CA1305932C - Process for the preparation of human antithrombin iii (atiii), vectors and host cells suitable for this purpose, biologically active atiii obtained in this way and medicaments containing the latter - Google Patents
Process for the preparation of human antithrombin iii (atiii), vectors and host cells suitable for this purpose, biologically active atiii obtained in this way and medicaments containing the latterInfo
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- CA1305932C CA1305932C CA000542428A CA542428A CA1305932C CA 1305932 C CA1305932 C CA 1305932C CA 000542428 A CA000542428 A CA 000542428A CA 542428 A CA542428 A CA 542428A CA 1305932 C CA1305932 C CA 1305932C
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
- C07K14/8121—Serpins
- C07K14/8128—Antithrombin III
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/108—Plasmid DNA episomal vectors
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Abstract
ABSTRACT OF THE DISCLOSURE
Human antithrombin III (ATIII) which has progressive in-hibitor activity and is stimulated by heparin is advant-geously obtained by expression after cotransfection of mammalian cells whose dihydrofolate reductase (DHFR) ac-tivity is deficient (dhfr-) with a) vectors which contain the ATIII cDNA, and b) vectors which carry the DHFR gene, in particular after gene amplification using methotrexate.
Human antithrombin III (ATIII) which has progressive in-hibitor activity and is stimulated by heparin is advant-geously obtained by expression after cotransfection of mammalian cells whose dihydrofolate reductase (DHFR) ac-tivity is deficient (dhfr-) with a) vectors which contain the ATIII cDNA, and b) vectors which carry the DHFR gene, in particular after gene amplification using methotrexate.
Description
13C;~S932 ~EHRIHGWERKE AKTIENGESELLSCHAFT HOE 86/~ 022 Dr.KL/St Specification A process for the preparation of human antithrombin III
(ATIII), vectors and host cells suitable for this purpose, biologically active ATIII obtained in this way, and medi-caments containing the latter.
The European Patent Application with the pubiication No.
O 090 505 discloses the preparation and characterizatio !
of the cDNA which codes for ATIII and the preparation of a human ATIII in E. coli. This Application also contains de-tails of how AT~II can be prepared in mammalian cell cul-tures. A suitable vector is stated to be a paR322 deriva-tive which contains a marker for selection in E. coli together with an E. coli origin of replication. The vec-tor is said also to contain the SV40 origin of replica-tion (derived from a 342bp PvuII-HindIII fragment).
In contrast, the present invention relates to a particu-larly advantageous process in which the host cell used is a mammalian cell which is able to produce dihydrofolate reductase either not at all or to an inadequate extent, and in which this host cell is cotransfected with two expression vectors, one ot which carries the ATIII gene and the other brings about the production of dihydrofol-ate reductase.
Thus the invention relates to a process for the prepara-tion of ATIII in mammalian cell culture using an expres-sion vector which contains the ATIII cDNA, which comprises ~ use as the mammalian cell of a dhfr cell which is co-;~; transfected not only with the said vector but also with another vector which carries a DHFR gene. Further aspects of the invention and its preferred embodiments are ex-;~ 35 pla~ined in detail hereinafter and defined in the patent claims.
~' ~",~, ,',''~ ~ :
, , 13~932 The cotransf-ction system according to th- invention thus makes use of t~o expression vectors the tirst being any desired vector, tor example a pPR322 deriv-tive in ~hich the ATII1 gene is under the control in m~nner kno~n S per se, of promoter ~hich is ctive in eukaryotic cells ~hereas in the second vector, uhich codes for dihydrofo-l~te reduetase and thus ~lso makes gene mplitic-tion possible, the expression ot the DHFR gene is contro(led by a DNA se~uence of the pBR322 ori region.
In n e-bod~ment of the ~nvent~on process for i prov~ng expression of proteln 18 pro~ided, vhich comprlses cotr-nsfectlng n~mmslian dhfr~ cell ~lth first vector cont-~nlng the cDNA for s-~t protein ~nd a secont vector ~hlch carrie~ a DHPR gene linket to the PvuII/EcoRI segment of pBR322 or promoter-active part~ therefrom in v~ per ltting ~e~k espres~io~ of ~lt DBF~-gene . .
The tigures shou particularly preferred embodiments of the invention by ~y ot example:
Figure 1, ~nd its continuation Figure la, describe the con~truction ot the vector pSVA S~OP 1 uhich cont-ins the pUR322 EcoRl-Pvull tragment, the Hindlll-Pvull tr~gment ~ith the origin of replication, and the promoter for the ~arly ~nd l~te transcripts, together uith the polyadeny-l~tion site from the UamHI-Hpal fragment trom the SV40 geno~e and, in addition, the translation stop codon from the plasmid pUt12 STOP;
figure 2 then sho~s the incorporation ot the DNA sequence ~hich codes for ATIII into the vector pSVA STOP 1;
Figure 3 describes the construction of the vectors pMTVA-dhfr ~which contains the MMTV-LTR promoter) and pSVOAdhfr (~hich has no eukaryotic or viral promoter) ; and Figure 4 sho~s ATIII expression rates of cell ~ines each of ~hich contain one of the t~o last-mentioned vectors ~ and uere obtained via gene amplification with methotrex-;-i ate.
'~'~';
.
It i5 expedient if not only the first vector, ~hich con-tains the\ATIII gene, but also the second vector ~;th the DUFR gene takes the form of a p~R322 derivative. Vectors of this type have the advantage that they can be amplified ,,~
i ,"~ .
13û~932 in E. coli and, at the same time, can be used as "shuttle vectors" in eukaryotic cells. It is advantageous to use as selection marker the ampicillin-resistance gene which is contained in the pBR322 EcoRI-PvuII fragment. Of course, it is possible in a manner known per se to incor-porate other, or additional, markers and to modify or eliminate inessential regions.
A further preferred embodiment of the vectors comprises the translation stop sequence from pUCi2 STOP (M. ~roker and E. Amann, Appl. Microbiol. ~iotechnol. 23 (1986) 294-296) being located downstream of the structural genes.
Translation stop codons in all three reading framesare located on this DNA segment, which permits the expression of modified genes which do not have their own translation stop s;gnal.
Mammalian cells which are able to produce dihydrofolate reductase either not at all or to an inadequate extent (dhfr ), and suitable vectors which are able to compen-sate for this defect, are generally known tErnst-L.
Winnacker, Gene und Klone, eine EinfUhrung in die Gen-technologie (Genes and Clones, an Introduction to Gene Manipulation), VCH Verlagsgesellschaft Weinheim, 1984, pages 267/268, 282, 289). Thus, for example, the isola-tion of CHO cell mutants which are deficient in dihydro-folate reductase activity is described by G. Urlaub and L.A. Chasin, Proc. Natl. Acad. Sci~ USA 77 (1980), 4216-4220. Cell lines of the dhfr type are obtained in this way. It is also possible analogously to prepare other mammalian cell lines in a reproducible manner. The mu-tants are triply auxotrophic and require glycine, a purine nucleotide such as hypoxanthine, and thymidine for growth.
Vectors which are able to insert the dihydrofolate reduc-tase gene into animal cells are described by, for example, F. Lee et al., Nature 294 (1981) 288. This entails use of the cDNA which codes for mouse dihydrofolate reductase ~, .
13~55~;~Z
(A.C.Y. Chang et al., Nature 275 (1978) 617-624)~ It is possible in an analogous manner to use other genes for dihydrofolate reductase which are functional ;n mammalian cells.
s The system is preferably the CH0 dhfr cell and the mouse DHFR gene.
Preferred expression vectors for ATIII make use of the early promoter from the DNA region of the SV40 origin of replication, of the human metallothioneine II promoter (M. Karin and R.I. Richards, Nature 299 (1982) 797-802), of the enhancer-promoter region of an immediate early gene from HCMV (human cytomegalovirus, M. 3Oshart et al., Cell 41 (1985) 521-530), or of the drosophila heat shock protein 70 (hsp 70) promoter (Holmgren et al., Cell 18 (1979) 1359-1370). The expression of dihydrofolate reduc-tase can take place under the control of a promoter which is weakly active in the mammalian cell line, such as the MMTV-LTR (mouse mammary tumor v;rus-long terminal repeat, F. Lee et al., loc. cit.) or even, which is a particular embodiment of the invention, without an eukaryotic promoter.
In the latter case the DHFR gene is located immediately upstream of a p3R322 DNA sequence which, in mammalian cells, is evidently recognized as a weak promoter (K.-D.
Langner et al., Proc. Natl. Acad. Sci. USA 83 (1986) 1598-1602).
Suitable express;on systems are also described in US Pat-ents 4 399 216 and 4 576 821 and in the European Patent Applications with the publication numbers 0 100 521 and 0 117 058 to 0 117 060.
A further embodiment of the invention comprises use of vectors which contain no introns or splice sites. It was surprising that such vectors are so very effecti~e, be-cause RNA splice sites are said in European Patent Application A2 090 505 to be necessary for vectors for the expression :L3G~S~3~
of ATI I I in cell cultures.
It is known that dihydrofolate reductase is inhibited by methotrexate. For this reason cells which grow in media containing no glycine, hypoxanthine and thymidine have their growth inhibited, or are killed, by methotrexate.
It is possible by varying the methotrexate concentration in the medium and the cell density to select cells which grow in the presence of methotrexate. These cells are, by reason of an amplification of the DHFR genes and an increased production of the enzyme DHFR associated there-with, resistant to the selected methotrexate concentra-tion. Surviving cells can again be exposed to increased methotrexate concentrations, which then results ;n cell lines having an even larger number of DHFR genes.
The gene amplification can be effected by two different techniques:
1. The cells are amplified by straightforward transfers into increasing methotrexate concentrations. This results in a genetically heterologous cell population which contains cells having different degrees of amplification.
(ATIII), vectors and host cells suitable for this purpose, biologically active ATIII obtained in this way, and medi-caments containing the latter.
The European Patent Application with the pubiication No.
O 090 505 discloses the preparation and characterizatio !
of the cDNA which codes for ATIII and the preparation of a human ATIII in E. coli. This Application also contains de-tails of how AT~II can be prepared in mammalian cell cul-tures. A suitable vector is stated to be a paR322 deriva-tive which contains a marker for selection in E. coli together with an E. coli origin of replication. The vec-tor is said also to contain the SV40 origin of replica-tion (derived from a 342bp PvuII-HindIII fragment).
In contrast, the present invention relates to a particu-larly advantageous process in which the host cell used is a mammalian cell which is able to produce dihydrofolate reductase either not at all or to an inadequate extent, and in which this host cell is cotransfected with two expression vectors, one ot which carries the ATIII gene and the other brings about the production of dihydrofol-ate reductase.
Thus the invention relates to a process for the prepara-tion of ATIII in mammalian cell culture using an expres-sion vector which contains the ATIII cDNA, which comprises ~ use as the mammalian cell of a dhfr cell which is co-;~; transfected not only with the said vector but also with another vector which carries a DHFR gene. Further aspects of the invention and its preferred embodiments are ex-;~ 35 pla~ined in detail hereinafter and defined in the patent claims.
~' ~",~, ,',''~ ~ :
, , 13~932 The cotransf-ction system according to th- invention thus makes use of t~o expression vectors the tirst being any desired vector, tor example a pPR322 deriv-tive in ~hich the ATII1 gene is under the control in m~nner kno~n S per se, of promoter ~hich is ctive in eukaryotic cells ~hereas in the second vector, uhich codes for dihydrofo-l~te reduetase and thus ~lso makes gene mplitic-tion possible, the expression ot the DHFR gene is contro(led by a DNA se~uence of the pBR322 ori region.
In n e-bod~ment of the ~nvent~on process for i prov~ng expression of proteln 18 pro~ided, vhich comprlses cotr-nsfectlng n~mmslian dhfr~ cell ~lth first vector cont-~nlng the cDNA for s-~t protein ~nd a secont vector ~hlch carrie~ a DHPR gene linket to the PvuII/EcoRI segment of pBR322 or promoter-active part~ therefrom in v~ per ltting ~e~k espres~io~ of ~lt DBF~-gene . .
The tigures shou particularly preferred embodiments of the invention by ~y ot example:
Figure 1, ~nd its continuation Figure la, describe the con~truction ot the vector pSVA S~OP 1 uhich cont-ins the pUR322 EcoRl-Pvull tragment, the Hindlll-Pvull tr~gment ~ith the origin of replication, and the promoter for the ~arly ~nd l~te transcripts, together uith the polyadeny-l~tion site from the UamHI-Hpal fragment trom the SV40 geno~e and, in addition, the translation stop codon from the plasmid pUt12 STOP;
figure 2 then sho~s the incorporation ot the DNA sequence ~hich codes for ATIII into the vector pSVA STOP 1;
Figure 3 describes the construction of the vectors pMTVA-dhfr ~which contains the MMTV-LTR promoter) and pSVOAdhfr (~hich has no eukaryotic or viral promoter) ; and Figure 4 sho~s ATIII expression rates of cell ~ines each of ~hich contain one of the t~o last-mentioned vectors ~ and uere obtained via gene amplification with methotrex-;-i ate.
'~'~';
.
It i5 expedient if not only the first vector, ~hich con-tains the\ATIII gene, but also the second vector ~;th the DUFR gene takes the form of a p~R322 derivative. Vectors of this type have the advantage that they can be amplified ,,~
i ,"~ .
13û~932 in E. coli and, at the same time, can be used as "shuttle vectors" in eukaryotic cells. It is advantageous to use as selection marker the ampicillin-resistance gene which is contained in the pBR322 EcoRI-PvuII fragment. Of course, it is possible in a manner known per se to incor-porate other, or additional, markers and to modify or eliminate inessential regions.
A further preferred embodiment of the vectors comprises the translation stop sequence from pUCi2 STOP (M. ~roker and E. Amann, Appl. Microbiol. ~iotechnol. 23 (1986) 294-296) being located downstream of the structural genes.
Translation stop codons in all three reading framesare located on this DNA segment, which permits the expression of modified genes which do not have their own translation stop s;gnal.
Mammalian cells which are able to produce dihydrofolate reductase either not at all or to an inadequate extent (dhfr ), and suitable vectors which are able to compen-sate for this defect, are generally known tErnst-L.
Winnacker, Gene und Klone, eine EinfUhrung in die Gen-technologie (Genes and Clones, an Introduction to Gene Manipulation), VCH Verlagsgesellschaft Weinheim, 1984, pages 267/268, 282, 289). Thus, for example, the isola-tion of CHO cell mutants which are deficient in dihydro-folate reductase activity is described by G. Urlaub and L.A. Chasin, Proc. Natl. Acad. Sci~ USA 77 (1980), 4216-4220. Cell lines of the dhfr type are obtained in this way. It is also possible analogously to prepare other mammalian cell lines in a reproducible manner. The mu-tants are triply auxotrophic and require glycine, a purine nucleotide such as hypoxanthine, and thymidine for growth.
Vectors which are able to insert the dihydrofolate reduc-tase gene into animal cells are described by, for example, F. Lee et al., Nature 294 (1981) 288. This entails use of the cDNA which codes for mouse dihydrofolate reductase ~, .
13~55~;~Z
(A.C.Y. Chang et al., Nature 275 (1978) 617-624)~ It is possible in an analogous manner to use other genes for dihydrofolate reductase which are functional ;n mammalian cells.
s The system is preferably the CH0 dhfr cell and the mouse DHFR gene.
Preferred expression vectors for ATIII make use of the early promoter from the DNA region of the SV40 origin of replication, of the human metallothioneine II promoter (M. Karin and R.I. Richards, Nature 299 (1982) 797-802), of the enhancer-promoter region of an immediate early gene from HCMV (human cytomegalovirus, M. 3Oshart et al., Cell 41 (1985) 521-530), or of the drosophila heat shock protein 70 (hsp 70) promoter (Holmgren et al., Cell 18 (1979) 1359-1370). The expression of dihydrofolate reduc-tase can take place under the control of a promoter which is weakly active in the mammalian cell line, such as the MMTV-LTR (mouse mammary tumor v;rus-long terminal repeat, F. Lee et al., loc. cit.) or even, which is a particular embodiment of the invention, without an eukaryotic promoter.
In the latter case the DHFR gene is located immediately upstream of a p3R322 DNA sequence which, in mammalian cells, is evidently recognized as a weak promoter (K.-D.
Langner et al., Proc. Natl. Acad. Sci. USA 83 (1986) 1598-1602).
Suitable express;on systems are also described in US Pat-ents 4 399 216 and 4 576 821 and in the European Patent Applications with the publication numbers 0 100 521 and 0 117 058 to 0 117 060.
A further embodiment of the invention comprises use of vectors which contain no introns or splice sites. It was surprising that such vectors are so very effecti~e, be-cause RNA splice sites are said in European Patent Application A2 090 505 to be necessary for vectors for the expression :L3G~S~3~
of ATI I I in cell cultures.
It is known that dihydrofolate reductase is inhibited by methotrexate. For this reason cells which grow in media containing no glycine, hypoxanthine and thymidine have their growth inhibited, or are killed, by methotrexate.
It is possible by varying the methotrexate concentration in the medium and the cell density to select cells which grow in the presence of methotrexate. These cells are, by reason of an amplification of the DHFR genes and an increased production of the enzyme DHFR associated there-with, resistant to the selected methotrexate concentra-tion. Surviving cells can again be exposed to increased methotrexate concentrations, which then results ;n cell lines having an even larger number of DHFR genes.
The gene amplification can be effected by two different techniques:
1. The cells are amplified by straightforward transfers into increasing methotrexate concentrations. This results in a genetically heterologous cell population which contains cells having different degrees of amplification.
2. At each methotrexate concentration level genetically uniform cell clones are isolated and analyzed, and only the cell lines with the best expression at each level are transferred to the next higher level.
It has emerged that in cell lines cotransfected according to the invention the gene amplification with methotrexate results not only in a multiplication of the DHFR genes but also in an amplification of the ATIII gene.
To propagate animal cells in culture it is generally necessary for serum to be present in the growth medium.
It has now been found that ATIII-producing cell lines can be maintained in serum-containing and in serum-free .
13(~S`32 medium by turns, during which no reduction in the ATIII
expression rates has been found over several transfers.
This embodiment of the process according to the invention is not on~y advantageous because it is possible to save on costly serum but it also considerably facilitates the removal of the ATIII if the last stage of production is carried out without serum~
The recombinant ATIII obtained according to the invention has full biological activity. It is, just as is the natu-ra~ product isolated from human plasma, stimulated by heparin. Thus, like the natural proàuct, it is suitable for the preparation of medicaments.
The invention is illustrated in detail in the examples vhich follow. The vector constructions are additional~y illustrated by the Figures 1 to 3, the drawings not, in general, being true to scale. In particular, the repre-sentations of small fragments and polylinker sequences are "stretched" as a rule.
Example 1 a) Construction of an expression vector for animal cells The plasmid pSV2dhfr ~Fig. 1) (Lee et al., loc. cit.) was cut with HindIII and EcoRI, and the 2.65 kb vector frag-ment ~hich carries the SV40 early promoter ~as iso~ated.
A 67 bp HindIII-EcoRI fragment from pUC12 STOP (Broker and Amann, loc. cit.) was ligated into the vector which had been pretreated in this ~ay, which resulted in the plasmid pSV2 STOP. Translation stop codons are located in all three reading frames of the 67 bp fragment from ~ pUC12 STOP. pSV2 STOP ~as Linearized with SacI, and the ;~ 35 resulting 3' protruding end ~as removed using the 3' 5' exonuclease activity of DNA polymerase I. EcoRI diges-tion ~as then carried out. After ligation to an EcoRI-HpaI fragment 133 bp in size from pBB3 (Fig. 1a) (B.
:,. . .
, " ~
, - ~3~3;~ .
30urachot et al., EMPO J. 1 ~1982) 895-900), which car-ries the SV40 polyadenylation signal for early trans-cripts, the expression vector pSVA STOP1 was obtained.
The polyadenylation site can also be isolated from the vector pIG6 t~ourachot et al., loc. cit.). It is poss-ible in the same way to isolate the 133bp BamHI-HpaI frag-ment from the SV40 genome, to fill in the BamHI cleavage site, and to attach an EcoRI linker.
Thus, pSVA STOP1 carries, between the SV40 early promoter and the SV40 polyadenylation signal for early transcripts, a cloning polylinker with three unique restriction sites (HindIlI-Sall-XbaI) and a sequence with translation stop codons in all three reading frames.
b) Construction of an ATIII expression vector.
ATIII cDNA is disclosed in European Application A2 O O9O 505 (or U.S. Patent No. 4,~17,294) and can be prepared as described there or synthesized by customary processes. Genman Patent Application P 36 18 638.4 proposes the plasmid p~AT6 (Figure 1) which contains the ATIII cDNA in the Smal cleavage site of the plasmid pUC13.
The subcloning plasmid for ATIII cDNA, p3AT6 (Fig. 2), was linearized by cutting at the unique EcoRI site. The resulting 5' protruding end was made blunt-ended by fill-ing in the complementary strand with DNA polymerase I(Klenow fragment) and then ligated with the SalI linker 5'd(pGGTCGACC) 3'.
Subsequent XbaI digestion allowed a DNA fragment which was about 1400 bp in size and coded for the complete pre-ATIII to be isolated. pSVATIlI was obtained by ligatingthis fragment into the expression vector pSVA STOP1 which had been cut with SalI and XbaI. The ATIII transcription unit on pSVATIII has no mRNA splice sites.
13~593Z
c) Construction of DHFR expression vectors for cotransfec-t i on .
The starting point for the D-HFR vectors used for the co-transfection was the plasmid pMTVdhfr (Lee et al., loc.
cit.). pMTVdhfr (Fig. 3) was cut with BglII, and the protruding 5' ends of the DNA were filled in with DNA
polymerase I (Klenow fragment). After digestion with EcoRI, a fragment 4.47 kb in size was isolated and liga-ted with a 133 bp EcoRI-HpaI fragment from pBa3 (Bourachot et al.~ loc. cit.). The new plasmid pMTVAdhfr carries the mouse DHFR cD~A flanked by MMTV-LTR and the SV40 poly-adenylation site for early transcripts.
pSVOAdhfr was obtained from pMTVAdhfr by deletion of a HindIII fragment ~hich is 1450 bp in size and carries MMTV-LTR.
Neither pMTVAdhfr nor pSVOAdhfr has mRNA splice sites.
Example 2 - The pLasmids pSVATlII and pMTVAdhfr or pSVOAdhfr w?re co-transfected by the caLcium phosphate precipitation method ~Graham and von der Eb, Virology 52 (1973) 456-467) into tHO dhfr cells. For this purpose, 20 ~9 of the plasmid pSVATIlI were mixed and coprecipitated with S ~9 of the DHFR expression pLasmids pMTVAdhfr or pSVOAdhfr. The co-precipitate of the plasmid mixture was transfected into CHO dhfr cells as described above (0.5 x 106 cells in 25 cm2 cuLture flask). After 3 days the celLs were trypsinized and transferred into several 60 mm Petri dishes, to which selection medium (containing no g~ycine, hypoxanthine and thymidine) was added. The only cells to survive under these conditions are those which have under-gone stable transfection with the DHFR gene.
tolonies composed of transfected cells become visible in ., ~, , . :
, ~, 13(P~932 _ 9 _ the Petri dishes after 1-3 weeks. It was poss;ble to ach;eve the follow;ng transfect;on rates ;n th;s:
pMTVAdhfr 5 x 10 6 pSVOAdhfr 1 x 10 5 SingLe colonies were isolated and propagated ;n medium containing no glycine, hypoxanthine and thymidine.
Culture supernatants of these new cell lines (CHO SVAT
III) were tested for recombinant ATIII us;ng a spec;f;c ELISA for detecting human ATIII. It was found from th;s that, in each case, 20% of the tested CHO dhfr cell lines secreted detectable amounts of human ATIII into the medi-um. In order to determine quant;tatively the express;on rate of the ATIII-producing lines, the following standard test procedure was carried out: 0.5 x 106 cells were pla-ted out in 5 ml of medium in 25 cm2 culture flasks. The medium was changed after 24 h (5 ml~. A further 24 h later the med;um was collected for the ELISA, and the cells were tryPs;n;zed and counted. The reported f;gures represent means of at least three transfers, with the ex-pression rates rema;ning constant w;th;n a range of +Z5%.
For all the express;on rates reported hereinafter (~9/106 cells/24 h), the cell count per 25 cm2 flask at the end of the test was 1+0.25 x 106 cells. The table which fol-lows reports the expression rates of the basic clones tested ;n the manner described:
CHO SVAT III-MTVAdhfr clone 1: 0.14 ~9/106 cells/24 h clone 2: 0.11 ~9/106 cells/24 h CHO SVAT III-SVOAdhfr clone 1: O.Oô ~9/106 cells/24 h 30clone 2: 0.14 ~9/106 cells/24 h.
Example 3 Amplification of the integrated ATIII sequences a) Amplification without isolat;on of ind;v;dual clones CHO SVAT III-MTVAdhfr (clones 1 and 2) and CHO SVAT
13~593;2 III-SVOAdhfr (clones 1 and 2) were successively transferred into increas;ng methotrexate (Mtx) concentrat;ons The expression rates (~9/10 cells/24h) determined by the stan-dard method described above were as follows:
Mtx CHO SVAT III-pMTVAdhfr CH0 SVAT III-SVOAdhfr Clone 1 Clone 2 Clone 1 Clone 2 0 0.14 0.11 0.08 0.14 10 0.050.34 0 54 _ _ 0.15 - 0.60 0,b4 2.7 0.3 0.55 1.15 - 3.3 0.6 1.18 2.00 0.86 1 - - 1.5 15 5 ~m - 4.00 - 8.5 b) Amplification with isolation of ind;vidual clones The Mtx concentration levels used were 0.1 ~m and 1 ~m.
Z0 Clones resistant to 0.1 ~m Mtx were isolated, 8 start;ng from CHO SVAT III-MTVAdhfr (clone 2) ("B" in Fig. 4) and 11 starting from CHO SVAT III-SVOAdhfr (clone 1) ("A" in Fig. 4), and were characterized on the basis of their ex-pression rates (Fig. 4); the ordinate shows the yield of ATIII in ~9/106 cells/24 h. All these clones produce a larger amount of ATIII than does the relevant starting clone (see above). The expression rates of the clones growing at 0.1 ~m Mtx were between 0.15 and 0.8 ~9/106 cells/24 h starting from CHO SVAT lII-MTVAdhfr (clone 2) (correspond;ng to an ;ncrease by a factor of 1.4 - 7) and between 0.35 and 3.2 ~9/106 cells/24 h starting from CHO
SVAT III-SVOAdhfr (clone 1) (corresponding to an increase by a factor of 4 - 40). It was shown by Southern blots (Southern, J. Mol. Biol. 98 (1975) 503) that a specific BamHI-HindIII fragment 1400 bp in size from the transfec-ted ATIII cDNA is amplified in all the analyzed clones growing at 0.1 ~m Mtx.
13~59;~;~
A further amplification round at 1 ~m Mtx was carried out with one of the best producing cell lines resis-tant to 0.1 ~m Mtx (CHO SVAT III-SVOAdhfr, clone 1 A2;
2.2 ~9/106 cells/Z4 h; identified as "A2" in Fig. 4).
It was Possible in this to isolate, on testing 9 clones in turn, a celL line (CHO SVAT III-SVOAdhfr clone 1 A27) which produces about 10 ~9/106 cells/24 h.
Example 4 Synthesis of ATIII in serum-free medium The CHO SVAT III-SVOAdhfr clone 1 A27 (1 ~m Mtx) was cultured in HamF12 selection medium (containing no gly-cine, thymidine and hypoxanthine) in 175 cm2 cultureflasks in the presence of 10~ (v/v) fetal calf serum to about 80% confluence, and the cells were subsequently washed with serum-free Iskoves medium and then maintained in this medium for 48 h to produce ATIII. The concentra-tion of ATIII in the medium after this time was 5 - 6 ~g/ml.
The cells were then maintained in serum-containing medium for Z4 - 48 h until another production round lasting 48 h was commenced in serum-free medium. It was possible to repeat the described production cycle severa~ times with-out a fall in the ATIII expression rates.
Example 5 Purification and characterization of ATIII from animal cells ATIII was concentrated both from serum-containing and from serum-free media by affinity chromatography on heparin-SEPHAROSE(R) (Miller-Anderssen et al., Thromb.
Res. 5 (1974) 439 - 452). The concentrated material show-ed in the Ouchterlony immunodiffusion test (Prog. Allergy 5 (1958) 1) immunological identity with ATIII obtained from human serum. Western blots with ATIII-specific 13~9~Z
' - 12 -antibodies also showed identity between the ATIIl from human plasma and the recombinant ATIII obtained from CHO
cells.
The ATIII secreted by CHO cells had both heparin cofactor (Hensen and Loeliger, Thromb. Diath. haemah. 9 (1963~
Suppl. 1, 18 - 29) and progressive inhibitor activity (Schrader et al., Arztl. Lab. 29 (1983) 35 - 39) to the full extent.
~:, ,.......
,
It has emerged that in cell lines cotransfected according to the invention the gene amplification with methotrexate results not only in a multiplication of the DHFR genes but also in an amplification of the ATIII gene.
To propagate animal cells in culture it is generally necessary for serum to be present in the growth medium.
It has now been found that ATIII-producing cell lines can be maintained in serum-containing and in serum-free .
13(~S`32 medium by turns, during which no reduction in the ATIII
expression rates has been found over several transfers.
This embodiment of the process according to the invention is not on~y advantageous because it is possible to save on costly serum but it also considerably facilitates the removal of the ATIII if the last stage of production is carried out without serum~
The recombinant ATIII obtained according to the invention has full biological activity. It is, just as is the natu-ra~ product isolated from human plasma, stimulated by heparin. Thus, like the natural proàuct, it is suitable for the preparation of medicaments.
The invention is illustrated in detail in the examples vhich follow. The vector constructions are additional~y illustrated by the Figures 1 to 3, the drawings not, in general, being true to scale. In particular, the repre-sentations of small fragments and polylinker sequences are "stretched" as a rule.
Example 1 a) Construction of an expression vector for animal cells The plasmid pSV2dhfr ~Fig. 1) (Lee et al., loc. cit.) was cut with HindIII and EcoRI, and the 2.65 kb vector frag-ment ~hich carries the SV40 early promoter ~as iso~ated.
A 67 bp HindIII-EcoRI fragment from pUC12 STOP (Broker and Amann, loc. cit.) was ligated into the vector which had been pretreated in this ~ay, which resulted in the plasmid pSV2 STOP. Translation stop codons are located in all three reading frames of the 67 bp fragment from ~ pUC12 STOP. pSV2 STOP ~as Linearized with SacI, and the ;~ 35 resulting 3' protruding end ~as removed using the 3' 5' exonuclease activity of DNA polymerase I. EcoRI diges-tion ~as then carried out. After ligation to an EcoRI-HpaI fragment 133 bp in size from pBB3 (Fig. 1a) (B.
:,. . .
, " ~
, - ~3~3;~ .
30urachot et al., EMPO J. 1 ~1982) 895-900), which car-ries the SV40 polyadenylation signal for early trans-cripts, the expression vector pSVA STOP1 was obtained.
The polyadenylation site can also be isolated from the vector pIG6 t~ourachot et al., loc. cit.). It is poss-ible in the same way to isolate the 133bp BamHI-HpaI frag-ment from the SV40 genome, to fill in the BamHI cleavage site, and to attach an EcoRI linker.
Thus, pSVA STOP1 carries, between the SV40 early promoter and the SV40 polyadenylation signal for early transcripts, a cloning polylinker with three unique restriction sites (HindIlI-Sall-XbaI) and a sequence with translation stop codons in all three reading frames.
b) Construction of an ATIII expression vector.
ATIII cDNA is disclosed in European Application A2 O O9O 505 (or U.S. Patent No. 4,~17,294) and can be prepared as described there or synthesized by customary processes. Genman Patent Application P 36 18 638.4 proposes the plasmid p~AT6 (Figure 1) which contains the ATIII cDNA in the Smal cleavage site of the plasmid pUC13.
The subcloning plasmid for ATIII cDNA, p3AT6 (Fig. 2), was linearized by cutting at the unique EcoRI site. The resulting 5' protruding end was made blunt-ended by fill-ing in the complementary strand with DNA polymerase I(Klenow fragment) and then ligated with the SalI linker 5'd(pGGTCGACC) 3'.
Subsequent XbaI digestion allowed a DNA fragment which was about 1400 bp in size and coded for the complete pre-ATIII to be isolated. pSVATIlI was obtained by ligatingthis fragment into the expression vector pSVA STOP1 which had been cut with SalI and XbaI. The ATIII transcription unit on pSVATIII has no mRNA splice sites.
13~593Z
c) Construction of DHFR expression vectors for cotransfec-t i on .
The starting point for the D-HFR vectors used for the co-transfection was the plasmid pMTVdhfr (Lee et al., loc.
cit.). pMTVdhfr (Fig. 3) was cut with BglII, and the protruding 5' ends of the DNA were filled in with DNA
polymerase I (Klenow fragment). After digestion with EcoRI, a fragment 4.47 kb in size was isolated and liga-ted with a 133 bp EcoRI-HpaI fragment from pBa3 (Bourachot et al.~ loc. cit.). The new plasmid pMTVAdhfr carries the mouse DHFR cD~A flanked by MMTV-LTR and the SV40 poly-adenylation site for early transcripts.
pSVOAdhfr was obtained from pMTVAdhfr by deletion of a HindIII fragment ~hich is 1450 bp in size and carries MMTV-LTR.
Neither pMTVAdhfr nor pSVOAdhfr has mRNA splice sites.
Example 2 - The pLasmids pSVATlII and pMTVAdhfr or pSVOAdhfr w?re co-transfected by the caLcium phosphate precipitation method ~Graham and von der Eb, Virology 52 (1973) 456-467) into tHO dhfr cells. For this purpose, 20 ~9 of the plasmid pSVATIlI were mixed and coprecipitated with S ~9 of the DHFR expression pLasmids pMTVAdhfr or pSVOAdhfr. The co-precipitate of the plasmid mixture was transfected into CHO dhfr cells as described above (0.5 x 106 cells in 25 cm2 cuLture flask). After 3 days the celLs were trypsinized and transferred into several 60 mm Petri dishes, to which selection medium (containing no g~ycine, hypoxanthine and thymidine) was added. The only cells to survive under these conditions are those which have under-gone stable transfection with the DHFR gene.
tolonies composed of transfected cells become visible in ., ~, , . :
, ~, 13(P~932 _ 9 _ the Petri dishes after 1-3 weeks. It was poss;ble to ach;eve the follow;ng transfect;on rates ;n th;s:
pMTVAdhfr 5 x 10 6 pSVOAdhfr 1 x 10 5 SingLe colonies were isolated and propagated ;n medium containing no glycine, hypoxanthine and thymidine.
Culture supernatants of these new cell lines (CHO SVAT
III) were tested for recombinant ATIII us;ng a spec;f;c ELISA for detecting human ATIII. It was found from th;s that, in each case, 20% of the tested CHO dhfr cell lines secreted detectable amounts of human ATIII into the medi-um. In order to determine quant;tatively the express;on rate of the ATIII-producing lines, the following standard test procedure was carried out: 0.5 x 106 cells were pla-ted out in 5 ml of medium in 25 cm2 culture flasks. The medium was changed after 24 h (5 ml~. A further 24 h later the med;um was collected for the ELISA, and the cells were tryPs;n;zed and counted. The reported f;gures represent means of at least three transfers, with the ex-pression rates rema;ning constant w;th;n a range of +Z5%.
For all the express;on rates reported hereinafter (~9/106 cells/24 h), the cell count per 25 cm2 flask at the end of the test was 1+0.25 x 106 cells. The table which fol-lows reports the expression rates of the basic clones tested ;n the manner described:
CHO SVAT III-MTVAdhfr clone 1: 0.14 ~9/106 cells/24 h clone 2: 0.11 ~9/106 cells/24 h CHO SVAT III-SVOAdhfr clone 1: O.Oô ~9/106 cells/24 h 30clone 2: 0.14 ~9/106 cells/24 h.
Example 3 Amplification of the integrated ATIII sequences a) Amplification without isolat;on of ind;v;dual clones CHO SVAT III-MTVAdhfr (clones 1 and 2) and CHO SVAT
13~593;2 III-SVOAdhfr (clones 1 and 2) were successively transferred into increas;ng methotrexate (Mtx) concentrat;ons The expression rates (~9/10 cells/24h) determined by the stan-dard method described above were as follows:
Mtx CHO SVAT III-pMTVAdhfr CH0 SVAT III-SVOAdhfr Clone 1 Clone 2 Clone 1 Clone 2 0 0.14 0.11 0.08 0.14 10 0.050.34 0 54 _ _ 0.15 - 0.60 0,b4 2.7 0.3 0.55 1.15 - 3.3 0.6 1.18 2.00 0.86 1 - - 1.5 15 5 ~m - 4.00 - 8.5 b) Amplification with isolation of ind;vidual clones The Mtx concentration levels used were 0.1 ~m and 1 ~m.
Z0 Clones resistant to 0.1 ~m Mtx were isolated, 8 start;ng from CHO SVAT III-MTVAdhfr (clone 2) ("B" in Fig. 4) and 11 starting from CHO SVAT III-SVOAdhfr (clone 1) ("A" in Fig. 4), and were characterized on the basis of their ex-pression rates (Fig. 4); the ordinate shows the yield of ATIII in ~9/106 cells/24 h. All these clones produce a larger amount of ATIII than does the relevant starting clone (see above). The expression rates of the clones growing at 0.1 ~m Mtx were between 0.15 and 0.8 ~9/106 cells/24 h starting from CHO SVAT lII-MTVAdhfr (clone 2) (correspond;ng to an ;ncrease by a factor of 1.4 - 7) and between 0.35 and 3.2 ~9/106 cells/24 h starting from CHO
SVAT III-SVOAdhfr (clone 1) (corresponding to an increase by a factor of 4 - 40). It was shown by Southern blots (Southern, J. Mol. Biol. 98 (1975) 503) that a specific BamHI-HindIII fragment 1400 bp in size from the transfec-ted ATIII cDNA is amplified in all the analyzed clones growing at 0.1 ~m Mtx.
13~59;~;~
A further amplification round at 1 ~m Mtx was carried out with one of the best producing cell lines resis-tant to 0.1 ~m Mtx (CHO SVAT III-SVOAdhfr, clone 1 A2;
2.2 ~9/106 cells/Z4 h; identified as "A2" in Fig. 4).
It was Possible in this to isolate, on testing 9 clones in turn, a celL line (CHO SVAT III-SVOAdhfr clone 1 A27) which produces about 10 ~9/106 cells/24 h.
Example 4 Synthesis of ATIII in serum-free medium The CHO SVAT III-SVOAdhfr clone 1 A27 (1 ~m Mtx) was cultured in HamF12 selection medium (containing no gly-cine, thymidine and hypoxanthine) in 175 cm2 cultureflasks in the presence of 10~ (v/v) fetal calf serum to about 80% confluence, and the cells were subsequently washed with serum-free Iskoves medium and then maintained in this medium for 48 h to produce ATIII. The concentra-tion of ATIII in the medium after this time was 5 - 6 ~g/ml.
The cells were then maintained in serum-containing medium for Z4 - 48 h until another production round lasting 48 h was commenced in serum-free medium. It was possible to repeat the described production cycle severa~ times with-out a fall in the ATIII expression rates.
Example 5 Purification and characterization of ATIII from animal cells ATIII was concentrated both from serum-containing and from serum-free media by affinity chromatography on heparin-SEPHAROSE(R) (Miller-Anderssen et al., Thromb.
Res. 5 (1974) 439 - 452). The concentrated material show-ed in the Ouchterlony immunodiffusion test (Prog. Allergy 5 (1958) 1) immunological identity with ATIII obtained from human serum. Western blots with ATIII-specific 13~9~Z
' - 12 -antibodies also showed identity between the ATIIl from human plasma and the recombinant ATIII obtained from CHO
cells.
The ATIII secreted by CHO cells had both heparin cofactor (Hensen and Loeliger, Thromb. Diath. haemah. 9 (1963~
Suppl. 1, 18 - 29) and progressive inhibitor activity (Schrader et al., Arztl. Lab. 29 (1983) 35 - 39) to the full extent.
~:, ,.......
,
Claims (12)
1. A process for improving expression of a protein, which comprises cotransfecting a mammalian dhfr cell with a first vector containing the cDNA for said protein and a second vector which carries a DHFR gene linked to the PvuII/EcoRI segment of pBR322 or promoter-active parts therefrom in a way permitting weak expression of said DHFR-gene.
2. A process according to claim 1, wherein the first vector contains the ATIII cDNA.
3. A process according to claim 2, wherein the first vector contains no splice sites.
4. A process according to claim 2, wherein the DHFR gene is amplified using methotrexate.
5. A process according to claim 4, wherein said ATIII
cDNA is co-amplified with said DHFR gene.
cDNA is co-amplified with said DHFR gene.
6. An expression vector for the expression of the DHFR
gene in eucaryotic cells comprising a weak promoter contained within the PvuII/EcoRI segment of pBR322, wherein said promoter is operatively linked to said DHFR gene.
gene in eucaryotic cells comprising a weak promoter contained within the PvuII/EcoRI segment of pBR322, wherein said promoter is operatively linked to said DHFR gene.
7. The vector according to claim 6 and specified as pSVOA dhfr or pMTVAdhfr as shown in Figure 3.
8. A mammalian dhfr cell cotransfected with a first vector containing the cDNA for a protein and a second vector which carries a DHFR gene linked to the PvuII/EcoRI segment of pBR322 or promoter-active parts therefrom in a way permitting weak expression of said DHFR-gene.
9. A mammalian cell according to claim 8, wherein the first vector contains the ATIII cDNA.
10. A mammalian cell according to claim 9, wherein the first vector contains no splice sites.
11. A mammalian cell according to claim 8, wherein the second vector is specified as pSVOAdhfr or pMTVAdhfr as shown in Figure 3.
12. A mammalian cell according to claim 8, 9, 10 or 11, wherein the DHFR gene is amplified using methotrexate.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DEP3624453.8 | 1986-07-19 | ||
| DE19863624453 DE3624453A1 (en) | 1986-07-19 | 1986-07-19 | METHOD FOR THE PRODUCTION OF HUMAN ANTITHROMBIN III (ATIII), VECTORS AND Host CELLS SUITABLE FOR THEM, BIOLOGICALLY ACTIVE ATIII, AND THE MEDICINAL PRODUCT CONTAINING THEM |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1305932C true CA1305932C (en) | 1992-08-04 |
Family
ID=6305549
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000542428A Expired - Lifetime CA1305932C (en) | 1986-07-19 | 1987-07-17 | Process for the preparation of human antithrombin iii (atiii), vectors and host cells suitable for this purpose, biologically active atiii obtained in this way and medicaments containing the latter |
Country Status (11)
| Country | Link |
|---|---|
| EP (1) | EP0256302B1 (en) |
| JP (2) | JP2721158B2 (en) |
| AT (1) | ATE85348T1 (en) |
| AU (1) | AU610830B2 (en) |
| CA (1) | CA1305932C (en) |
| DE (2) | DE3624453A1 (en) |
| DK (1) | DK174993B1 (en) |
| ES (1) | ES2053476T3 (en) |
| FI (1) | FI94425C (en) |
| GR (1) | GR3007700T3 (en) |
| PT (1) | PT85346B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03215430A (en) * | 1990-01-19 | 1991-09-20 | Kita Kiyoshi | Anti-coagulant for articular cavity |
| DE3901917A1 (en) * | 1989-01-24 | 1990-07-26 | Behringwerke Ag | MUTANTS OF HUMAN ANTITHROMBIN III |
| IL161909A0 (en) * | 2001-11-28 | 2005-11-20 | Sandoz Ag | Method for producing a recombinant polypeptide |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS597693B2 (en) * | 1978-01-07 | 1984-02-20 | 株式会社ミドリ十字 | Antithrombin preparation and its manufacturing method |
| JPS5846100A (en) * | 1981-09-11 | 1983-03-17 | Agency Of Ind Science & Technol | Plasmid vector having three kinds of marker and its preparation |
| EP0090505B1 (en) * | 1982-03-03 | 1990-08-08 | Genentech, Inc. | Human antithrombin iii, dna sequences therefor, expression vehicles and cloning vectors containing such sequences and cell cultures transformed thereby, a process for expressing human antithrombin iii, and pharmaceutical compositions comprising it |
| DE3382068D1 (en) * | 1982-03-15 | 1991-01-31 | Univ Columbia | METHOD FOR INTRODUCING CLONED AMPLIFICABLE GENES IN EUKARYOTIC CELLS AND FOR THE PRODUCTION OF PROTEIN PRODUCTS. |
| IL73854A0 (en) * | 1983-12-27 | 1985-03-31 | Genetics Inst | Vectors containing accessory dna for transformation of eucaryotic cells |
| IL71691A (en) * | 1984-04-27 | 1991-04-15 | Yeda Res & Dev | Production of interferon-ypsilon |
| US4656254A (en) * | 1985-12-02 | 1987-04-07 | Miles Laboratories, Inc. | Method of preparing alpha-1-proteinase inhibitor and antithrombin III |
-
1986
- 1986-07-19 DE DE19863624453 patent/DE3624453A1/en not_active Withdrawn
-
1987
- 1987-07-11 DE DE8787110027T patent/DE3783968D1/en not_active Expired - Lifetime
- 1987-07-11 AT AT87110027T patent/ATE85348T1/en not_active IP Right Cessation
- 1987-07-11 EP EP87110027A patent/EP0256302B1/en not_active Expired - Lifetime
- 1987-07-11 ES ES87110027T patent/ES2053476T3/en not_active Expired - Lifetime
- 1987-07-16 FI FI873153A patent/FI94425C/en not_active IP Right Cessation
- 1987-07-16 PT PT85346A patent/PT85346B/en unknown
- 1987-07-17 DK DK198703751A patent/DK174993B1/en not_active IP Right Cessation
- 1987-07-17 AU AU75773/87A patent/AU610830B2/en not_active Expired
- 1987-07-17 CA CA000542428A patent/CA1305932C/en not_active Expired - Lifetime
- 1987-07-18 JP JP62179879A patent/JP2721158B2/en not_active Expired - Lifetime
-
1993
- 1993-04-27 GR GR930400430T patent/GR3007700T3/el unknown
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- 1996-10-16 JP JP8273823A patent/JP2798659B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| FI94425C (en) | 1995-09-11 |
| JPH09117293A (en) | 1997-05-06 |
| DK375187A (en) | 1988-01-20 |
| DK174993B1 (en) | 2004-04-13 |
| EP0256302B1 (en) | 1993-02-03 |
| EP0256302A3 (en) | 1989-11-02 |
| JP2798659B2 (en) | 1998-09-17 |
| ES2053476T3 (en) | 1994-08-01 |
| PT85346B (en) | 1990-06-29 |
| PT85346A (en) | 1987-08-01 |
| JP2721158B2 (en) | 1998-03-04 |
| AU610830B2 (en) | 1991-05-30 |
| DE3783968D1 (en) | 1993-03-18 |
| FI873153A (en) | 1988-01-20 |
| FI873153A0 (en) | 1987-07-16 |
| DE3624453A1 (en) | 1988-01-28 |
| ATE85348T1 (en) | 1993-02-15 |
| FI94425B (en) | 1995-05-31 |
| EP0256302A2 (en) | 1988-02-24 |
| GR3007700T3 (en) | 1993-08-31 |
| DK375187D0 (en) | 1987-07-17 |
| AU7577387A (en) | 1988-03-03 |
| JPS6344898A (en) | 1988-02-25 |
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