WO1997009434A1 - Nucleases specifiques de la structure de l'adn - Google Patents
Nucleases specifiques de la structure de l'adn Download PDFInfo
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- WO1997009434A1 WO1997009434A1 PCT/GB1996/002218 GB9602218W WO9709434A1 WO 1997009434 A1 WO1997009434 A1 WO 1997009434A1 GB 9602218 W GB9602218 W GB 9602218W WO 9709434 A1 WO9709434 A1 WO 9709434A1
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- nucleotide sequence
- sequence according
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- codon
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to novel enzymes, in particular DNA structure-specific nucleases, and their use in diagnostics.
- the phage resolving enzymes are primarily DNA structure- specific nucleases that cleave branched structures in DNA, such as helical junctions and bulges, (Duckett, D.R. , et al., Eur. J. Biochem. (1992) , 207, 285-295) . They also exhibit some activity against single-base mismatches (Solaro, P.C, et al., J. Molec. Biol. (1993) , 230, 868-877) . There has recently been considerable interest in these proteins as a means of detecting mismatches, and hence mutations, in heteroduplexed DNA. (Youil, R. ,
- the random mutagenesis approach may not prove to be universal and indeed, attempts to use it in the context of other enzymes, notably the T4 endonuclease VII enzyme, has failed.
- the wild-type gene of this enzyme is known (Tomaschewski et al, Nucl. Acids Res. (1988) 15, 3632- 3633) .
- the invention provides a nucleotide sequence which encodes a DNA structure-specific nuclease but in which a codon for at least one amino acid residue has been changed as compared to the sequence of a wild-type nuclease and wherein the nuclease encoded by the sequence is less toxic to a host cell than said wild-type enzyme, and/or said nuclease does not have the catalytic activity of the wild-type enzyme but which retains the structure- selective DNA binding properties of the wild-type enzyme; provided that said DNA molecule does not encode a mutant of T7 endonuclease I selected from P54L P59S (double mutant) , E65K V64I (double mutant) , D55N, E35K or P59K.
- the reduced toxicity of the nuclease means that the nucleotide sequence may be overproduced in a host cell without being toxic to that cell.
- the said corresponding wild-type enzyme comprises T4 endonuclease VII and so the nucleotide sequence encodes a mutant of this enzyme.
- the codon change involves the replacement of a codon for an acidic amino acid residue with a codon for a non-acidic residue.
- a codon for a non-acidic residue For example, an asparatate or a glutamate residue may be replaced by a non-acidic residue such as alanine.
- Such changes have been found to give rise to mutant nucleases which have both low toxicity (and can therefore be over expressed) and which lack catalytic activity.
- a particular example of such nucleases are mutants of T4 endonuclease VII wherein the glutamate residue at position 86 in the amino acid sequence shown in Figure 1 is replaced with a non-acidic residue, in particular alanine.
- An alternative codon change involves the replacement of a codon for a histidine residue with a threonine residue.
- Nucleases obtained by expression of such nucleotide sequences have reduced toxicity as compared to wild-type.
- it has been for a mutant of T4 endonuclease VII which has a threonine in place of the histidine at position 38 in the amino acid sequence as set out in Figure 1.
- T4 endonuclease VII point mutants are also within the scope of the present invention either individually or in combination. They all have low toxicity when expressed in E. coli and exhibit structure selective binding to DNA junctions. In this way, the T4 endonuclease VII point mutants are useful as binding probes.
- the mutation is referred to in the standard way by: the amino acid (in single letter code) to be replaced, the position in the amino acid sequence shown in Figure 1 and the amino acid (in single letter code) to be substituted.
- D40A PA inactive 20 ⁇ 19 binds DNA junctions full zinc content
- MBP binds DNA junctions
- additional codon changes have been made in the sequence of the wild-type protein in order to optimise codon choice for the particular host cell.
- optimisation for expression in E. coli include the replacement of leucine codons such as TTA or CTA are replaced by CTG, CTT or CTC. Other such changes would be apparent to the skilled person. Such optimisation will increase the levels of protein expression.
- the nucleotide sequence of the invention may form part of a larger sequence.
- the larger sequence encodes a translational fusion protein which include peptides which enable purification of the product to be effected using a single step affinity chromatography step.
- a fusion protein which includes a maltose binding protein (MBP) can be readily purified on an amylose column.
- a fusion protein which includes protein A may be readily separated using an Ig sepharose column.
- a preferred nucleotide sequence of the invention comprises a sequence which comprises part or all of the altered sequence shown in Figure 1 (SEQ ID NO 2) as well as sequences which hybridise to such sequences under stringent conditions, other than wild-type sequences.
- Codon changes may be introduced into the DNA sequence of a wild-type gene which expresses a DNA site-specific nuclease using site directed mutagenesis techniques which are well known in the art.
- the DNA sequence may be constructed entirely or partly synthetically, also as is conventional in the art.
- the invention further provides a process for producing a DNA structure-specific nuclease which comprises transforming host cells with an expression vector which includes a nucleotide sequence as described above, culturing said cell and recovering DNA structure-specific nuclease produced.
- Expression vectors including such sequences as well as host cells transformed with said nucleotide sequence form further aspects of the invention.
- Host cells may be prokaryotic such as bacterial cells for instance E. coli or eukaryotic cells such as mammalian cell lines, yeast or fungal cells.
- the invention also provides a DNA structure-specific nuclease which is encoded by a nucleotide sequence as described above.
- mutant resolving enzymes which express to higher levels can be used in any application for the wild-type enzymes as described above.
- the catalytically inactive non-cleaving mutants have a possible use for the detection of mismatches in a different way. As has been suggested (Dean supra) if such a mutant binds to a mismatch without cleavage, an antibody directed against the protein would allow detection of the mismatch in a microtitre well thus removing the need for electrophoretic separation.
- Figure 1 shows the gene sequence of wild-type T4 endonuclease VII (SEQ ID NO 1) and the changes introduced in order to produce a synthetic gene (encoding wild-type protein) to serve as a starting point for generating mutants (SEQ ID NO 2) ;
- FIG. 1 illustrates the elements used in gene design
- Figure 3 shows maps of the plasmids used in the preparation of mutant nucleases
- Figure 4 shows gel electrophoresis of endonuclease VII E86A.
- Track 1 shows a mixture of proteins serving as molecular weight standards (molecular weights indicated at left in kDa) .
- Track 2 shows protein A-endonuclease VII E86A (calculated molecular weight 36kDa) ;
- Track 3 shows oligohistidine-endonuclease VII E86A (calculated molecular weight 20kDa) and
- track 4 shows endonuclease VII E86A with oligohistidine peptide removed (calculated molecular weight 18 kDa) ;
- Figure 5 shows a map of endonuclease VII showing three potential structural domains.
- a N-terminal zinc-binding domain a C-terminal domain with sequence similarity to T4 endonuclease V, and a central domain with some sequence similarity to the functionally related T7 endonuclease I .
- the latter region may form a significant part of the active site of the protein and contains the E86A mutation;
- Figure 6 shows a plasmid generated by the gene encoding endonuclease VII E86A being subcloned into pQE30;
- Figure 7 shows a map of endonuclease VII showing the location of point mutants identified in Table I .
- Figure 8 Binding isotherms for endonuclease VII and derived mutant proteins binding to a four-way DNA junction. Extent of protein binding to four-way junctions as a function of total protein concentration was estimated by gel electrophoresis. The fraction of DNA junction bound to protein was calculated for each protein concentration, and plotted against the protein concentration (calculated for a dimeric species, M)on a logarithmic scale. The data were fitted to a model for the binding process (see below) , from which the binding affinities were calculated. The points plotted are experimental data, and the lines are simulations derived using the association constants derived from the fits.
- Example 1 A synthetic gene encoding T4 endonuclease VII
- a gene was constructed from synthetic DNA to express T4 endonuclease VII in Escherichia coli. The purpose was two-fold. To increase levels of protein expression by optimised codon choice, and to introduce unique restriction sites throughout the gene to facilitate mutagenesis.
- the complete gene sequence is presented in Figure 1, with the changes introduced into the synthetic gene shown below the wild-type sequence. A total of 53 codons were changed from the wild-type T4 sequence. The major changes were in the leucine codons where the TTR and CTA codons (18 codons) , were replaced by CTG, CTT or CTC. 17 restriction sites were created within the gene.
- the gene was designed in 6 elements as shown in Figure 2. To allow expression of each part of the gene separately, a methionine codon was added at the N-terminal end and a stop codon at the C-terminal end, as well as a factor Xa cleavage site (the sequence IEGR) . Restriction sites at the 5' and 3' ends were added to enable initial cloning into pAT153. Each part was constructed with two oligonucleotides (for D and F elements) or four oligonucleotides (elements A, B, C, E) . These oligonucleotides ranged in size from 52 to 108 bases.
- T4 endonuclease VII in _.. coli In order to examine numerous mutants of endonuclease VII an acceptable level of expression together with a simple purification procedure was sought. A tightly controlled expression system was also used, because of the toxicity of the protein in bacterial cells. (Kosak, H.G. et al., Eur. J. Biochem. (1990) , 194, 779-784) . To facilitate purification over expression systems that produce translational fusions with peptides, enabling purification using a single step of affinity chromatography were employed. Two such systems were employed, with fusions to protein A and the maltose binding protein (MBP) .
- MBP maltose binding protein
- the vector pMAL-p (Maina CV. , et al, Gene (1988) , 74, 365-373) contains the inducible P tac promoter controlling the expression of a malE-lacZa fusion.
- the signal sequence of the malE gene directs the synthesised protein into the cell periplasm.
- a cloning site is located in a linker between the malE gene and the lacZa sequence, which also includes a cleavage site for the protease factor Xa.
- the plasmid carries the lacl gene encoding the Lac repressor. Expression of the fusion protein is induced by addition of IPTG to the growth medium.
- the synthetic endonuclease VII gene was recovered from pATSEVII by digestion with BamHI and inserted into the two BamHI sites of the polylinker.
- the ligated plasmid was transformed into E. coli TBI.
- the plasmid map is shown in Figure 3A. Cells containing the plasmid were induced with IPTG, broken open by sonication and the protein purified by chromatography on an amylose resin column.
- the plasmid pK19PRA (Zueco et al. , Anal. Biochem.
- E. coli strains JM101 and TBI containing recombinant pK19 or pMAL-p plasmids respectively were grown at 37 * C to an absorbance of 0.6 at 660 nm from inoculation of 1 li of LB medium supplemented with 1 mM ZnCl 2 and the appropriate antibiotic.
- Transcription of the T4 endonuclease VII gene was induced by addition of IPTG to a final concentration of 1 mM and incubation for a further 2 hours at 37 * C
- the cells were harvested by centrifugation and resuspended in 400 ml of 30 mM Tris, HCl (pH8) , 20% sucrose. EDTA was added to a final concentration of 1 mM and the mixture incubated for 5-10 min at room temperature. After centrifugation at 10,000 rpm for 20 min at 4 * C, the supernatant was removed and the pellet resuspended in 400 ml of ice-cold 5 mM MgS0 4 and incubated for 10 min in an ice bath.
- the sample was then centrifuged as before, the pellet resuspended in 8 ml 0.5 M sodium phosphate (pH 7.2) and loaded on an amylose resin column eluted under gravity. After washing the column with three volumes of 10 mM sodium phosphate (pH 7.2) , 0.5M NaCl, 10 mM ⁇ -mercaptoethanol, 1 mM EGTA, 0.25 % Tween and five volumes of 10 mM sodium phosphate (pH 7.2) 0.5M NaCl, 10 mM ⁇ -mercaptoethanol, 1 mM EGTA, the protein was eluted with 10 mM maltose in 10 mM phosphate buffer (pH 7.2) , 0.5 M NaCl. 1 mM EGTA. Fractions containing the protein were pooled and the solution dialysed in 50 mM Tris.HCl (pH7.6) 1 mM DTT and 50% glycerol for 2 h
- Endonuclease VII-E86A as an oligohistidine fusion protein was prepared from 1 litre of E. coli strain M15 (pREP4) transformed with pQE30EVII-E86A.
- the cells were grown to an A660 of 0.5 and then induced with IPTG to a final concentration of 1 mM for 2 hours. Cells were harvested and lysed as above.
- the protein was purified by affinity chromatography using nickel- TA (nitrilo-tri-acetate) resin (Qiagen) .
- the cell extract was applied to the column in 50 mM phosphate, pH 8.0, 300 mM NaCl, and eluted using a gradient of imidazole from 0-500 mM in the same buffer plus 5% glycerol.
- Oligohistidine-endonuclease VII-E86A elutes in approximately 250 mM imidazole. Following this step the protein is usually >70% pure.
- the oligohistidine sequence was removed by digestion with 1:500 (w/w) Factor Xa protease at 4°C for 16 h.
- the resulting endonuclease VII-E86A was purified by chromatography on a Mono-S column.
- T4 endonuclease VII H38T Over expression of T4 endonuclease VII H38T: a low toxicity mutant Wild-type T4 endonuclease VII is highly toxic when over expressed in E. coli , and can only be expressed to low levels as a consequence. In the course of analysing the role of histidine residues by mutation, one with considerably lowered toxicity was found which can be expressed to higher levels. This mutant has histidine 38 changed to threonine. Despite the reduced toxicity, the enzyme is fully active as MBP and protein A fusions. For example, the rate constants for cleavage of DNA junctions at O'C were found to be the same for mutant and wild-type enzyme, within the experimental error imposed by protein concentration determination. T4 endonuclease VII H38T therefore has the significant practical advantages of over expression to much larger levels without any detectable alteration in properties below 30°C.
- Mutagenesis Site-directed mutagenesis was performed using the method of Kunkel (1985) (Proc. Natl. Acad. Sci. USA 82, 488-492), which involves the use of single-stranded template DNA that contains a small percentage of uracil bases in place of thymine generated in dut ung E. coli (Strain CJ236) DNA synthesis from a mutagenic primer using normal deoxyribonucleoside triphosphates regenerates the double-stranded plasmid in which the parental strand contains dU. Transformation into a dut* ung * strain leads to selective degradation of this strand, and repair synthesis of the plasmid effectively enriches for the mutagenised strand. Yields of up to 90% of the desired mutants were achieved.
- pUC119SEVII was transformed into E. coli strain CJ236 (dut ung) .
- Single-stranded DNA was prepared by transfection of a 100 ml culture of CJ236 containing pUC119SEVII in LB supplemented with appropriate antibiotics by helper phage M13K07. This was incubated at 37°C overnight with vigorous shaking to ensure good aeration.
- the culture was harvested by centrifugation at 15,000 rpm at 4°C, and the supernatant was decanted into 0.25 volumes of phage-precipitation solution (3.75 M ammonium acetate, pH 8.0, 20% polyethylene glycol (MW 8,000)) , mixed well, and left on ice for 30 minutes. After centrifugation, the pellet was resuspended in 400 ⁇ l of TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) . The phage were lysed by the addition of 400 ⁇ l of chloroform: isoamyl alcohol (24:1) followed by vortexing for 1 min. The solution was centrifuged for 5 min in an Eppendorf centrifuge, and the upper aqueous phase was extracted with phenol/chloroform until there was no material visible at the interface. The upper phase containing the DNA was recovered by precipitation with ethanol.
- a mutagenic oligonucleotide was designed to effect the change of glutamate 86 to alanine in the gene for endonuclease VII.
- the sequence was
- Second-strand synthesis was performed by the addition of 10 ⁇ l of PE3 buffer (20 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 1 mM DTT, 5 mM dNTPs) , 5 U T4 DNA ligase and 3 U T4 DNA polymerase for 5 min on ice, 5 min at room temperature, 2 h at 37°C and finally overnight at room temperature. Half of this mixture was transformed (Cohen, et al.,Proc.
- the BamHI fragment of pK19SEVII-E86A was excised and ligated into the BamHI site in the multiple cloning region of plasmid pQE30 (Bujard, et al., (1987)Meth. Enzymol. 155, 416-433) (Quiagen), to generate PQE30EVII-E86A.
- This was transformed into E. coli strain M15 (pREP4) .
- the resulting colonies were screened for the presence of the insertion, and the orientation of the insertion was determined by restriction analysis (data not shown) . Positive colonies were used for subsequent preparations of the mutant protein.
- Endonuclease VII E86A was cloned as a translational fusion with proteinA in the plasmid pK19PRA, transformed in E. coli JM101.
- the gene was under the control of the lac promoter, and was induced by addition of IPTG.
- the cell lysate was applied to an IgG-sepharose column and the fusion protein eluted in 0.5 M acetic acid, pH 3.4.
- the peak fraction was then applied to a Mono-S ion exchange column and eluted with a gradient of NaCl.
- the purity of the protein was analysed by polyacrylamide gel electrophoresis in a buffer containing SDS, and found to contain a single polypeptide migrating at the position expected for a calculated mass of 36 kDa ( Figure 4).
- Endonuclease VII E86A was also expressed as a fusion with an oligohistidine sequence (6 consecutive N-terminal histidine residues) .
- the complete coding sequence including the cleavage site for Factor Xa protease, was excised from pK19SEVII-E86A and ligated into the vector pQE30 (Bujard, et al., supra), to give a single translational reading frame that included the oligohistidine sequence ( Figure 5) .
- the gene is transcribed from a T5 promoter, under the control of two lac operator sequences.
- the resulting plasmid (pQE30EVII-E86A) ( Figure 6) was transformed into 5 E.
- oligohistidine-endonuclease VII E86A was purified by chromatography on a nickel-containing
- the endonuclease VII E86A (as fusions with either protein 25. A or oligohistidine peptide, or following removal of the oligohistidine peptide) failed to cleave four-way DNA junctions under any conditions tested (data not shown) . However, while not cleaving DNA junctions, both proteinA-endonuclease VII E86A and endonuclease VII E86A 30 acted as a inhibitors of active proteinA-endonuclease VII H38T enzyme. In the absence of other proteins, proteinA-endonuclease VII H38T cleaves a four-way DNA junction as observed previously for active endonuclease VII preparations.
- the ratio of DNA junction and 35 proteinA-endonuclease VII H38T was kept constant, and increasing concentrations of endonuclease VII E86A were added to the incubation. As the concentration of the inactive endonuclease VII E86A increased, the extent of cleavage diminished, eventually leading to complete suppression of the cleavage reaction. This indicates that endonuclease VII E86A competes for substrate with the active enzyme. These results are consistent with a normal binding of DNA by the inactive mutant, thus preventing access by the catalytically active protein (proteinA- endonuclease VII H38T) .
- Colorimetry (All mutants measured this way) Colorimetric assays were performed as described by Giedroc et al. Proc. Natl. Acad. Sci. USA. 83, (1986). The proteins were dialysed for two hours in 20 M Tris.HCl (pH 8), 600 mM NaCl and 5% glycerol. 4-(2-pyridylazo) resorcinol (PAR) was added to a final concentration of 0.1 mM to 800 ⁇ l of protein solution ranging from 1 to 2 ⁇ M. The protein-resorcinol solution was titrated with p- hydroxymercuriphenylsulphonic acid (PMPS) .
- PMPS p- hydroxymercuriphenylsulphonic acid
- PMPS is a reducing agent that binds to the cysteine residues and therefore releases the zinc.
- the zinc released by PMPS is then chelated by the resorcinol in the solution.
- the titration is followed by measuring the absorbance of Zn(II)PAR 2 at 500nm (Hunt et al, J. Biol. Chem. 259, 1984) . When this absorbance reaches a plateau, indicating that all the zinc has been released, the concentration of protein bound zinc can be calculated by using a titration of standard ZnCl 2 solutions with resorcinol obtained under the same conditions. Absorption measurements were performed on a CARY IE UV- visible spectrophotometer using 1 ml polystyrene cuvettes.
- Atomic absorption spectroscopy (H41T measured this way as check on the other method) 1.5 ml of a solution of each protein at a concentration ranging between 1 and 5 ⁇ M was dialysed overnight in 200 mM NaCl, 20 mM Tris.HCl (pH 8), 1 mM DTT. Spectroscopy was performed on a UNICAM 939/959 atomic absorption spectrometer equipped with a zinc lamp, using an acetylene flame.
- Varying amounts of each mutant and wild-type protein were incubated with 24.2 nM [5 32 P]labelled junction for 10 min at room temperature in lO ⁇ l of binding buffer 50 mM Tris- HCl (pH7.4), 100 mM NaCl, 1 mM dithiothreitol and either 1 mM EDTA or 200 ⁇ M MgCl 2 .
- K D the dissociation constant
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Abstract
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6110684A (en) * | 1998-02-04 | 2000-08-29 | Variagenics, Inc. | Mismatch detection techniques |
WO2001005800A2 (fr) * | 1999-07-18 | 2001-01-25 | Borries Kemper | Procede d'enrichissement d'heteroduplexes et son utilisation dans la detection de mutations |
WO2001062968A2 (fr) * | 2000-02-25 | 2001-08-30 | General Atomics | Enzymes de liaison nucleique mutantes et leur application dans le diagnostic, la detection et la purification |
EP1185675A1 (fr) * | 2000-03-31 | 2002-03-13 | Korea Advanced Institute of Science and Technology | Souche d'escherichia coli secretant le facteur stimulant les colonies de granulocytes humains (g-csf) |
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US5308762A (en) * | 1989-09-01 | 1994-05-03 | Vanderbilt University | T4 endonuclease V DNA repair enzyme having specific activity at low salt concentrations |
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1996
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US5308762A (en) * | 1989-09-01 | 1994-05-03 | Vanderbilt University | T4 endonuclease V DNA repair enzyme having specific activity at low salt concentrations |
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US6110684A (en) * | 1998-02-04 | 2000-08-29 | Variagenics, Inc. | Mismatch detection techniques |
EP1053352A1 (fr) * | 1998-02-04 | 2000-11-22 | Variagenics, Inc. | Procedes de detection d'un mauvais appariement |
EP1053352A4 (fr) * | 1998-02-04 | 2001-03-21 | Variagenics Inc | Procedes de detection d'un mauvais appariement |
WO2001005800A2 (fr) * | 1999-07-18 | 2001-01-25 | Borries Kemper | Procede d'enrichissement d'heteroduplexes et son utilisation dans la detection de mutations |
WO2001005800A3 (fr) * | 1999-07-19 | 2001-05-10 | Borries Kemper | Procede d'enrichissement d'heteroduplexes et son utilisation dans la detection de mutations |
WO2001062968A2 (fr) * | 2000-02-25 | 2001-08-30 | General Atomics | Enzymes de liaison nucleique mutantes et leur application dans le diagnostic, la detection et la purification |
WO2001062968A3 (fr) * | 2000-02-25 | 2002-08-08 | Gen Atomics | Enzymes de liaison nucleique mutantes et leur application dans le diagnostic, la detection et la purification |
EP1185675A1 (fr) * | 2000-03-31 | 2002-03-13 | Korea Advanced Institute of Science and Technology | Souche d'escherichia coli secretant le facteur stimulant les colonies de granulocytes humains (g-csf) |
EP1185675A4 (fr) * | 2000-03-31 | 2004-05-12 | Korea Advanced Inst Sci & Tech | Souche d'escherichia coli secretant le facteur stimulant les colonies de granulocytes humains (g-csf) |
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