WO1995011982A1 - Systeme d'expression pour lignees cellulaires eucaryotes - Google Patents

Systeme d'expression pour lignees cellulaires eucaryotes Download PDF

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Publication number
WO1995011982A1
WO1995011982A1 PCT/EP1994/003488 EP9403488W WO9511982A1 WO 1995011982 A1 WO1995011982 A1 WO 1995011982A1 EP 9403488 W EP9403488 W EP 9403488W WO 9511982 A1 WO9511982 A1 WO 9511982A1
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Prior art keywords
csf
vector system
protein
expression
cells
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English (en)
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Antonio Mele
Luigi Rotondaro
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Menarini Ricerche SpA
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Menarini Ricerche SpA
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Priority to AU79395/94A priority Critical patent/AU7939594A/en
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Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a process for the production of recombinant proteins in eukaryotic cell lines.
  • it relates to the application of an expression vector system to the production of stable eukaryotic cell lines, which are able to synthesize large quantities of active proteins, correctly processed in the post-translation phase and having a relevant therapeutic value.
  • State of the art Techniques for directing the synthesis of therapeutically important eukaryotic proteins in bacteria and yeasts are well established. Such methods are efficient but involve problems due to incorrect post- translational modifications (protein processing, glycosylation, myristilation, gamma-glutamyl-carboxylation, etc.). In addition, many proteins are incorrectly folded and may form insoluble aggregates.
  • eukaryotic cell lines [COS cells, Chinese hamster ovary (CHO) cells , NIH-3T3 cells] as hosts for the production of recombinant proteins.
  • Vectors useful for the transfer and expression of heterologous genes in eukaryotic cells usually carry viral promoters and enhancers in order to direct transcription.
  • the cDNA sequence that one intends to express is usually equipped with a polyadenylation signal, and in some vectors, with an intron, because splicing (the process of removing introns and joining of exons) seems to be essential, in some cases, for the DNA expression.
  • Polyadenilation signal and intron sequences are the sequences involved in the processing of transcripts.
  • eukaryotic vectors usually contain a prokaryotic replication origin and markers which enable selection both in eukaryotic and prokaryotic cells.
  • a problem facing many eukaryotic expression systems is the low yield of recombinant protein if compared to that of prokaryotic systems.
  • a large number of variables may contribute to poor production of the recombinant protein: poor vector stability; low vector copy number; inefficient promoters or enhancers; strong promoter/enhancer combinations that may interfere with vector replication and cause instability; lack of polyadenylation sites; lack of RNA splice sites; inefficient transport of mRNA from nucleus to cytoplasm; undesirable RNA secondary structures; unsuitable selective markers resulting in low selective pressure for plasmid maintenance; mRNA instability; protein degradation; incorrect protein processing or folding.
  • rhG-CSF recombinant human G-CSF
  • the recombinant DNA construct according to the present invention is useful for obtaining a high level of eukaryotic gene expression in the CHO dhfr " cell line, and comprises at least the following features:
  • promoter comprises both the promoter itself (nucleotide sequences recognized by RNA-polymerase molecules starting RNA synthesis) and the promoter/enhancer combination (enhancer refers to cis-acting nucleotide sequences which act on a promoter to increase its transcriptional efficiency), whereas, in case the enhancer sequences are suitably deleted, that will be clearly indicated;
  • RNA transcripts comprising the second intron and the beta-globin gene polyadenylation site; these sequences facilitate the transport from nucleus to cytoplasm and the translation of the mRNA;
  • SV4 ⁇ early region promoter is suitably modified in order to remove the SV " 4 ⁇ enhancer, (this has the effect of reducing the DHFR transcription so that efficient gene amplification can be obtained in CHO dhfr " cells).
  • said sequence hereinafter named SV1DHFR, contains the SV-+0 small-t antigen intron and the SV-10 early region polyadenylation site to facilitate the transport from nucleus to cytoplasm and the translation of mRNA encoding the DHFR enzyme.
  • the eukaryotic expression vector according to the present invention contains a DNA fragment, derived from pSV2dhfr, containing the origin of replication and ampicillin resistence gene derived from pBR322. This facilitates replication and selection of E.coli transformants.
  • the vector according to the present invention contains a DNA sequence encoding the desired protein.
  • said vector contains a cDNA, encoding an eukaryotic protein of interest, which has been modified in order to remove the sequences responsible for the destabilization of the corresponding mRNA.
  • Such sequences are located in the 3' untranslated region of the mRNA and contain repeats of the AUUUA motif which is believed to be responsible for destabilization of short-lived mRNA (Shaw and Kamen, Cell, 46, 659-667, 1986).
  • the murine cytomegalovirus (MCMV) major immediate early promoter is preferably used (Dorsch-Hasler et al., Proc. Natl. Acad. Sci., USA, 82,
  • the second intron and the polyadenylation site preferably derive from rabbit betal-globin gene (Rohrbaugh et al., Mol. Cell. Biol. 5, 147-160, 1985).
  • the sequence used as marker is preferably a DNA fragment derived from the commercially available pSV2dhfr vector (ATCC 37146) , and containing the dihydrofolate reductase (DHFR) cDNA coupled to the SV4 ⁇ early promoter for expression in eukaryotic cells.
  • a further embodiment of the present invention provides a stably transformed CHO dhfr " cell line containing multiple copies (up to 2000 per cell) of the transfected sequences and able to synthesize the desired eukaryotic protein at high levels.
  • eukaryotic protein expression include human granulocyte colony stimulating factor (hG-CSF) as well as human GM-CSF and human erythropoietin.
  • a method for producing the said eukaryotic protein which comprises culturing the previously described stably transformed CHO dhfr " cell line, containing multiple copies of the expression vector, and recovering the said protein.
  • a further aspect of the present invention relates to the preparation of the vector using the human cytomegalovirus (HCMV) major immediate early promoter and then using the obtained vector in a suitable cell line [for example, CHOdhfr " or cells of the Hep G2 line (ATCC HB8065)].
  • HCMV human cytomegalovirus
  • suitable cell line for example, CHOdhfr " or cells of the Hep G2 line (ATCC HB8065)].
  • Figure 1 illustrates plasmid pMCMV ⁇ #51-2 containing the origin of replication (ORI) and ampicillin resistence gene (Amp R) of pBR322; the MCMV major immediate early promoter (comprised between AccI and Hindlll); the sequence, for processing of the RNA transcripts, obtained from rabbit betal-globin gene (comprised between Sad and BamHI) comprising the betal-globin second intron, and polyadenylation site; and a multiple cloning site (polylinker) suitable for insertion of the desired DNA fragment encoding a desired heterologous protein.
  • Figure 2 illustrates plasmid pMCMV #53 ⁇ 2.
  • pMCMV #53-2 contains: a) a 3-7 Kbp from AccI to Bglll DNA fragment obtained from pSV2dhfr (ATCC 371-+6) containing the origin of replication and ampicillin resistence gene of pBR322, and the SV4 ⁇ small-t antigen intron and the SV40 early region polyadenylation site; b) the MCMV major immediate early promoter (a DNA fragment identical to that used in the pMCMV ⁇ #51-2 vector construction) ; c) a polylinker suitable for insertion of the desired DNA fragment encoding a desired heterologous protein.
  • the polylinker was obtained by annealing the following two complementary synthetic DNA sequences reported in SEQ ID NO: 1 and SEQ ID NO: 2.
  • Figure 3 illustrates plasmid pMCMV ⁇ G-CSF #55-4, derived from plasmid pMCMV ⁇ #51-2 by insertion of the sequence encoding hG-CSF.
  • Figure 4 illustrates plasmid pMCMV G-CSF # 7-12 derived from plasmid pMCMV #53-2 by insertion of the sequence encoding hG-CSF.
  • Figure 5 illustrates plasmid pMCMV ⁇ G-CSF-SVl-DHFR #69-1 derived from plasmid pMCMV ⁇ G-CSF #55-4 by addition of the SV1DHFR transcription unit.
  • Figure 6a illustrates the preparation of plasmid pSV2-G-CSF.
  • Figure 6b illustrates the preparation of plasmid pSV2(G-CSF-dhfr) .
  • the invention relates to a system for producing correctly processed and post-translationally modified eukaryotic proteins in CHO cells by using recombinant DNA technology.
  • these proteins are of human therapeutic or diagnostic value, such as growth Factors, lymphokines, interferons, enzymes or antigens. It will be remarked that although the production of mature proteins is preferred, hybrid proteins or protein fragments having the relevant activity may also be produced.
  • the pMCMV ⁇ #51-2 DNA construct shown in Figure 1 contains, in addition to the 2.9 Kbp from AccI to BamHI DNA fragment obtained from pSV2dhfr (ATCC 37146) , the origin of replication and ampicillin resistence gene of pBR322, and contains also the MCMV major immediate early promoter comprising nucleotides from -492 to +39 as described by Dorsch-Hasler et al. (Proc. Natl. Acad. Sci., USA, 82, 8325-8329, 1985) .
  • Such DNA fragment was obtained by PCR amplification using plasmid pON4 ⁇ 2 (mentioned in the reference of Manning and Mocarski, Virology, 167, 477-*+84, 1988) as the template.
  • a 5' primer containing the sequence SEQ ID NO:3 and a 3' primer of sequence SEQ ID NO:4 the 5 1 and 3' primers contain respectively an AccI restriction site and a Hindlll site in order to facilitate the vector preparation by using standard recombinant DNA technology (Maniatis et al. , Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1982).
  • Plasmid pMCMV ⁇ #51-2 also contains a polylinker obtained by annealing two synthetic complementary DNA sequences: SEQ ID N0:5 and SEQ ID N0:6. The presence of this multiple cloning site facilitates subsequent insertion of the DNA fragment encoding the protein of interest. Plasmid pMCMV ⁇ #51-2 also contains a DNA fragment containing the rabbit betal- globin gene second intron and the polyadenylation site corresponding to nucleotides from +478 to +1633 using the nucleotide numbering system of Rohrbaugh et al (cited before) . This fragment was obtained thanks to standard PCR technology using plasmid pUK4.7 as template.
  • Plasmid pUK4.7 contains the 4.7 Kbp fragment of Kpnl ends from rabbit betal-globin gene (Lacy et al., Cell 18, 1273-1283, 1979) inserted in the Kpnl site of plasmid pUC19 (kindly provided by courtesy of Dr. R. Hardison. The Pennsylvania State University, PA, USA).
  • the former primer contains a Sad restriction site and the latter primer contains a BamHI restriction site in order to facilitate vector construction by using standard cloning technology.
  • Plasmid pMCMV ⁇ #51-2 represents a basic eukaryotic expression system, having a strong promoter and enhancer derived from MCMV and the second intron splice site and polyadenylation signal derived from rabbit betal- globin gene. It contains a polylinker to facilitate subsequent insertion of the fragment encoding the desired protein, as well as sites for the insertion of the transcription unit SV1DHFR derived from commercially available plasmid pSV2dhfr. It should be noted that the plasmid lacks an origin of replication for mammalian cells and thus it will not replicate unless it becomes integrated into the mammalian chromosome.
  • the cells it transformed which amplified the number of copies of said plasmid, are selected using increasing levels of methotrexate (MTX) , a dihydrofolate reductase inhibitor. It is possible to obtain an amplification of about 2000 copies per cell and this results in a high efficient expression of the desired protein.
  • MTX methotrexate
  • the vector using HCMV major immediate early promoter contains the nucleotides sequence from -737 to +116 (Boshart et al., Cell, 4l, 521- 530, 1985) which was amplified by PCR, using plasmid pRL103 as template. Said plasmid is described in Pizzorno et al., J. Virol., 62, 1167-1179, 1988).
  • the PCR 5' primer includes a AccI site and has the sequence SEQ ID N0:9; the PCR 3' primer includes a Hindlll site and has the sequence SEQ ID N0:10.
  • hG-CSF expression vector The complete coding sequence of hG-CSF was amplified by standard PCR technology using plasmid pG-CSF6 (Tweardy et al, Oncogene Research 1, 209-220, 1987) as template.
  • the sequence of the 5' PCR primer is reported in SEQ ID NO:11 and that of the 3' PCR primer is reported in SEQ ID NO:12.
  • Both primers contain a Xbal site enabling the cloning of the PCR fragment, containing the hG-CSF coding sequence, into the Xbal site of pBluescript SK plasmid (Stratagene, La Jolla, CA, USA) to obtain plasmid pBS G-CSF.
  • Plasmid pMCMV ⁇ G-CSF #55-4 lacks a selection marker for eukaryotic cells and this was provided by using the SV1DHFR transcription unit obtained from the commercially available plasmid pSV2dhfr. Said unit comprises the SV4 ⁇ early region promoter (from which the enhancer has been removed by digestion with SphI) , the DHFR cDNA, the SV4 ⁇ small-1 antigen intron and early region polyadenylation site. Deletion of enhancer sequences from SV4 ⁇ early region promoter in order to reduce the level of recombinant gene expression was previously described (Gorman et al, Mol. Cell Biol. 2, 1044-1051, 1982). The lack of an enhancer leads to inefficient transcription of the DHFR cDNA so that cells having multiple integrated copies of the DHFR gene are preferably selected by methotrexate (MTX) selection.
  • MTX methotrexate
  • the authors of the present invention isolated, from pSV2dhfr, the 1.8 Kbp from SphI to BamHI fragment containing an enhancerless SV40 early promoter, the DHFR cDNA and the SV4 ⁇ small-1 antigen intron and polyadenylation site.
  • This fragment was cloned into the pUC131 vector, between the SphI and BamHI sites.
  • This cloning step positioned a new EcoRV site next to the SphI site and both a Sail and a PstI site next to the BamHI site.
  • the DHFR transcription unit was isolated as EcoRV to PstI end fragment and cloned into the pUC130 vector, between Hindi and PstI sites. From the last construct the DHFR transcription unit, named SV1DHFR, can be isolated as a BamHI fragment.
  • Plasmids pUC130 and pUC131 were constructed by replacing the PvuII fragment into pUC ⁇ with that containing the polylinker from M13tgl30 or M13tgl31, respectively (Kieny et al. Gene 26, 91-99, 1983).
  • the SV1DHFR transcription unit contained between two BamHI sites was ligated to plasmid pMCMV ⁇ CSF #55-4 which has also been BamHI cleaved, as shown in Fig.5.
  • the resulting plasmid pMCMV ⁇ G-CSF-SV1DHFR #69-1 (Fig.5) contains all the elements necessary for integration, selection, amplification, and high rhG-CSF expression.
  • the CHO host cell is a CHO dhfr " cell line described by Urlaub G. and Chasin L.A. , PNAS 77:4216-4220, 1980 and obtained from Dr. P.Reddy at the istar Institute Philadelphia, PA, USA.
  • CHOdhfr cells were transformed by transfection with plasmid pMCMV ⁇ G-
  • CSF-SV1DHFR #69-1 (Fig.5) and dhfr + transformants were selected in nucleoside free medium.
  • rhG-CSF stable transformants in which amplification of the transfected sequences occurred were selected in a nucleoside free maximn containing MTX at concentration 160-320 mM; MTX resistant clones were subsequently isolated and some of them shown to express rhG-CSG at higher levels, compared to those shown by the transformants previously selected in the nucleoside free medium.
  • the MCMV promoter-based vectors i.e. pMCMV ⁇ G- CSF #55-4, pMCMV G-CSF #57-12, pMCMV ⁇ G-CSFSVIDHFR #69"D as claimed her, allow a G-CSF productivity of about 6-100 times higher than that obtained with the SV40 early promoter-based vector, the pSV2 (G-CSF-dhfr) as described in Table 3- Said vector was constructed by fusing parts of two vectors; pSV2neo (ATCC 37149) and pSV2dhfr (Subramani et al, Mol. Cell Biol. 1, 854-864, 1981).
  • pSV2 (G-CSF-dhfr) the hG-CSF cDNA from pG-
  • CSF6 plasmid (Tweardy et al), contains in the 3' untranslated region AU destabilizing sequences, is under the transcriptional control of SV40 early region promoter and uses processing signals for transcripts from
  • SV4 ⁇ (SV40 small-t antigen intron and early region polyadenylation site) .
  • the vector pSV2 (G-CSF-dhfr) is the nearest if related to the constructs described in the prior art for G-CSF expression. It should be noted, moreover, that since plasmid pMCMV ⁇ G-CSF #55-4 and pMCMV #57-12 both contain: 1) the same promoter; 2) the same cDNA of the G-CSF gene deleted of destabilizing sequences; and 3) signals for 3' transcripts processing (intron and polyadenylation site from beta-globin and SV4 ⁇ , respectively) it is surprising the 10-times difference in the efficiency of expression observed between the two vectors mentioned above.
  • Table 3 shows the unpredictable increase of rhG-CSf yield obtained when using vector pMCMV ⁇ G-CSF #55-4(and vector pMCMV ⁇ G-CSFSVIDHFR #69-1).
  • telomeres were cultivated in MEM alpha medium containing nucleotides and deoxyribonucleosides (Gibco BRL) , supplemented with 5% fetal calf serum. Cells were plated in a 60mm dish and were transfected with the indicated amount of plasmid DNA using the cationic lipid Lipofectin Reagent (Gibco BRL) according to the manifacture's instruction.
  • vectors pMCMVG-CSF #57"12 and 93/4-1 are equally efficient in the expression of rhG-CSF. In this case removing the mRNA destabilizing sequences is not sufficient in order to increase the expression level of the cytokine.
  • vectors pMCMV ⁇ G-CSF #55-4 and 93/4-1 higher levels of expression are obtained with the plasmid pMCMV ⁇ G-CSF #55-4 not containing the mRNA destabilizing sequences showing that the removal of these sequences is a necessary step in order to obtain an efficient expression system.
  • This fragment was cloned between the Nael and Smal sites of pMCMV ⁇ G-CSF #55-4 and pMCMVG-CSF #57-12 (both deleted by digestion of the 0.13 kbp Nael to Smal fragment) obtaining the vectors 93/2-9 and 93/4-1, respectively.
  • the vectors 93/2-9 and 93/4-1 contain a hG-CSF cDNA which includes the complete coding sequence and ATTTA repeated motif (mRNA destabilizing sequences) under the transcriptional control of the MCMV major immediate early promoter and use sequences for processing the RNA transcripts either from rabbit betal-globin gene or SV4 ⁇ , respectiwely.
  • Table 4 shows the hG-CSF yields obtained in transient expression experiments performed in CHOdhfr " cells using supercoiled plasmid DNA of pMCMV ⁇ G-CSF #55"4, pMCMVG-CSF #57-12, 93/2-9 and 93/4-1.
  • CHOdhfr cells were cultivated in MEM alpha medium containing nucleotides and deoxyribonucleosides (Gibco BRL) , supplemented with 5% fetal calf serum. Cells were plated in a 6 ⁇ mm dish and were transfected with the p indicated amount of plasmid DNA using the cationic lipid Lipofectin Reagent (Gibco BRL) according to the manifacture's instruction.
  • the NH2-terminal amino acid sequence of purified rhG-CSF has been determined.
  • the primary structure analysis of rhG-CSf was performed using a protein sequencer (Millipore Mod.6625). As a result, the amino acid sequence of 14 residues beginning from the NH2-terminus was determined and it is reported in SEQ ID N0:13.
  • G-CSF preferably hG-CSF
  • the present invention also relates to a pharmaceutical composition comprising as active principle a therapeutically effective amount of G- CSF (preferably hG-CSF) according to the present invention, optionally in combination with suitable pharmaceutically acceptable carriers and excipients.
  • G-CSF may be used directly for the treatment of tumors.
  • hG-CSF is used as active principle, in association with a specific carrier specific for tumoral cells, for preparing an effective pharmaceutical composition in anti-tumor theraphy.

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Abstract

L'invention se rapporte à un procédé de préparation du facteur de stimulation de colonie de granulocytes humain (G-CSF) recombinant, consistant à utiliser un système vectoriel comprenant au moins l'un des éléments suivants: (1) un promoteur de cytomégalovirus (CMV) en amont d'une séquence d'ADN codant la protéine requise, (2) une séquence destinée au traitement de produits de transcription d'ARN, et (3) une séquence marqueur destinée à la sélection et à l'amplification. L'invention se rapporte également à l'utilisation de G-CSF humains recombinants de ce type pour la préparation de compositions pharmaceutiques favorisant l'hématopoïèse, renforçant le système immunitaire contre les infections et stimulant des effecteurs cellulaires à amorçage fonctionnel agissant contre les affections malignes de façon à entraîner l'hyperproduction de ces effecteurs.
PCT/EP1994/003488 1993-10-25 1994-10-24 Systeme d'expression pour lignees cellulaires eucaryotes Ceased WO1995011982A1 (fr)

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AU79395/94A AU7939594A (en) 1993-10-25 1994-10-24 Expression system for eukaryotic cell lines

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ITFI93A000212 1993-10-25
ITFI930212A IT1262545B (it) 1993-10-25 1993-10-25 Sistema di espressione per linee cellulari eucariotiche

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5916773A (en) * 1994-05-27 1999-06-29 Menarini Ricerche S.P.A. Recombinant production of fusion proteins comprising erythropoietin and GM-CSF components
WO2000025827A3 (fr) * 1998-10-30 2000-08-10 Menarini Ricerche Spa Composition pharmaceutique contenant des fragments d'un adn codant pour une proteine antigene, a effet antitumoral
JP2010042006A (ja) * 2009-09-14 2010-02-25 Lonza Biologics Plc Ｃｈｏ細胞において組換えタンパク質を発現する方法
US7932087B2 (en) 2002-07-18 2011-04-26 Lonza Biologics Plc Method of expressing recombinant protein in CHO cells

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0265874A2 (fr) * 1986-10-23 1988-05-04 Green Cross Corporation Méthode de production de pro-urokinase humaine
WO1989001036A1 (fr) * 1987-07-23 1989-02-09 Celltech Limited Vecteurs d'expression a base d'adn recombinant

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0265874A2 (fr) * 1986-10-23 1988-05-04 Green Cross Corporation Méthode de production de pro-urokinase humaine
WO1989001036A1 (fr) * 1987-07-23 1989-02-09 Celltech Limited Vecteurs d'expression a base d'adn recombinant

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DAVID J. TWEARDY ET AL.: "Molecular cloning and characteritation of a cDNA for human G-CSF...", ONCOGENE RESEARCH, vol. 1, pages 209 - 220 *
FRED A. M. ASSELBERGS ET AL.: "A two-plasmid system for transient expression of cDNAs in primate cells", ANALYTICAL BIOCHEMISTRY, vol. 209, pages 327 - 331 *
GRAY SHAW ET AL.: "A conserved AU sequence from the 3' untranslated region of GM-CSF mRNA mediastes selective mRNA degradation", CELL, vol. 46, pages 659 - 667 *
IDEM *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5916773A (en) * 1994-05-27 1999-06-29 Menarini Ricerche S.P.A. Recombinant production of fusion proteins comprising erythropoietin and GM-CSF components
WO2000025827A3 (fr) * 1998-10-30 2000-08-10 Menarini Ricerche Spa Composition pharmaceutique contenant des fragments d'un adn codant pour une proteine antigene, a effet antitumoral
US7932087B2 (en) 2002-07-18 2011-04-26 Lonza Biologics Plc Method of expressing recombinant protein in CHO cells
JP2010042006A (ja) * 2009-09-14 2010-02-25 Lonza Biologics Plc Ｃｈｏ細胞において組換えタンパク質を発現する方法

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AU7939594A (en) 1995-05-22
IT1262545B (it) 1996-07-02
ITFI930212A0 (it) 1993-10-25

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