：
5’?-GACTGCATGCGGGGAGGA GAATCATGAGTAAAGGAGAAGAAC-3’
#18842：
5’-CGTGCTCGAGAATTCGGATCCCTTTAGTGTCAATTGG-3’
The fragment of amplification is connected on the plasmid pSJ4295 of BamHI+SphI enzymic digestion with BamHI and SphI enzymic digestion, produces plasmid pSJ4313 and pSJ4314 (Fig. 8).
E)pSJ4530
Use the following step and make up actual amplification vector plasmid:
(i) (see US 5,737,753, embodiment 1 and Fig. 4 with BglII and HindIII digestion PSJ1985
Describe), the fragment of separating 1.7kb.
(ii), separate the 3.9kb fragment with BamI and HindIII digestion pSJ4313.
(iii) these two fragments are mixed then and connect, connect mixture then EcoRI and
The HindIII enzymic digestion, the fragment of from agarose gel electrophoresis, separating 2.9kb.
(see WO 96/23073, embodiment 6 retouches (iv) then isolating fragment to be connected to PSJ2739
State) the EcoRI-HindIII fragment of 5.4kb on, connect mixture and transform into subtilis
In (Bacillus subtilis) DN1885 bacterial strain, select erythromycin (5 μ g/ml) at 30 ℃.So divide
From to the bacterial strain SJ4530 that contains plasmid pSJ4530 (Fig. 9).
Engage the structure of F+strain
To be made into competence and, obtain bacterial strain SJ4541 and SJ4542 as WO 96/23073 embodiment 4 described subtilis (B.subtilis) bacterial strain PP289-5 with plasmid pSJ4530 conversion.On the flat board that contains D-L-Ala (100 μ g/ml) in the resistance of 30 ℃ of screenings to erythromycin (5 μ g/ml) and tsiklomitsin (5 μ g/ml).
Plasmid is to the transfer of Bacillus licheniformis (B.lichenifomis):
Bacterial strain SJ4541 and SJ4542 are used as the F+strain of engaging process the amplification vector plasmid is changed in Bacillus licheniformis (Bacillus lichenifomis) F-strain, and engaging process is undertaken by WO96/23073 is described basically.These F-strains contain the alpha-amylase gene amyL of a chromosome copies.
The conversion zygote that obtains has the resistance to erythromycin (5 μ g/ml).
Amplification box chromosomal integration:
To transform the zygote bacterium colony and on the LBPSG flat board that contains erythromycin (5 μ g/ml), rule, and, grow the amplification vector plasmid integration and go into chromosomal bacterium colony 50 ℃ of cultivations.Then with the single colony inoculation on these 50 ℃ of flat boards to not containing in the antibiotic liquid growth medium, 30 ℃ of propagation, so that plasmid replication recovers, the final excision that the result is a plasmid from the karyomit(e), lacking under the situation of selective pressure, the plasmid of cutting-out will be lost from cell.Required recombination event has taken place in this process, be that Amy-L C-does not hold the integration of coding region and in the more excision of catchment (or opposite), these cells contain the F64L-S65T-GFP gene of the downstream insertion that follows the amyL gene coding region closely, can identify based on the F64L-S65T-GFP protein expression.These cells make the flow cytometry of apparatus cell sorting function traditionally, isolate from other cells such as the FASCalibur flow cytometry.
Separation with bacterial strain of several gene copies
From the bacterial strain that as above makes up, separate and contain the coding α-Dian Fenmei of several copies and the Bacillus licheniformis of F64L-S65T-GFP gene (Bacillus lichenifomis) bacterial strain.
In the growth medium that F64L-S65T-GFP can express, breed as starting culture with the bacterial strain that as above makes up.
Take a sample from culture, suitably dilution is usually to 5 * 10 5With 5 * 10 6Between the cells/ml.
Analyze the sample of dilution on the FASCalibur flow cytometry, wherein particle is bright in the exposure at 488nm place by Argon ion laser.
Adjust the FASCalibur flow cytometry so that can be, lateral light scattering (SSC) and at about 530nm place green/yellow-green fluorescence (FL1) signal detection cell according to its forward light scattering (FSC).
With FSS or SSC is one, and FL1 sets up point diagram for another axle.
Corresponding to setting up reject gate (sort gate) than the cell of the higher FL1 fluorescence of ordinary cells tool, and the cell of the flow cytometry signal that has in this go-on-go door sub-elected from whole cell masses.
The cell that sub-elects is coated on the LBPSG flat board, cultivated 1-2 days at 37 ℃.
Cell is scraped off dull and stereotyped going up and collection.This has constituted new cell,intermediate mass.
Use new cell,intermediate mass as parent material, whole breeding, flow cytometry with cell sorting output capacity that FL1 fluorescence is more higher than ordinary cells, the cell that cultivation sub-elects repeats certain number of times until the cell mass that can measure with the FASCalibur flow cytometry compared with the significantly high FL1 cell fluorescence of beginning culture tool to produce another intermediate cell colony.
Compared with sorting cells the cell colony of the significantly high FL1 cell fluorescence of beginning culture tool, coated plate on the LBPSG flat board was cultivated 1-2 days at 37 ℃, separated single bacterium colony from these.
Cultivate bacterial strain in the growth medium that allows α-Dian Fenmei and F64L-S65T-GFP to express, single bacterium colony and former starting culture are relatively.When FL1 cell fluorescence of go up measuring as FASCalibur and α-Dian Fenmei output all increase than former starting culture, just obtained to contain the bacterial strain of several gene clones.

Claims (10)

1. structure does not contain the lay eggs method of white matter cell of the microorganism multiple copied that inserts the selected marker, comprising:
A) one section sequence is imported in the karyomit(e) of microorganism cells, make this cell chromosome contain dna structure A-P-S-A thus, wherein
A represents homologous DNA sequence,
The dna sequence dna of P presentation code target protein matter,
The S presentation code can screen the protein DNA sequence
B) breed described cell;
C) screening produces the proteinic cell of selectivity of increasing amount; And
D) cell of being identified recovery c); And randomly
E) cell that reclaims in the use step d) is as parent material repeating step b-d.
2. be fluorescigenic protein under suitable exposure according to the process of claim 1 wherein that this can screen protein.
3. according to the method for claim 2, wherein this fluorescing proteins is green fluorescent protein (GFP) or its mutation.
4. be by making the flow cytometry of apparatus cell sorting ability according to the method for claim 2 or 3, screening step C wherein), screening fluoresces, and the cell that can screen proteinic amount and increase carries out.
5. according to the method for claim 4, wherein this fluorescing proteins is green fluorescent protein (GFP) or its mutation, in the screening step, makes GFP or its mutation send fluorescence under suitable exposure.
6. according to any means of claim 1-5, wherein this microorganism cells is a bacterial cell.
7. according to the method for claim 6, bacterial cell wherein is the cell that genus bacillus (Bacillus) belongs to, subtilis (Bacillus subtilis) particularly, slow genus bacillus (Bacillus lentus), bacillus brevis (Bacillus brevis), bacillus acidocldarius (Bacillus stearothermophus), Alkaliphilic bacillus (Baci ' lusalkalophlus), bacillus amyloliquefaciens (Bacillus amyloliquefaciens), Bacillus coagulans (Bacillus coagulans), Bacillus circulans (Bacilluscirculans), bacillus lautus (Bacillus lautus), bacillus thuringiensis (Bacillus thuringiensis) is put under house arrest the cell of genus bacillus (Bacillus clausii) or Bacillus licheniformis (Bacillus lichenifomis).
8. the method for aforementioned any claim, wherein this target protein matter is a kind of enzyme, particularly the protein enzyme, lipase, amylase, tilactase, Starch debranching enzyme, cellulase, glucose isomerase, protein disulfide isomerase, cyclodextrin glucanotransferase (CGTase, cyclodextrin gluconotransferase), phytase, glucose oxidase, Transglucosylase, laccase, or zytase.
9. according to the obtainable microorganism multiple copied of any method of the claim 1-8 white matter cell of laying eggs, it is characterized in that this cell contains a plurality of copies and expresses gene and a plurality of copy of target protein matter (" P ") and express the gene that can screen protein (" S ").
10. produce the method for at least one target protein matter in microorganism cells, this method comprises:
(i) under the condition that allows target protein matter to produce, cultivate according to the microorganism multiple copied of the claim 9 white matter cell of laying eggs; And
(ii) from laying eggs the white matter cell, the multiple copied of the nutrient solution that obtains or microorganism reclaims this target protein matter.
CN 98806873 1997-07-03 1998-07-02 Protein producing cell containing multiple copies of a desired gene and a screenable marker but no selection marker Pending CN1261918A (en)

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