HK1086031B - Neomycin phosphotransferase genes and method for the selection of high-producing recombinant cells - Google Patents

Description

Neomycin phosphotransferase gene and method for selecting high-yield recombinant cells

Technical Field

The present invention relates to novel modified neomycin phosphotransferase genes and their use in methods of selecting high-producing recombinant cells. Accordingly, the present invention also relates to novel expression vectors comprising the modified neomycin phosphotransferase gene according to the invention, preferably in combination with a gene of interest functionally linked to a heterologous promoter. The invention further relates to methods for producing heterologous gene products using corresponding highly productive recombinant cells.

Background

Mammalian cells are the preferred host cells for the production of complex biopharmaceutical proteins because their post-translational modifications are not only functionally but also pharmacokinetic compatible with humans. Commercially relevant cell types are hybridomas, myelomas, CHO (chinese hamster ovary) cells and BHK (baby hamster kidney) cells. The cultivation of host cells is increasingly carried out under serum-free and protein-free production conditions. The reasons for this are the associated reduced costs, reduced interference in the purification of the recombinant protein and a reduced potential for the introduction of pathogens (e.g. prions, viruses). The use of CHO cells as host cells has become more widespread because this cell is adapted to growth in suspension in serum and protein free medium and is also recognized and accepted by regulatory authorities as a safe production cell.

To produce a stable mammalian cell line expressing a heterologous gene of interest (GOI), the heterologous gene is introduced into the desired cell line, typically by transfection, along with a selectable marker gene, such as neomycin phosphotransferase. The heterologous gene and the selectable marker gene may be expressed simultaneously or together from a single vector, or from separate vectors co-transfected. 2 to 3 days after transfection, cells are transferred to a medium containing a selection agent, for example G418 in the case of the neomycin phosphotransferase gene (NPT gene), and cultured for several weeks under this selection condition. Emerging resistant cells can then be isolated and investigated for expression of the desired gene product (GOI).

In establishing cell lines with highly expressed desired proteins, a major problem arises from the random and unguided integration of recombinant vectors into transcriptionally active or inactive sites of the host cell genome. The result is a population of cells with disparate heterologous gene expression rates, the productivity of which generally follows a normal distribution. Therefore, in order to identify cell clones with very high heterologous gene expression of interest, a large number of clones must be examined and tested, resulting in time, labor and cost expenditure. The optimization of the vector system used for transfection therefore aims at increasing the high-producing fraction in the transfected cell population by means of an appropriate selection strategy and with the aid of reducing the costs in clone confirmation. The development of this expression system is the subject of the present invention.

The gene of aminoglycoside-3' -phosphotransferase II enzyme (neomycin phosphotransferase) (EC27195) which is related to transposon 5-in Escherichia coli is used as a selectable marker in many organisms such as bacteria, yeast, plants and mammalian cells. This enzyme confers resistance to many aminoglycoside antibiotics such as neomycin, kanamycin and G418, which are inactivated by transfer of the ATP terminal phosphate to the 3' hydroxyl group of the aminocaproic ring I. In addition to wild-type neomycin phosphotransferase (wt NPT), some mutants are known to have reduced phosphotransferase activity and thus reduced tolerance to aminoglycoside antibiotics in bacteria (Blazquez et al, 1991; Kocab iyik et al, 1992; Yenofsky et al, 1990) and in leaves (Yenofsky et al, 1990).

One of the mutants (Glu182 Asp) was used as a marker for selection of rat embryonic stem cells to incorporate the neomycin phosphotransferase gene into the c-myc gene by targeted homologous recombination (gene targeting) (Hanson et al, 1995). The authors limited the use of modified enzymes to gene targeting.

Patent application WO 99/53046 describes the expression of a modified neomycin phosphotransferase gene (Asp261Asn) in mammalian cells in connection with production. The authors describe a non-cloning method to express genes of interest in mammalian cells. Cells targeted under selective pressure can be cultured by co-transfecting the cells with three individual DNA fragments encoding a promoter element, a gene of interest, and a selectable marker coupled to an Internal Ribosome Entry Site (IRES) element, wherein all three DNA fragments are combined as a functional bicistronic transcription unit (gene of interest promoter-IRES-neomycin phosphotransferase gene). The alignment of the elements only occurs in transfected cells, so only a few cells show the correct alignment of the elements. Furthermore, after gene amplification, high producing clones cannot be generated using amplifiable selectable markers. The cells produced after repeated selection and gene amplification exhibited yields of up to 6pg protein/cell/day.

None of the documents discloses a modified neomycin phosphotransferase gene that is particularly suitable for the preparation of high expression vector systems for mammalian cells, which allows the development of high yielding cells containing one or more complete functional transcription units of both the gene or genes of interest and the modified neomycin phosphotransferase gene with reduced antibiotic resistance for the preparation of recombinant biopharmaceutical proteins. The DNA construct described in WO 99/53046 contains only the neomycin gene functionally linked to a dihydrofolate reductase (DHFR) promoterless gene.

It is therefore necessary to obtain a neomycin phosphotransferase gene which is suitably modified, in particular for the development of corresponding high expression vector systems for biological processes. It is therefore an object of the present invention to provide corresponding novel modified neomycin phosphotransferase genes, expression vectors containing the modified neomycin phosphotransferase genes and a gene of interest functionally linked to a heterologous promoter, methods for selecting highly productive recombinant cells, preferably mammalian cells, and methods for producing heterologous gene products.

Surprisingly, it has been possible within the scope of the present invention to prepare and identify novel modified highly selective neomycin phosphotransferase genes, characterized in that they are particularly suitable for selecting highly productive cells.

Summary of The Invention

The present invention provides novel modified neomycin phosphotransferase genes. Surprisingly, it has been found that enrichment of transfected mammalian cells with high expression rates of co-integrated genes of interest can be achieved by using the modified neomycin phosphotransferase gene described below as a selectable marker. After transfection with one of the novel neomycin phosphotransferase genes according to the invention, the cells exhibited a 1.4 to 14.6 fold increase in the productivity of the protein (antibody) compared to the use of the wild-type neomycin phosphotransferase as selectable marker.

The modified neomycin phosphotransferase gene according to the invention is preferably a mutant which encodes an amino acid which differs from the wild-type gene in amino acid position 91, 182, 198, 227, 240 or 261. In a preferred embodiment, the neomycin phosphotransferase gene according to the invention is the mutant Glu182Gly, Glu182Asp, Trp91Ala, Val198Gly, Asp227Ala, Asp227Val, Asp227Gly, Asp261Asn, Asp261Gly or Phe240 Ile. For the selection of highly productive mammalian cells, it has proved particularly suitable to use the mutants Trp91Ala, Asp227Val, Asp261Asn, Asp261Gly and Phe240Ile, while the mutants Asp227Val and Asp261Gly on the other hand give the highest productivity to the cell clone and are therefore particularly preferred.

The highly productive cells are obtained by using eukaryotic expression vectors containing a heterologous gene of interest functionally linked to a heterologous promoter and a modified neomycin phosphotransferase gene according to the invention. The expression vector preferably contains other regulatory elements, such as one or more enhancers functionally linked to the promoter. In addition, expression vectors containing a fluorescent protein gene functionally linked to the gene of interest and a heterologous promoter, preferably via an Internal Ribosome Entry Site (IRES), are also preferred, allowing bicistronic expression of the gene encoding the fluorescent protein and the gene encoding the protein/product of interest under the control of the heterologous promoter. Particularly preferred are expression vectors in which the heterologous gene of interest is under the control of the ubiquitin (ubiquitin)/S27a promoter.

The invention also relates to expression vectors which, in place of the gene of interest, contain sequence regions which, for incorporation into the multiple cloning sites of this gene, have multiple recognition sequences for restriction endonucleases.

In another aspect, the present invention relates to a recombinant mammalian cell containing one of the aforementioned modified neomycin phosphotransferase genes according to the present invention. Furthermore, the invention relates to a recombinant mammalian cell which has been transfected with one of the expression vectors according to the invention. These are preferably recombinant rodent cells, with recombinant hamster cells such as CHO cells or BHK cells being particularly preferred. In another preferred embodiment, the recombinant cell is additionally transfected with a gene for an amplifiable selectable marker, such as the gene for dihydrofolate reductase (DHFR).

The present invention also relates to a method for enriching recombinant mammalian cells expressing a modified neomycin phosphotransferase gene, characterized in that (i) a collection of mammalian cells is transfected with a modified neomycin phosphotransferase gene having an activity of only 1 to 80%, preferably only 1 to 60%, more preferably only 1.5 to 30%, still more preferably only 1.5 to 26% compared to the wild-type neomycin phosphotransferase and/or one of the above modifications; (ii) culturing a mammalian cell under conditions that allow for expression of the modified neomycin phosphotransferase gene; and (iii) culturing the mammalian cells in the presence of at least one selective agent which selectively acts on the growth of the mammalian cells and confers a growth advantage on the cells expressing the neomycin phosphotransferase gene.

The invention also relates to a method for expressing at least one gene of interest in recombinant mammalian cells, characterized in that (i) a collection of mammalian cells is transfected with at least one gene of interest and with a modified neomycin phosphotransferase gene which has only an activity of 1 to 80%, preferably only 1 to 60%, more preferably only 1.5 to 30%, still more preferably only 1.5 to 26% compared to the wild-type neomycin phosphotransferase and/or one of the above modifications; (ii) culturing the cells under conditions that allow expression of the gene of interest and the modified neomycin phosphotransferase gene; (iii) culturing mammalian cells in the presence of at least one selective agent that selectively acts on the growth of mammalian cells and confers a growth advantage on cells expressing the neomycin phosphotransferase gene; and (iv) obtaining the protein of interest from the mammalian cells or from the culture supernatant.

The invention further relates to a method for obtaining and selecting recombinant mammalian cells expressing at least one heterologous gene of interest, characterized in that (i) the recombinant mammalian cells are transfected with an expression vector according to the invention which, in addition to the gene of interest and the modified neomycin phosphotransferase gene, encodes a fluorescent protein; (ii) culturing mammalian cells under conditions that allow expression of the gene of interest, the gene encoding the fluorescent protein, and the modified neomycin phosphotransferase gene; (iii) culturing mammalian cells in the presence of at least one selective agent that selectively acts on the growth of mammalian cells and confers a growth advantage on cells expressing the neomycin phosphotransferase gene; and (iv) selecting the mammalian cells by flow cytometry analysis.

If the mammalian cells have additionally been transfected with an amplifiable selectable marker gene, such as the DHFR gene, it is possible to culture the mammalian cells under conditions in which the amplifiable selectable marker gene is also expressed, and to add a selection agent to the culture medium to cause amplification of the amplifiable selectable marker gene.

Preferably, the method according to the invention is carried out on mammalian cells adapted for growth in suspension, i.e. mammalian cells cultured in suspension culture. Other embodiments relate to methods of culturing mammalian cells, preferably adapted for suspension growth, under serum-free conditions.

Drawings

FIG. 1 shows a schematic preparation of the basic vector used to express recombinant proteins in CHO-DG44 cells. "P/E" is a combination of CMV enhancer and hamster ubiquitin/S27 a promoter, only "P" represents a promoter element and "T" is a termination signal for transcription, which is necessary for polyadenylation of transcribed mRNA. Within each transcription unit the position and direction of transcription initiation is indicated by arrows. For cloning of heterologous genes, sequence regions with multiple cleavage positions for restriction endonucleases (multiple cloning site — MCS) are inserted after the promoter element. The amplifiable selectable marker dihydrofolate reductase is abbreviated as "dhfr" and the selectable marker neomycin phosphotransferase is abbreviated as "npt" (npt wild-type or npt mutant). The "IRES" element from encephalomyocarditis virus serves as an internal ribosome entry site within the dicistronic transcription unit and allows subsequent translation of the green fluorescent protein "GFP".

FIG. 2 shows a schematic preparation of eukaryotic expression vectors encoding single chain proteins (FIG. 2A) or subunits of monoclonal antibodies (FIG. 2B) and used to transfect CHO-DG44 cells. "P/E" is a combination of CMV enhancer and hamster ubiquitin/S27 a promoter, only "P" represents a promoter element and "T" is a termination signal for transcription, which is necessary for polyadenylation of transcribed mRNA. Within each transcription unit the position and direction of transcription initiation is indicated by arrows. The amplifiable selectable marker dihydrofolate reductase is abbreviated as "dhfr" and the selectable marker neomycin phosphotransferase is abbreviated as "npt". NPT mutants E182G (SEQ ID NO: 3), E182D (SEQ ID NO: 19), W91A (SEQ ID NO: 5), D190G (SEQ ID NO: 23), V198G (SEQ ID NO: 7), D208G (SEQ ID NO: 25), D227A (SEQ ID NO: 9), D227V (SEQ ID NO: 11), D227G (SEQ ID NO: 21), D261G (SEQ ID NO: 13), D261N (SEQ ID NO: 15), and F240I (SEQ ID NO: 17) contain point mutations that result in altered amino acids (represented by the 1-letter code) at the indicated positions. The "IRES" element from encephalomyocarditis virus serves as an internal ribosome entry site within the dicistronic transcription unit and allows subsequent translation of the green fluorescent protein "GFP". "MCP-1" encodes monocyte chemoattractant protein-1 (monocyte chemoattractant protein-1), while "HC" and "LC" encode the heavy or light chain, respectively, of humanized monoclonal IgG2 antibody.

FIG. 3 shows the partial sequence of the neomycin phosphotransferase (npt) gene in which a PCR insertion point mutation has been performed by means of a mutagenic primer. Capital letters represent the nucleotide sequence of the NTP coding region and lowercase letters represent flanking non-coding nucleotide sequences. The amino acid sequence (3-letter code) predicted from the nucleotide sequence is shown above the coding nucleotide sequence. The arrows represent the direction, length and position of the primers used, the arrow has a solid line for the mutagenic forward primer, the arrow has a dashed line for the mutagenic reverse primer, the arrow has a dotted line for the primer Neovalve 5(SEQ ID NO: 27) or Neovalve 2(SEQ ID NO: 29) located upstream of the npt gene or the mutation position, respectively, and the arrow has a dotted line for the primer Neorev5(SEQ ID NO: 28) or IC49(SEQ ID NO: 30) located downstream of the npt gene or the mutation position, respectively. Nucleotides replaced relative to the wild-type sequence are highlighted above or below the arrow.

FIG. 4 shows conserved domains within the NPT amino acid sequence and the location of the inserted NPT mutation. Based on sequence homology between different aminoglycoside-modified enzymes, different conserved domains (shown in grey) were identified within the NPT protein sequence. Three motifs (motif) in the C-terminal region of the enzyme clearly have particular utility. Motifs 1 and 2 may be involved in ATP-catalyzed or catalyzed transfer of the terminal phosphate in nucleotide binding, while motif 3 is considered to have the function of ATP hydrolysis and/or to change the configuration of the enzyme-aminoglycoside complex. Amino acids occurring in at least 70% of the aminoglycoside-modified enzymes are highlighted in bold. The amino acids of the single lower line were assigned to the same group based on their similarity and occurred with at least 70% aminoglycoside modified enzyme. Amino acids marked with asterisks represent the position of the mutation.

FIG. 5 shows the effect of NPT mutations on the selection of stably transfected MCP-1 expressing cells. For this purpose, CHO-DG44 cells were transfected with the vectors pBIN-MCP1, pKS-N5-MCP1 or pKS-N8-MCP1 (FIG. 2A), which contained the NTP Wild Type (WT) or the NPT mutant Glu182Asp or Asp227Gly as selectable marker. To select for stably transfected cells, 400. mu.g/ml or 800. mu.g/ml G418 was added to the medium as a selection agent. The concentration of the produced recombinant protein MCP-1 in the cell culture supernatant was determined by ELISA and the specific productivity per cell and per day was calculated. The bars represent the mean of specific productivity or titer of 18 pools from four cultures in 6-well plates.

The effect of NTP mutation on the selection of cells expressing stably transfected monoclonal antibodies was investigated in figure 6. For this purpose, CHO-DG44 cells were transfected with pBIDG-HC/pBIN-LC (NPT wild type), pBIDG-HC/pKS-N5-LC (NPT mutant Glu182Asp) or pBIDG-HC/pKS-N8-LC (NPT mutant Asp227Gly) (FIG. 6A) in plasmid combinations or with pBIDG-HC/pBIN-LC (NPT wild type), pBIDG-HC/pBIN1-LC (NPT mutant Glu182Asp), pBIDG-HC/pBIN2-LC (NPT mutant Trp91Ala), pBIDG-HC/pBIN3-LC (NPT mutant Val198G), pBIDG-HC/pBIN4-LC (NPT mutant Asp227Ala), pBIDG-HC/pBIN5-LC (NPT mutant Asp227 Asp), pBIDG-HC/pBIN 2-261, pBIN 23-Asp 240-Asp (NPT mutant Asp) or pBIDG-HC-Asp (NPT mutant Asp) in Asp) (NPT mutant Asp 240-Asp) (NPT-Asp) in combination Fig. 6B), which differ from each other only in the NTP gene (wild type or mutant) used as a selectable marker. The concentration of recombinant monoclonal IgG2 antibody produced in the cell culture supernatant was determined by ELISA and specific productivity per cell and per day was calculated. In total, 5-9 pools were established per vector combination. The bars represent the mean value of specific productivity or titer of all pools from the test of 6 cultures performed in 75 cm square flasks. To calculate relative titers or relative specific productivities, the average of pools selected with NPT wild-type genes was considered as 1.

FIG. 7 shows the enrichment of cells with higher GFP expression in a pool of transfected cells by using NPT mutants according to the invention as selectable markers. For this purpose, CHO-DG44 cells were transfected with plasmid combinations pBIDG-HC/pBIN-LC (NTP wild type), pBIDG-HC/pKS-N5-LC (NPT mutant Glu182Asp), pBIDG-HC/pKS-N8-LC (NPT mutant Asp227Gly), pBIDG-HC/pBIN1-LC (NPT mutant Glu182Gly), pBIDG-HC/pBIN2-LC (NPT mutant Trp91Ala), pBIDG-HC/pBIN3-LC (NPT mutant Vall98G), pBIDG-HC/pBIN4-LC (NPT mutant Asp227Ala), pBIDG-HC/pBIN5-LC (NPT mutant Asp227Val), pBIDG-HC/pBIN6-LC (NPT mutant Asp261Gly), pBIDG-HC/7-LC (NPT mutant Asp 261) or pBIDG-N240-Asp (NPT mutant Asp 240) into the pool of pBIDG-HC/pBIDIN 8, which differ from each other only in the use of the NTP gene (wild type or mutant) as a selectable marker. Furthermore, the pBIDG vector also contained GFP as a marker gene. The transfected cell pool was selected for 2-3 weeks in HT-free medium supplemented with G418, and then measured for GFP fluorescence by FACS analysis. Each figure represents the mean value of GFP fluorescence from each pool transfected with the same plasmid combination, except for the non-transfected CHO-DG44 cells as negative controls.

FIG. 8 shows the increase in monoclonal antibody productivity achieved by dhfr-mediated gene amplification, exemplified by a cell mass obtained from CHO-DG44 transfected with vector combinations pBIDG-HC/pBIN-LC (NPT wild type) or pBIDG-HC/pBIN5-LC (NPT mutant D227V). Dhfr-mediated gene amplification was performed by adding 100nM and then 500nM MTX to the medium after the first selection in hypoxanthine/thymidine free CHO-S-SFMII medium in the presence of G418. The concentration of monoclonal antibodies in the cell culture supernatants of the pools was determined by ELISA and specific production rates per cell and per day (pg/cell day) were calculated. Each data point represents the average of 6 cultures performed in 75 cm square flasks.

FIG. 9 shows the enzyme activity of NPT mutants according to the invention compared to NPT wild-type in a dot assay. For this purpose, cell extracts of two differently monoclonal antibody expressing cell aggregates (cell aggregates 1 and 2) which had been transfected and selected with the NPT wild-type gene (SEQ ID NO: 1) or with the NPT mutants E182G (SEQ ID NO: 3), E182D (SEQ ID NO: 19), W91A (SEQ ID NO: 5), V198G (SEQ ID NO: 7), D227A (SEQ ID NO: 9), D227V (SEQ ID NO: 11), D227G (SEQ ID NO: 21), D261G (SEQ ID NO: 13), D261N (SEQ ID NO: 15) and F240I (SEQ ID NO: 17) were prepared. As a negative control, untransfected CHO-DG44 cells were used. G418 was used as a substrate in the phosphorylation assay. The extract was filtered through a sandwich of P81 phosphocellulose and nitrocellulose membranes in a 96-well vacuum manifold. Proteins phosphorylated by protein kinases and unphosphorylated proteosomes bind to nitrocellulose, while phosphorylated and unphosphorylated G418 passes through nitrocellulose and binds to phosphocellulose (fig. 9A). The radioactive signals were detected and quantified using a PhosphoImager (PhosphoImager). The signal obtained with 5 μ g of extract was used to calculate the percent enzyme activity. The percent enzyme activity represents the average of pooled NTP mutants from 2 expressing monoclonal antibodies, where the enzyme activity of wild-type NPT was taken as 100% (fig. 9B).

FIG. 10 shows northern blot analysis of NPT expression and NPT gene copy number in transfected cell pools. For this purpose, total RNA of two differently expressing monoclonal antibody cell aggregates which had been transfected and selected with the NPT wild-type gene (SEQ ID NO: 1) or with the NPT mutants E182G (SEQ ID NO: 3), E182D (SEQ ID NO: 19), W91A (SEQ ID NO: 5), V198G (SEQ ID NO: 7), D227A (SEQ ID NO: 9), D227V (SEQ ID NO: 11), D227G (SEQ ID NO: 21), D261G (SEQ ID NO: 13), D261N (SEQ ID NO: 15) and F240I (SEQ ID NO: 17) was prepared. As a negative control, untransfected CHO-DG44 cells were used. And (3) mixing 30: mu.g of each RNA was hybridized with FITC-dUTP labeled PCR product containing the coding region of the NPT gene. In all transfected cells, a specific single NPT transcript of approximately 1.3kb was detected. In order to determine the copy number of the npt gene, genomic DNA was isolated from the aforementioned cell mass expressing the monoclonal antibody in dot blot analysis. 10, 5, 2.5, 1.25, 0.63 or 0.32. mu.g of genomic DNA was hybridized with FITC-dUTP-labeled PCR products containing the coding region of the NPT gene. As a negative control, untransfected CHO-DG44 cells were used. Plasmid pBIN-LC was used as standard (320pg, 160pg, 80pg, 40pg, 20pg, 10pg, 5pg, 2.5 pg). The copy number of the npt gene in the cell pool was calculated using a standard series determined by the signal intensity measured on the calibrated plasmid DNA.

FIG. 11 shows the isolation of cell aggregates of high-expressing monoclonal antibodies by GFP-based selection using FACS, using two-cell aggregates (cell aggregates 5 and 8). These pools of pBID-HC and pBING-LC co-transfected cells were subjected to sequential GFP-based FACS selection. The concentration of IgG2 antibody in the cell culture supernatants of the pools was determined by ELISA after each sorting step and specific productivity per cell and per day (pg/cell day) was calculated. A total of 6 picks were performed, of which in each case 5% of the cells with the highest GFP fluorescence were selected. Each data point represents the average of at least 6 cultures performed in 75 cm square flasks.

FIG. 12 shows the increase in monoclonal antibody productivity achieved by combining GFP-based selection and MTX amplification steps, using cell aggregates 5 and 8 as an example (compare FIG. 11). Two weeks after co-transfection of CHO-DG44 with the vectors pBID-HC and pBING-LC, 5% of the cells with the highest GFP fluorescence were selected from cell aggregates 5 and 8. Dhff-mediated gene amplification was then performed by adding 100nM Methotrexate (MTX, Methotrexate) to the medium. The concentration of monoclonal antibodies in the pooled cell culture supernatants was determined by ELISA and specific production rates per cell and per day (pg/cell day) were calculated. Each data point represents the average of at least 6 incubations performed in 75 cm square flasks.

FIG. 13 shows the correlation between antibody productivity and GFP fluorescence in the case of cell aggregates 5 and 8 (compare FIG. 11). These cell aggregates were obtained by transfection of CHO-DG44 with the vector combination pBID-HC and pBING-LC. They were subjected to serial GFP-based FACS selection, each of which selected 5% of the cells with the highest GFP fluorescence. The concentration of IgG2 antibody in the pooled cell culture supernatants was determined by ELISA after each sorting step and specific production rates per cell and per day (pg/cell day) were calculated. Each data point represents the average of at least 6 cultures performed in 75 cm square flasks.

Detailed description of the invention and preferred embodiments

The following amino acid position information is in each case referred to the amino acid sequence represented by the amino acid sequence having SEQ ID NO: 1, or a wild-type neomycin phosphotransferase gene. By "modified neomycin phosphotransferase gene" is meant a nucleic acid encoding a polypeptide having neomycin phosphotransferase activity, having an amino acid different from that of the wild-type protein at least one of the amino acid positions more fully described in the specification, which is a residue of a protein having the amino acid sequence of SEQ ID NO: 2 is homologous. In this context, the word "homologous" means that the region of the sequence carrying the mutation can be compared with a reference sequence, in this case the sequence according to SEQ ID NO: 2, using the so-called standard "alignment" algorithms, such as, for example, "BLAST" (Altschul, S.F., Gish, W, Miller, W., Myers, E.W. & Lipman, D.J. (1990) "basic local alignment search tool" J.Mol.Biol.215: 403. 410; Gish, W. & States, D.J. (1993) "identifying protein coding regions by database similarity search" Nature Genet.3: 266. 272; Madden, T.L., Tatusov, R.L. & Zhang, J. (1996) "the use of the Web BLAST server" Meth.Enzymol.266: Nu 141; Zhang, J. & Madden, T.L (1997) "Popowerr: A. or New Automation search and sequence analysis of proteins and the library of the Webson, Zhang.7. 1997, Zhang, Sp." Alppel.7. Shu.7. Shu., Zhang, Sp. J. "protein search database similarity search tool" and database similarity search database ". 10. 1997)". When they match their sequence order and can be identified using standard "alignment" algorithms, the sequences are identical.

The present invention provides novel modified neomycin phosphotransferase genes and methods for making and selecting mammalian cell lines that allow for high expression of heterologous gene products, preferably biopharmaceutically related polypeptides or proteins. The method according to the invention is based firstly on the selection of cells which, in addition to the gene of interest, also express the neomycin phosphotransferase gene according to the invention, which gives the transfected cells a selective advantage over the untransfected cells. Surprisingly, it has been found that the use of the modified neomycin phosphotransferase gene (mNPT gene) according to the invention which has been described herein has a significant selective advantage over the wild-type neomycin phosphotransferase gene (wtNPT gene). This is particularly relevant for the use of mutants with lower enzymatic activity than wtNPT.

Modified neomycin phosphotransferase gene according to the present invention

It has proved to be particularly suitable to use modified NTP genes which encode NPT having only a wtNPT enzymatic activity of 1 to 80%, preferably only 1 to 60%. Preferred NTP mutants are those having only 1 to 30% wtNPT enzyme activity, with those having only 1.5 to 26% wtNPT enzyme activity being particularly preferred. The enzymatic activity of NTPs can be determined, for example, in a dot assay as described in example 4 and referred to as method 5.

The phrase "wild-type neomycin phosphotransferase" is intended to mean a neomycin phosphotransferase gene encoding an aminoglycoside-3' -phosphotransferase II enzyme (EC2.7.1.95), which gene is associated with the native transposon 5-in E.coli and contains, for example, the sequence shown in SEQ ID NO: 2, or by the amino acid sequence shown in SEQ ID NO: 1. This enzyme confers resistance to many aminoglycoside antibiotics such as neomycin, kanamycin and G418, which are inactivated by transfer of the ATP terminal phosphate to the 3' hydroxyl group of the hexosamine ring I. By the word wtNPT is also meant a polypeptide having an amino acid sequence identical to that represented by SEQ ID NO: 1, the enzyme activity to which the encoded NPT is comparable. This includes in particular those NPTs in which the enzymatic active center, which catalyzes the transfer of the terminal phosphate from ATP to the substrate, is present in the same or almost the same morphology (Shaw et al, 1993; Hon et al, 1997; Burk et al, 2001) and thus has an amino acid sequence which is identical to the sequence of the amino acid sequence SEQ ID NO: 2, comparable enzymatic activity. wtNPT has comparable enzymatic activity if it exhibits about 81 to 150%, preferably 90 to 120%, of the sequence defined by SEQ ID NO: 2, and the activity can be determined in a spot assay, e.g., as described in example 4 and referred to as method 5.

Basically, mutants are preferred, wherein the reduced enzymatic activity compared to wtNPT is based on modifications of the amino acid sequence, such as based on substitutions, insertions or deletions of at least one or more amino acids. Deletion, insertion and substitution mutants can be generated by "site-specific mutagenesis" and/or "PCR-based mutagenesis techniques". Corresponding methods are described, for example, by Lottspeich and Zorbas (1998) (Chapter 36.1 and other references).

Surprisingly, it has been found that it is possible to achieve a particularly efficient enrichment of transfected mammalian cells with a co-integrated gene of interest having a high expression rate if a neomycin phosphotransferase mutant is used as selectable marker, wherein less than the amino acid tryptophan at amino acid position 91, the amino acid glutamic acid at amino acid position 182, the amino acid valine at amino acid position 198, the amino acid aspartic acid at amino acid position 227, the amino acid aspartic acid at amino acid position 261 or the amino acid phenylalanine at amino acid position 240 has been altered compared to wtNPT. Thus, mutants with respect to the amino acids at positions 91, 182, 198, 227, 261 and/or 240 are preferred. Particularly advantageous are substitution mutants, i.e.mutants in which the amino acid occurring at this position in the wild type has been replaced by another amino acid. Even more preferred are the corresponding substitution mutants, wherein changing the corresponding amino acids results in a reduction of the enzyme activity compared to wtNPT to 1 to 80%, preferably to 1 to 60%, more preferably to 1.5 to 30%, still more preferably to 1.5 to 26%. Particularly preferred are modified NPT genes in which amino acids 91, 227, 261 and/or 240 have been modified so as to result in an enzymatic activity of only 1 to 80%, preferably only 1 to 60%, more preferably only 1.5 to 30%, most preferably only 1.5% to 26% compared to wtNPT. More preferred are substitution mutants wherein the amino acid has been modified at amino acid position 227 such that the enzymatic activity of the modified NPT is less than 26%, preferably between 1 and 20%, more preferably between 1 and 16% compared to wtNPT.

According to another embodiment of the invention, advantageous mutants are those which encode glycine, alanine, valine, leucine, isoleucine, phenylalanine or tyrosine at amino acid position 91, 182 or 227 in comparison with wtNPT. Furthermore, glutamic acid can be substituted at amino acid position 182 with aspartic acid, asparagine, glutamine, or any other preferred negatively charged amino acid. Also preferred are modified NPT genes which, in comparison with wtNPT, encode glycine, alanine, leucine, isoleucine, phenylalanine, tyrosine or tryptophan at amino acid position 198. Also preferred are modified NPT genes which encode glycine, alanine, valine, isoleucine, tyrosine or tryptophan at amino acid position 240 as compared to wt NPT. Also preferred are modified NPT genes which, in comparison with wtNPT, encode glycine, alanine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine or aspartic acid at amino acid position 261. In particular, it has been found that transfected mammalian cells enriched for the co-integrated gene of interest with a high expression rate are possible with the mutants Glu182Gly, Glu182Asp, Trp91Ala, Val198Gly, Asp227Ala, Asp227Val, Asp227Gly, Asp261Asn and Phe240Ile as selectable markers, with the result that these mutants are particularly preferred. Even more preferred are the mutants Asp227Val, Asp227Gly, Asp261Asn, Phe240Ile and Trp91Ala, so that optimum enrichment rates are achieved by these mutants. The mutant Asp227Val is particularly preferred.

In contrast, the Asp190Gly and Asp208Gly mutants have been shown to be inappropriately labeled for selection of transfected CHO-DG44 cells under serum-free culture conditions. As a result of the potentially greatly reduced enzyme function of these mutants (Asp190Gly, Asp208Gly), only a few cells are obtained after the selection period, which seriously impair their growth and viability.

The amino acids at positions 182 and 227, which are based on the wild type, are non-conserved amino acids, which are located outside the three conserved motifs of the C-terminal region of the aminoglycoside-3' -phosphotransferase. The amino acid at position 91 also belongs to a non-conserved amino acid and is located outside one of the conserved motifs of the N-terminal region of the aminoglycoside-3' -phosphotransferase. In contrast, amino acids at positions 198 and 240 are conserved amino acids in the C-terminal region of NPT, but outside of the conserved motif. In contrast, the amino acid at position 261 is a conserved amino acid in the third motif of the C-terminal region (Shaw et al, 1993, Hon et al, 1997; Burk et al, 2001).

Compared to the use of wtNPT as selectable marker, the cells showed a fold increase in productivity of 1.4-2.4 in the case of the Glu182Gly, Glu182Asp and Val198Gly mutants, 1.6-4.1 in the case of the Asp227Gly mutant, 2.2 or 4 in the case of the Asp227Ala or Trp91Ala mutant, 5.7 or 7.3 in the case of the Phe240Ile or Asp261Asn mutant and even 9.3 or 14.6 in the case of the Asp261Gly or Asp227Val mutant. For expression of the multi-chain protein (antibody), co-transfection was performed. Both protein chains are expressed each in their own vector, one vector encoding additionally the NPT gene and the other encoding the amplifiable, selectable dihydrofolate reductase gene.

The present invention therefore relates to a method for enriching recombinant mammalian cells expressing a modified neomycin phosphotransferase gene, characterized in that (i) a collection of mammalian cells is transfected with a modified neomycin phosphotransferase gene which has only 1 to 80%, preferably only 1 to 60%, more preferably only 1.5 to 30%, still more preferably only 1.5 to 26% of the activity and/or one of the modifications specified herein compared to the wild-type neomycin phosphotransferase; (ii) culturing a mammalian cell under conditions that allow for expression of the modified neomycin phosphotransferase gene; and (iii) culturing the mammalian cells in the presence of at least one selective agent which selectively acts on the growth of the mammalian cells and confers a growth advantage on the cells expressing the neomycin phosphotransferase gene.

Particularly preferred are corresponding methods which are used for the modified NPT genes described in more detail in the present application, in particular if the modified NPT gene used encodes a modified NPT which, in comparison with the wild-type gene, encodes alanine at amino acid position 91, glycine or aspartic acid at amino acid position 182, glycine at amino acid position 198, alanine, glycine or valine at amino acid position 227, glycine or asparagine at amino acid position 261 or isoleucine at amino acid position 240. Even more preferred is the NPT gene which encodes valine at amino acid position 227 and/or glycine and/or asparagine at amino acid position 261, compared to the wild type gene. Particularly preferred are those NPT genes which, in comparison with the wild-type gene, code for isoleucine at amino acid position 240 or for valine at amino acid position 227, while the NPT gene which, in comparison with the wild-type gene, codes for valine at amino acid position 227 is particularly preferred. Naturally, the invention also encompasses modified NPT genes and the use of modified NPT genes according to the invention, which comprise a combination of corresponding amino acid exchanges.

The present invention further relates to eukaryotic expression vectors comprising (i) a heterologous gene of interest functionally linked to a heterologous promoter and (ii) a modified neomycin phosphotransferase gene according to the present invention encoding a neomycin phosphotransferase having a lower enzymatic activity than the wild-type neomycin phosphotransferase. By "low" or "lower" enzyme activity for the purposes of the present invention is meant an enzyme activity which corresponds to a wtNPT enzyme activity of at most 80%, preferably 1 to 80%, more preferably only 1 to 60%. According to a particular embodiment of the invention, a "lower enzymatic activity" represents an enzymatic activity of 1 to 30%, preferably 1.5 to 26%, compared to the wild-type neomycin phosphotransferase.

Preferably the expression vector contains a modified NPT gene encoding a modified NPT having a wtNPT enzymatic activity of only 1 to 80%, preferably only 1 to 60%. Also preferred are expression vectors with a modified NPT gene, which encode a mutant with a wtNPT enzyme activity of only 1 to 30%. Particularly preferred are expression vectors containing a modified NPT gene which encodes a mutant with a wtNPT enzyme activity of only 1.5 to 26%, as determined by the point assay in example 4 and referred to as method 5.

In another embodiment of the invention, the expression vector contains a gene which modifies NPT, compared with wtNPT, in amino acid positions Trp91, Glu182, Val198, Asp227, Phe240 or in position Asp 261. In this context, the NPT mutant is preferably modified at position Trp91, Glu182, Val198, Asp227, Phe240 or Asp261 and has a wtNPT enzyme activity of only 1 to 80%, preferably only 1 to 60%, more preferably only 1.5 to 30%, particularly preferably only 1.5% to 26%. Preferably, amino acids Trp91, Glu182 or Asp227 can each be substituted with glycine, alanine, valine, leucine, isoleucine, phenylalanine or tyrosine at the corresponding position. Preferably, the glutamic acid at position 182 can also be substituted with aspartic acid, asparagine, glutamine or another, preferably negatively charged, amino acid. Also preferred are modified NPT genes which encode glycine, alanine, leucine, isoleucine, phenylalanine, tyrosine or tryptophan at amino acid position 198 in comparison to wtNPT. Furthermore, the modified NPT gene preferably encodes glycine, alanine, valine, isoleucine, tyrosine or tryptophan in comparison with wtNPT at amino acid position 240. Also preferred is a modified NPT gene which encodes a glycine, alanine, leucine, isoleucine, phenylalanine, tyrosine, tryptophan, asparagine, glutamine or aspartic acid, particularly preferably using a mutant compared to wtNPT with amino acid 261 in which the aspartic acid at position 227 is replaced by glycine, alanine, valine, leucine or isoleucine and the aspartic acid at position 261 is replaced by alanine, valine, leucine, isoleucine or glutamine, particularly by glycine or asparagine.

Particularly preferred are expression vectors containing a modified NPT gene encoding a Glu182Gly, Glu182Asp, Trp91Ala, Val198Gly, Asp227Ala, Asp227Val, Asp227Gly, Asp261Asn or Phe240Ile mutant containing in the case of the Glu182Gly mutant the amino acid sequence SEQ ID NO: 4, containing the amino acid sequence SEQ ID NO: 20, containing the amino acid sequence SEQ id no: 6, in the case of the Val198Gly mutant comprising the amino acid sequence SEQ ID NO: 8, in the case of the Asp227Ala mutant comprising the amino acid sequence SEQ ID NO: 10, in the case of the Asp227Val mutant comprising the amino acid sequence SEQ ID NO: 12, in the case of the Asp227Gly mutant comprising the amino acid sequence SEQ ID NO: 22, in the case of the Asp261Gly mutant comprising the amino acid sequence SEQ ID NO: 14, in the case of the Asp261Asn mutant comprising the amino acid sequence SEQ ID NO: 16 and in the case of the Phe240Ile mutant comprises the amino acid sequence SEQ id no: 18. most preferred are expression vectors utilizing the Asp227Val, Asp227Gly, Asp261Asn, Phe240Ile or Trp91Ala mutants, particularly if they contain the amino acid sequences shown in SEQ id nos: 12. SEQ ID NO: 22. SEQ ID NO: 14. SEQ ID NO: 16. SEQ ID NO: 18 or SEQ ID NO: 6 or a nucleotide sequence as set forth in SEQ ID NO: 11. SEQ ID NO: 21. SEQ ID NO: 13. SEQ ID NO: 15. SEQ ID NO: 17 or SEQ ID NO: 5 encodes or contains these sequences.

Furthermore, the present invention provides for the first time modified neomycin phosphotransferase genes and gene products thereof which encode different amino acids compared to wtNPT at amino acid positions Trp91, Val198 or Phe 240. The invention provides for the first time, in particular, Trp91, Val198 or Phe240 mutants which have a reduced enzymatic activity compared with wtNPT. The modified NPT described here and which can be made available within the scope of the present invention preferably codes for alanine at amino acid position 91, glycine at position 198 and isoleucine at position 240. Furthermore, the present invention provides for the first time NPT mutants which, in comparison with wtNPT, encode a glycine at position 182, an alanine or valine at position 227 and a glycine at position 261. Both genes and gene products (enzymes) are provided for the first time within the scope of the present invention. Herein, the present invention provides for the first time a modified NPT having an amino acid sequence according to SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14 and SEQ ID NO: 18. furthermore, the present invention provides a modified NPT gene having a DNA sequence according to SEQ id no: 3. SEQ ID NO: 5. SEQ ID NO: 7. SEQ ID NO: 9. SEQ ID NO: 11. SEQ ID NO: 13 and SEQ ID NO: 17.

target gene

The gene of interest contained in the expression vector according to the present invention includes a nucleotide sequence of any length encoding a product of interest. The gene product or "product of interest" is typically a protein, polypeptide, peptide, or fragment or derivative thereof. However, it may also be RNA or antisense RNA. The gene of interest may exist in its full-length, shortened form, as a fusion gene or as a tagged gene. It may be genomic DNA or, preferably, cDNA or a corresponding fragment or fusion. The gene of interest may be a native gene sequence, or may be mutated or otherwise modified. Such modifications include codon optimization and humanization to accommodate established host cells. The gene of interest may encode a secreted, cytoplasmic, nuclear, membrane-bound or cell surface-bound polypeptide.

The term "nucleotide sequence" or "nucleic acid sequence" refers to oligonucleotides, nucleotides, polynucleotides and fragments thereof, as well as DNA or RNA of genomic or synthetic origin, which exist in single or double strands and may represent the coding or non-coding strand of a gene. Nucleic acid sequences can be modified using standard techniques, such as position-specific mutagenesis or PCR-mediated mutagenesis (e.g., as described in Sambrook et al, 1989 or Ausubel et al, 1994).

"encoding" means the property or property of a nucleotide of a particular sequence in a nucleic acid, e.g., a gene in a chromosome or mRNA, as a substrate in a biological process for synthesizing other polymers and macromolecules, e.g., rRNA, tRNA, mRNA, other RNA molecules, cDNA or polypeptides. Thus, a gene encodes a protein if the desired protein is produced in a cell or other biological system by transcription and subsequent translation of mRNA. Both the coding strand, which is identical in nucleotide sequence to the mRNA sequence and is normally also provided in sequence databases, such as EMBL or GenBank, and the non-coding strand of the gene or cDNA as a transcription matrix, can be referred to as being used to encode a product or protein. Nucleic acids encoding proteins also include nucleic acids having a different nucleotide sequence order based on the degenerate genetic code, but which result in the same amino acid sequence of the protein. The nucleic acid sequence encoding the protein may also contain introns.

The term "cDNA" refers to deoxyribonucleic acid, which is prepared by reverse transcription and synthesis of a second DNA strand from mRNA or other RNA produced by a gene. If the cDNA is present as a double-stranded DNA molecule, it contains both coding and non-coding strands.

The term "intron" refers to a non-coding nucleotide sequence of any length. They occur naturally in many eukaryotic genes and are removed from previously transcribed mRNA precursors by a process known as splicing. This requires precise excision of the intron at the 5 'and 3' ends and proper ligation of the resulting mRNA ends to allow for the production of a mature processed mRNA with the correct reading frame for successful protein synthesis. Many splice donor and splice acceptor positions involved in the splicing process, i.e., sequences located directly adjacent to exon-intron or intron-exon, have been characterized. For a general overview, see Ohshima et al, 1987.

Protein of interest

Proteins/polypeptides of biopharmaceutical importance include, for example, but are not limited to, antibodies, enzymes, cytokines, lymphokines, adhesion molecules, receptors, and derivatives or fragments thereof. In general, all polypeptides that are agonists or antagonists and/or have therapeutic or diagnostic utility are of value.

The term "polypeptide" is used with respect to amino acid sequences or proteins, and means a polymer of amino acids of any length. The term also includes proteins that are modified post-translationally by reactions such as glycosylation, phosphorylation, acetylation, or protein processing. The structure of a polypeptide can be modified, for example, by substituting, deleting or inserting amino acids, and fusing with other proteins, while still retaining their biological activity. The term "polypeptide" thus also includes, for example, fusion proteins consisting of an immunoglobulin component, such as an Fc portion, and a growth factor, such as an interleukin.

Examples of therapeutic proteins are insulin, insulin-like growth factors, human growth hormone (hGH) and other growth factors, tissue plasminogen activator (tPA), Erythropoietin (EPO), cytokines, e.g., Interleukins (IL), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, Interferon (IFN) - α, - β, - γ, - ω or- τ, Tumor Necrosis Factor (TNF), such as TNF- α, β or- γ, TRAIL, G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF. Further examples are monoclonal, polyclonal, multispecific and single-chain antibodies, and fragments thereof, such as, for example, Fab ', F (ab')₂Fc and Fc' fragments, light (L) and heavy (H) immunoglobulin chains, and constant, variable or hypervariable regions thereof, as well as Fv and Fd fragments (Chamov et al, 1999). The antibody may be of human or non-human origin. Humanized and chimeric antibodies are also possible.

Fab fragments (antigen binding fragments ═ Fab) consist of two chain variable regions, which are held together by adjacent constant regions. They may be produced, for example, by treatment with proteases, such as papain, from conventional antibodies or by DNA cloning. Other antibody fragments are F (ab')₂Fragments which can be produced by proteolytic digestion with pepsin.

It is also possible to prepare, by gene cloning, truncated antibody fragments consisting only of the variable regions of the heavy (VH) and light (VL). It is called Fv fragment (variable fragment-fragment of the variable part). Because in these Fv fragments it is not possible to covalently bind via the cysteine groups of the constant chains, Fv fragments are generally stabilized by some other means. For this purpose, the variable regions of the heavy and light chains are generally joined together by short peptide fragments of about 10 to 30 amino acids, particularly preferably 15 amino acids. This results in a single polypeptide chain in which VH and VL are linked together by a peptide linker. Such antibody fragments are also referred to as single chain Fv fragments (scFv). Examples of scFv antibodies are known and described, for example, in Huston et al, 1988.

In recent years, various strategies for the production of multimeric scFv derivatives have been developed. The intent is to produce recombinant antibodies with improved pharmacokinetic properties and increased avidity of the bound antibody. To achieve multimerization of scFv fragments (Multimerizing), they can be produced as fusion proteins with multimerization domains. The multimerization domain may be, for example, the CH3 region of IgG or a helix ("coiled coil"), such as a leucine-zipper domain. In another strategy, interactions between the VH and VL regions of scFv fragments are used to carry out multimerization (e.g., minibifunctional antibodies (diabodies), minitrifunctional antibodies (tribodies), and minipentafunctional antibodies (pentadies)).

The term "minibifunctional antibody" is used by the person skilled in the art to denote a bivalent homodimeric scFv derivative. Peptide linkers are shortened to 5-10 amino acids in scFv molecules, resulting in homodimers by overlapping VH/VL chains. The minibifunctional antibody may also be stabilized by inserting a disulfide bridge. Examples of minibifunctional antibodies can be found, for example, in Perisic et al, 1994.

The skilled person uses "miniantibodies" (minibodies), representing bivalent homodimeric scFv derivatives. It consists of a fusion protein which contains, as dimerization domain, the CH3 domain of an immunoglobulin, preferably of an IgG, particularly preferably of an IgGl. This is linked to the scFv fragment by a hinge region, which also belongs to IgG, and a linker region. Examples of such miniantibodies are described by Hu et al, 1996.

The skilled person uses "minitrifunctional antibodies", representing trivalent homotrimeric scFv derivatives (Kortt et al, 1997). Direct fusion of VH-VL does not require the use of linker sequences, resulting in trimer formation.

Fragments, which are referred to by the person skilled in the art as miniantibodies (mini antibodies), have a di-, tri-or tetravalent structure and are also derivatives of scFv fragments. Multimerization is achieved by the "coiled-coil" structure of a di-, tri-or tetramer (Pack et al, 1993 and 1995; Lovejoy et al, 1993).

Gene encoding fluorescent protein

In another embodiment, the expression vector according to the invention comprises a gene encoding a fluorescent protein, preferably functionally linked to the gene of interest. Preferably, both genes are transcribed under the control of a single heterologous promoter, such that the protein/product of interest and the fluorescent protein are encoded by the bicistronic mRNA. This allows the identification of cells that produce large amounts of the protein/product of interest by the expression rate of the fluorescent protein.

The fluorescent protein may be, for example, a green, cyan, blue, yellow, or other colored fluorescent protein. One particular example is Green Fluorescent Protein (GFP) obtained from aequorea victoria or jellyfish reniformis (Renilla reniformis), and mutants developed therefrom, see e.g., Bennet et al, 1998; chalfie et al, 1994; WO 0I/04306 and the references mentioned therein.

Other fluorescent proteins and the genes encoding them are described in WO 00/34318, WO 00/34326, WO 00/34526 and WO 01/27150, incorporated herein by reference. These fluorescent proteins are fluorophores from non-bioluminescent organisms of the species coralloides (Anthozoa), such as, for example, mahalanobis (Anemonia majano), corallinum (Clavularia sp.), buttonhallium (Zoanthus sp.) I, buttonhallium II, sea anemone (Discosomastiata), Cornus edodes (Discosoma sp.) "Red", Cornus edodes "Green", Cornus edodes "magenta", and sea anemone rugosa (Anemonia sulcata).

In addition to wild-type proteins, the fluorescent proteins used according to the invention also comprise mutants and variants, which are produced naturally or by genetic techniques, fragments, derivatives thereof or variants which have been fused, for example, with other proteins or peptides. Mutations used, for example, may alter the excitation or emission spectrum, chromophore formation, extinction coefficient or stability of the protein. In addition, codon optimization can be used to improve expression in mammals or other species. According to the invention, it is also possible to use a fluorescent protein fused to a selectable marker, preferably an amplifiable selectable marker, such as dihydrofolate reductase (DHFR).

The fluorescence emitted by the fluorescent protein allows the protein to be detected, for example, by a flow cytometer with Fluorescence Activated Cell Sorter (FACS) or by fluorescence microscopy.

Other regulating elements

The expression vector contains at least one heterologous promoter which allows gene expression of the gene of interest and preferably of the fluorescent protein.

The term "promoter" denotes a polynucleotide sequence which allows and controls the transcription of a gene or sequence to which it is functionally linked. The promoter contains a recognition sequence for RNA polymerase binding, and a transcription initiation site (transcription initiation site). In order to express the desired sequence in certain cell types or host cells, an appropriate functional promoter must be selected. Those skilled in the art are familiar with a variety of promoters from a variety of sources, including constitutive, inducible, and repressible promoters. They are deposited in databases, such as GenBank, and are available as individual elements, or as elements cloned into polynucleotide sequences obtained from commercial or industrial sources. In inducible promoters, the activity of the promoter may be decreased or increased in response to a signal. An example of an inducible promoter is the tetracycline (tet) promoter. The promoter contains the tetracycline operator sequence (tetO), which is inducible by the tetracycline-regulated transactivator protein (tTA). When tetracycline is present, the binding of tTA to tetO is inhibited. Examples of other inducible promoters are jun, fos, metallothionein and heat shock promoters (see also Sambrook et al, 1989; Gossen et al, 1994). Promoters particularly suitable for high expression in eukaryotes are, for example, the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the long terminal repeat of the Rous sarcoma virus, and the early promoter of the human cytomegalovirus. Examples of other heterologous mammalian promoters are actin, immunoglobulin or heat shock.

Promoters particularly suitable for high expression in eukaryotes are, for example, the SV40 early promoter, the adenovirus major late promoter, the mouse metallothionein-I promoter, the long terminal repeat of the Rous sarcoma virus, and the early promoter of the human cytomegalovirus. Examples of other heterologous mammalian promoters are actin, immunoglobulin or heat shock promoters.

The corresponding heterologous promoter may be functionally linked to other regulatory sequences in order to increase/regulate the transcriptional activity in the expression cassette.

For example, a promoter may be functionally linked to an enhancer sequence in order to increase transcriptional activity. For this, several copies of one or more enhancers and/or enhancer sequences may be used, such as the CMV or SV40 enhancer. Thus, in another embodiment, the expression vector according to the invention contains one or more enhancer/enhancer sequences, preferably the CMV or SV40 enhancer.

The term "enhancer" refers to a polynucleotide sequence that acts in cis position on the activity of a promoter and thus stimulates transcription of a gene functionally linked to the promoter. Unlike promoters, enhancers are not position and orientation dependent, and thus can be placed before or after the transcription unit, within an intron, or even within the coding region. Enhancers can be located in close proximity to the transcription unit and at a considerable distance from the promoter. Physical and functional overlap with the promoter is also possible. Those skilled in the art know that many enhancers from various sources (and deposited in databases, such as GenBank, for example SV40 enhancer, CMV enhancer, polyoma enhancer, adenovirus enhancer) are available as separate elements or as elements cloned into polynucleotide sequences (for example deposited at ATCC, available from commercial and industrial sources). Many promoter sequences also contain enhancer sequences, such as the frequently used CMV promoter. The human CMV enhancer is one of the strongest enhancers identified to date. An example of an inducible enhancer is the metallothionein enhancer, which can be stimulated by glucocorticoids or heavy metals.

Other possible modifications are, for example, the introduction of multiple Spl binding sites. The promoter sequence may also be associated with regulatory sequences which allow the control/regulation of the transcriptional activity. Thus, promoters can be made repressible/inducible. For example, it can be performed by linking it to a sequence of the binding site of a positively-or negatively-regulated transcription factor. The transcription factor Spl mentioned above, for example, has a positive effect on the transcription activity. Another example is the binding site of the activation protein AP-1, which can act positively and negatively on transcription. AP-1 activity can be controlled by a wide variety of factors, such as growth factors, cytokines, and serum (Faisst et al, 1992, and references therein). Transcription efficiency can also be increased by looking at altering the promoter sequence through a mutation (substitution, insertion or deletion) of one, two, three or more bases, which then determines in a reporter gene assay whether this increases promoter activity.

Basically, additional regulatory elements include promoters other than the hamster ubiquitin/S27 a promoter, enhancers, termination and polyadenylation signals, and other expression control elements. Inducible and constitutive regulatory sequences are known for various cell types.

"transcriptional regulatory elements" typically include a promoter upstream of the gene sequence to be expressed, transcription initiation and termination sites, and polyadenylation signals.

The term "transcription start site" means a nucleic acid in a construct that corresponds to the first nucleic acid to be incorporated into the major transcript, i.e., the mRNA precursor. The transcription start position may overlap with the promoter sequence.

The term "transcription termination site" means a nucleotide sequence which is normally at the 3' end of the gene of interest or gene segment to be transcribed and which causes termination of transcription by RNA polymerase.

A "polyadenylation signal" is a signal sequence which causes cleavage at a specific position at the 3 'end of eukaryotic mRNA and incorporates approximately 100-200 adenine nucleotides (poly A tail) at the cleaved 3' end after transcription. The polyadenylation signal comprises the sequence AATAAA, located approximately 10-30 nucleotides upstream of the cleavage site, and the sequence located downstream. Various polyadenylation elements are known, such as tk polyadenylation, SV40 late and early polyadenylation, or BGH polyadenylation (described in e.g. US5,122,458).

In a preferred embodiment of the invention, each transcription unit has a promoter or promoter/enhancer element, a gene of interest and/or a marker gene, and a transcription termination element. In another preferred embodiment, the transcription unit further comprises a translation regulatory unit.

"translational regulatory elements" include, for each polypeptide to be expressed, the translation initiation site (AUG), the stop codon, and the polyadenylation signal. For optimal expression, it may be advisable to remove, add or alter the 5 '-and/or 3' -untranslated regions of the nucleic acid sequence to be expressed, in order to exclude any additional translation initiation codons that may be inappropriate, or other sequences that may affect the level of transcription or expression. To facilitate expression, a ribosome consensus binding site may be inserted separately immediately upstream of the start codon. To produce a secreted polypeptide, the gene of interest typically contains a signal sequence that encodes a signal precursor peptide, which transports the polypeptide that has been synthesized to and through the ER membrane. The signal sequence is usually, but not always, located at the amino terminus of the secretory protein and can be cleaved by a signal peptidase after the protein has been inserted into the ER membrane. A gene sequence typically, but not necessarily, contains its own signal sequence. If the native signal sequence is not present, a heterologous signal sequence can be introduced in a known manner. Many signal sequences of this species are known to those skilled in the art and are deposited in sequence databases, such as GenBank and EMBL.

A particularly important regulatory element according to the present invention is the Internal Ribosome Entry Site (IRES). IRES elements include the 5' -terminal methylguanosine upstream of the independent gene? The sequence of the (guanosinium) CAP (CAP structure) functionally activates translation initiation and allows translation of two cistrons (open reading frames) from a single transcript in animal cells. The IRES element provides an independent ribosome entry site for translation of the open reading frame immediately downstream. In contrast to bacterial mRNA, which may be polycistronic, i.e., it may encode more different polypeptides or products, most mRNA from animal cells are monocistronic and encode only one protein or product by translation of the mRNA one after the other. In the case of polycistronic transcripts in eukaryotic cells, translation will start from the closest upstream translation start position and will be stopped by the first stop codon, followed by release of the transcript from the ribosome. Thus, only the first polypeptide or product encoded by the mRNA will be produced during translation. In contrast, polycistronic transcripts having an IRES element functionally linked to a second or subsequent open reading frame in the transcript, allowing for subsequent translation of the open reading frame coding construct located downstream thereof, to produce two or more polypeptides or products encoded by the same transcript in eukaryotic cells.

IRES elements can be of various lengths and of various origins, and can originate, for example, from encephalomyocarditis virus (EMCV) or other picornaviruses. Various IRES sequences and their use in constructing vectors are described in the literature, see e.g., Pelletier et al, 1988; jang et al, 1989; davies et al, 1992; adam et al, 1991; morgan et al, 1992; sugimoto et al, 1994; ramesh et al, 1996; mosser et al, 1997.

The gene sequence located downstream is functionally linked to the 3' end of the IRES element, i.e. the separation distance is chosen such that the expression of the gene is not or only slightly affected, or is sufficiently expressed for the intended purpose. The optimal permissive distance for sufficient expression between the IRES element and the start codon of the gene downstream thereof can be determined by simple experimentation by varying the spacing and determining the rate of expression as a function of the spacing using reporter gene measurements.

By means of said measurements it is possible to obtain optimal expression cassettes which are valuable for the expression of heterologous gene products. Accordingly, expression cassettes obtained by one or more of these measurements are also a further object of the invention.

Hamster ubiquitin/S27 a promoter

In another embodiment, the expression vector according to the invention comprises the hamster ubiquitin/S27 a promoter, preferably functionally linked to the gene of interest, more preferably functionally linked to the gene of interest and the gene encoding the fluorescent protein.

The hamster ubiquitin/S27 a promoter is a potent homologous promoter, described in WO 97/15664. Such promoters have preferably at least one of the following characteristics: a GC-rich sequence region, an Spl binding site, a polypyrimidine element, lacking a TATA box. Particularly preferred are promoters with an Spl binding site but lacking a TATA box. Furthermore preferred are constitutively active promoters, and in particular promoters which are equally active in serum-containing, low-serum and serum-free culture conditions. In another embodiment, it is an inducible promoter, in particular a promoter activated by removal of serum.

A particularly advantageous embodiment is a promoter having the nucleotide sequence contained in FIG. 5 of WO 97/15664. Particularly preferred in this case are promoters which contain the sequence from positions-161 to-45 in FIG. 5.

The promoters used in the examples of the present specification contain the sequences of the sequences having SEQ ID NOs: 55 from position 1923 to 2406. This sequence corresponds to the fragment-372 to +111 from FIG. 5 of WO97/15664 and represents a preferred promoter, i.e.a preferred promoter should comprise this sequence region. Another suitable promoter fragment contains the sequence from positions 2134 to 2406 (corresponding to-161 to +111 in FIG. 5 of WO 97/15664). The promoter containing only the sequence at positions 2251 to 2406 is no longer functional (corresponding to positions-45 to +111 in FIG. 5 of WO 97/15664). It is possible to extend the promoter sequence in the 5' direction starting from position 2143.

It is also possible to use functional subfragments of the complete hamster ubiquitin/S27 a promoter sequence, as well as functional mutant variants of the complete sequence or subfragments thereof, which have been modified, for example, by substitutions, insertions or deletions. The corresponding subfragments, mutants or variants are also referred to hereinafter as "modified promoters".

Modified promoters, preferably in combination with other regulatory elements, preferably having transcriptional activity, which correspond to the sequences shown in SEQ ID NO: the promoter fragment of nucleotide sequence positions 1923 to 2406 provided in 55, -372 to +111 of figure 5 of WO 97/15664). Modified promoters were found to be useful in the sense of the present invention if they have transcriptional activity in a comparative reporter assay of at least 50%, preferably at least 80%, more preferably at least 90%, and even more preferably at least 100% of the 1923 to 2406 fragments (-372 to +111 fragments). Particularly preferred are modified promoters which are specific for the wild-type sequence of the hamster ubiquitin/S27 a promoter SEQ ID NO: 1 has a minimum sequence homology of at least 80%, preferably at least 85%, preferably at least 90%, even more preferably at least 95%, and especially preferably at least 97%, and has a corresponding promoter activity in a comparative reporter assay.

In a corresponding comparative reporter assay, the promoter fragment to be tested comprises reference sequences cloned in front of respective promoterless reporter genes encoding, for example, luciferase, secreted alkaline phosphatase or Green Fluorescent Protein (GFP). This construct (promoter sequence + reporter gene) is subsequently introduced into test cells, for example CHO-DG44, by transfection, and the reporter gene expression induced by the respective promoter fragment is determined by measuring the protein content of the reporter gene. Corresponding tests are also exemplified in, for example, Ausubel et al, Current Protocols in Molecular Biology, 1994, update.

Promoter sequences and modified promoters of the hamster ubiquitin/S27 a promoter, which may also include, for example, the 5' untranslated region or a fragment selected therefrom, and the coding region of the ubiquitin/S27 a gene or a fragment selected therefrom, can be used by the skilled worker from a knowledge of the sequences described in WO97/15664, for example in Sambrook et al, 1989; obtained by various standard methods described in Ausubel et al, 1994. For example, one can start with the sequence described in WO97/15664, select an appropriate fragment, and chemically synthesize an oligonucleotide probe containing the sequence of the fragment. For example, such probes may be used, for example, to clone the ubiquitin/S27 a gene or its 5' untranslated region or other fragment from a hamster genome library by hybridization. Using the reporter gene assay described above, one skilled in the art would be able to identify fragments having promoter activity without undue effort and for the purposes of the present invention. The 5' untranslated region or its specific fragment can be easily obtained by PCR amplification using the corresponding primer from genomic DNA or genomic library. Fragments of the 5' untranslated region can also be obtained from larger DNA fragments by restriction exonuclease III digestion. Such DNA molecules may also be synthesized chemically, or produced by ligation from chemically synthesized fragments.

Deletion, insertion and substitution mutants can be produced by "site-specific mutagenesis" and/or "PCR-based mutagenesis techniques". Comparable methods are mentioned, for example, in chapter 36.1 of Lottspeich and Zorbas 1998 and in further references.

By hybridization with probes derived from the 5 'untranslated region of the hamster ubiquitin/S27 a gene or from the S27a portion or the 3' untranslated region of the hamster ubiquitin/S27 a gene, it is also possible to identify and isolate appropriate promoter sequences from other, preferably mammalian, corresponding homologous genes. Corresponding techniques are described in e.g. Lottspeich and Zorbas 1998 chapter 23. For the purposes of the present invention, "homology" is a gene which is "homologous" if its nucleotide sequence exhibits at least 70%, preferably at least 80%, preferably at least 90%, more preferably at least 95%, and particularly preferably at least 97% identity to the nucleotide sequence of the gene.

By the described method an optimal expression cassette can be obtained which is of high value for the expression of heterologous gene products. The expression cassettes obtained by one or more of these methods are therefore likewise subject matter of the invention.

Preparation of an expression vector according to the invention

In principle, the expression vectors according to the invention can be prepared by conventional methods customary to those skilled in the art, as described, for example, by Sambrook et al (1989). Functional components of the vector are also described, such as appropriate promoters (in addition to the hamster ubiquitin/S27 a promoter), enhancers, stop and polyadenylation signals, antibiotic resistance genes, selectable markers, origins of replication and splice signals. They can be prepared using conventional cloning vectors, such as plasmids, phages, phagemids, cosmids or viral vectors, such as baculovirus, retrovirus, adenovirus, adeno-associated virus and herpes simplex virus, and artificial chromosomes/minichromosomes. Eukaryotic expression vectors, which also typically contain prokaryotic sequences such as origins of replication and antibiotic resistance genes, allow the vector to replicate and select in bacteria. Many eukaryotic expression vectors containing multiple cloning sites for introducing polynucleotide sequences are known and some are available from various companies, such as Stratagene, La Jolla, CA, USA; invitrogen, Carlsbad, CA, USA; promega, Madison, Wis., USA or BD Biosciences Clontech, Palo Alto, Calif., USA.

The hamster ubiquitin/S27 a promoter, the gene of interest, the gene encoding the fluorescent protein, preferably also an amplifiable selectable marker gene, for example dihydrofolate reductase, and optionally additional regulatory elements, such as an Internal Ribosome Entry Site (IRES), enhancer or polyadenylation signal, are introduced into the expression vector in one of the ways familiar to the person skilled in the art. The expression vector according to the present invention contains at least a heterologous promoter, a gene of interest and a modified neomycin phosphotransferase gene. Preferably, the expression vector also contains a gene encoding a fluorescent protein. The ubiquitin/S27 a promoter is particularly preferably used according to the invention as heterologous promoter. Particularly preferred expression vectors are those in which the heterologous promoter (preferably the ubiquitin/S27 a promoter), the gene of interest and the gene encoding the fluorescent protein are functionally linked together or functionally linked and the neomycin phosphotransferase gene is located in the same or separate transcription units.

Within the context of this specification, the term "functionally linked" or "functionally linked" means two or more nucleic acid sequences or partial sequences which are positioned such that they perform their intended function. For example, a promoter/enhancer is functionally linked to a coding gene sequence if it can control or regulate the transcription of the linked gene sequence in an antegrade position. Typically, but not necessarily, functionally linked DNA sequences are in close proximity and, if two coding gene sequences are linked, or in the case of a secretory signal sequence, in the same reading frame. Although the functionally linked promoter is usually located upstream of the coding gene sequence, it does not necessarily have to be in close proximity thereto. Enhancers also need not be in close proximity, so long as they facilitate transcription of the gene sequence. For this purpose, they can be upstream and downstream of the gene sequence, optionally at a distance therefrom. The polyadenylation site is functionally linked to the gene sequence and, if it is located 3' to the gene sequence, allows transcription via the coding sequence to the polyadenylation signal. The ligation may be performed according to conventional recombination methods, for example by PCR techniques, by ligation at appropriate restriction cleavage sites, or by splicing. If no suitable cleavage sites are available, synthetic oligonucleotide linkers or adapters may be used in a known manner. According to the invention, it is preferred that the functional linkage is not via an intron sequence.

In one illustrative embodiment, a heterologous promoter (preferably the ubiquitin/S27 a promoter), a gene of interest, and a gene encoding a fluorescent protein are functionally linked. This means, for example, that the gene of interest and the gene encoding the fluorescent protein are expressed from the same heterologous promoter. In a particularly preferred embodiment, this functional linkage is performed via an IRES element, allowing the synthesis of a bicistronic mRNA from two genes. The expression vectors according to the invention may additionally contain enhancer elements which function functionally with respect to one or more promoters. Particularly preferred are expression vectors in which the ubiquitin/S27 a promoter or modified form thereof is linked to an enhancer element, such as the SV40 enhancer or CMV enhancer element.

Basically, the expression of a gene in an expression vector can be carried out starting from one or more transcription units. A transcription unit is defined as a region containing one or more genes to be transcribed. The genes within a transcription unit are functionally linked to each other such that all genes within such unit are under the transcriptional control of the same promoter or promoter/enhancer. The result of the transcriptional ligation of the gene is that more than one protein or product can be transcribed from a single transcriptional unit and expressed thereby. Each transcription unit may contain the regulatory elements necessary for the transcription and translation of the gene sequences contained therein. Each transcription unit may contain the same or different regulatory elements. Functional linkage of genes in a transcription unit can be performed using IRES elements or introns.

The expression vector may contain a single transcription unit for expression of the gene of interest, the modified NPT gene and optionally the gene encoding the fluorescent protein. Alternatively, the genes may be arranged in two or more transcription units. Various combinations of genes in the transcription unit are possible here. In another embodiment of the invention, more than one expression vector consisting of one, two or more transcription units can be introduced into the host cell by co-transfection, or in sequential transfections in any order. Any combination of regulatory elements and genes on each vector may be selected so long as sufficient expression of the transcription unit is ensured. If desired, additional regulatory elements and genes, such as additional genes of interest or selectable markers, can be placed on the expression vector.

Thus, the expression vector according to the invention may contain, in one or in two separate transcription units, a gene encoding the gene of interest and a gene encoding the modified neomycin phosphotransferase. Each transcription unit can transcribe and express one or more gene products. If two genes are contained in a single transcription unit, they are under the control of the same promoter or promoter/enhancer, and an IRES is preferably used to ensure functional linkage of all components. If the gene encoding the fluorescent protein and the amplifiable selectable marker gene are contained in two separate transcription units, they may be under the control of the same or different promoters/enhancers. However, for the selectable marker gene, it is preferred to use its native or weaker heterologous promoter, such as the SV40 early promoter, and preferably no enhancer. In the context of the present invention, expression vectors with two separate transcription units are preferred. One (bicistronic) transcription unit contains the gene of interest and the gene encoding the fluorescent protein, while the other transcription unit contains the amplifiable selectable marker gene. Preferably, each transcription unit is restricted at the 3' end by a sequence encoding a poly A signal, preferably tk poly A, BGH poly A or SV40 poly A.

Also preferred according to the invention are those vectors in which the gene of interest is replaced by a multiple cloning site which allows cloning of the gene of interest via a recognition sequence for a restriction endonuclease. From the prior art, numerous recognition sequences for all kinds of restriction endonucleases are known, as well as the restriction endonucleases belonging thereto. It is preferable to use a sequence consisting of at least six nucleotides as a recognition sequence. A list of suitable recognition sequences can be found, for example, in Sambrook et al, 1989.

Host cell

For transfection with the expression vectors according to the invention, eukaryotic host cells, preferably mammalian cells, and more particularly rodent cells, such as mouse, rat and hamster cell lines, are used. Transfection of corresponding cells with the expression vectors according to the invention results in transformed, genetically modified, recombinant or transgenic cells, which are also an object of the invention.

Preferred host cells within the scope of the present invention are hamster cells, such as BHK21, BHK TK^-CHO, CHO-K1, CHO-DUKX B1 and CHO-DG44 cells, or derivatives/progeny of these cell lines. Particularly preferred are CHO-DG44, CHO-DUKX, CHO-K1 and BHK21 cells, in particular CHO-DG44 and CHO-DUKX cells. Mouse myeloma cells are also suitable, with NS0 and Sp2/0 cells being preferred, as well as derivatives/progeny of these cell lines.

Examples of hamster and mouse cells that can be used according to the invention are provided in table 2 below. However, derivatives and progeny of these cells, other mammalian cells, including but not limited to human, mouse, rat, simian, rodent cell lines, or eukaryotic cells, including but not limited to yeast, insect, and plant cells, may also be used as host cells for the production of biopharmaceutical proteins.

Table 1: hamster and mouse producer cell lines

Cell lines	Accession number
		NS0	ECASS No. 85110503
Sp2/0-Ag14	ATCC CRL-1581
		BHK21	ATCC CCL-10
BHK TK	ECACC No. 85011423
		HaK	ATCC CCL-15
2254-62.2 (BHK-21-derivatives)	ATCC CRL-8544
		CHO	ECACC No. 8505302
CHO-K1	ATCC CCL-61
		CHO-DUKX(＝CHOduk，CHO/dhfr)	ATCC CRL-9096
CHO-DUKX B1	ATCC CRL-9010
		CHO-DG44	Urlaub et al; cell 33[2 ]]，405-412，1983
CHO Pro-5	ATCC CRL-1781
		V79	ATCC CCC-93
B14AF28-G3	ATCC CCL-14
		CHL	ECACC No. 87111906

Transfection of eukaryotic host cells with a polynucleotide or one of the expression vectors according to the invention is carried out by conventional methods (Sambrook et al, 1989; Ausubel et al, 1994). Suitable methods for transfection include, for example, lipoid-mediated transfection, calcium phosphate co-precipitation, electroporation, polycation (e.g., DEAE dextran) -mediated transfection, protoplast fusion, microinjection, and viral infection. According to the invention, stable transfection is preferably carried out, wherein the construct is integrated into the genome of the host cell, or into the artificial chromosome/minichromosome, or is contained in the host cell in episomal form in a stable manner. Transfection methods which provide the best transfection frequency and expression of the heterologous gene in the respective host cell are preferred here. By definition, each sequence or each gene inserted into a host cell is referred to as a "heterologous sequence" or "heterologous gene" with respect to the host cell. Even if the sequence to be introduced or the gene to be introduced is identical to the endogenous sequence or endogenous gene of the host cell, it is applicable. For example, the hamster actin gene introduced into hamster host cells is also a heterologous gene by definition.

According to the invention, particular recombinant mammalian cells, preferably rodent cells, particularly preferably hamster cells such as CHO or BHK cells, are transfected with an expression vector as described herein according to the invention.

In the recombinant production of heterodimeric proteins, for example monoclonal antibodies (MAbs), transfection of appropriate host cells can in principle be carried out by two different methods. Monoclonal antibody's of this class are composed of a number of subunits, heavy and light chains. The genes encoding these subunits can be placed in separate or polycistronic transcription units on a single plasmid, which is then used to transfect host cells. This attempts to ensure stoichiometric expression of the gene after integration into the host cell genome. However, in the case of independent transcription units, it must be ensured thereby that the mrnas encoding the different proteins exhibit the same stability, as well as the transcription and translation efficiency. In the second case, the expression of the gene in the polycistronic transcription unit is carried out by a single promoter, and only one transcript is formed. By using IRES elements, correct and efficient internal translation initiation of genes in the second and subsequent cistrons is obtained. However, the expression rate of these cistrons is lower than that of the first cistrons, which is substantially more efficient than IRES-dependent translation initiation by so-called "cap" -dependent translation initiation of the pre-initiation complex. In order to achieve true equimolar expression of the cistrons, additional intercistronic elements may for example be introduced which together with the IRES element lead to a uniform expression rate (WO 94/05785).

A further and preferred possibility for the simultaneous preparation of a plurality of heterologous proteins according to the invention is co-transfection, in which genes are integrated in separate expression vectors. This has the advantage that certain ratios of genes and gene products can be adjusted to each other, thereby balancing the differences in mRNA stability, as well as in transcription and translation efficiency. In addition, the expression vectors are more stable because of their smaller size and easier to manipulate on cloning and transfection.

Thus, in a particular embodiment of the invention, the host cell is additionally transfected, preferably co-transfected, with one or more vectors having a gene encoding one or more other proteins of interest. The other vector or vectors used for co-transfection encode, for example, another protein or proteins of interest, and at least one other selectable marker, such as dihydrofolate reductase, under the control of the same promoter/enhancer combination.

According to the invention, adaptation and culture of the host cells is preferably established in the absence of serum, optionally in a medium free of animal proteins/peptides. Examples of commercially available media include Ham ' S F12(Sigma, Deisenhofen, DE), RPMI-1640(Sigma), Dulbecco ' S modified Eagle ' S Medium (DMEM; Sigma) minimal essential Medium (MEM; Sigma), eskoff (Iscove ' S) modified Dulbecco ' S Medium (IMDM; Sigma), CD-CHO (Invitrogen. Carlsbad, CA, USA), CHO-S-SFMII (Invitrogen), serum-free CHO-medium (Sigma), and protein-free CHO-medium (Sigma). Each of these media is optionally supplemented with various compounds, such as hormones and/or other growth factors (e.g., insulin, transferrin, epidermal growth factor, insulin-like growth factor), salts (e.g., sodium chloride, calcium, magnesium, phosphate), buffered solutions (e.g., HEPES), nucleosides (e.g., adenosine, thymidine), glutamine, glucose or other equivalent nutrients, antibiotics, and/or trace elements. Although a serum-free medium is preferred according to the invention, it is also possible to use a medium which has been mixed with a suitable amount of serum to culture the host cells. To select genetically modified cells expressing one or more selectable marker genes, one or more appropriate selection agents are added to the medium.

The term "selective agent" means a substance that interferes with the growth and survival of a host cell lacking the selectable marker gene in question. Within the scope of the present invention, it is preferred to use geneticin (G418) as a media additive to select heterologous host cells carrying the modified neomycin phosphotransferase gene. Preferably, a G418 concentration of between 100 and 800. mu.g/ml of medium is used, particularly preferably between 300 and 400. mu.g/ml of medium. If the host cell is to be transfected with a plurality of expression vectors, i.e.if several genes of interest are to be introduced into the host cell separately, they generally have different selectable marker genes.

A "selectable marker gene" is a gene which allows the specific selection of cells containing the gene by adding the corresponding selection agent to the medium. More specifically, antibiotic resistance genes can be used as positive selection markers. Only cells which have been transformed with this gene are able to grow in the presence of the corresponding antibiotic and are therefore selected. In contrast, untransformed cells are unable to grow or survive under these selection conditions. There are positive, negative and bifunctional selectable markers. Positive selection markers allow for selection and enrichment of transformed cells by resistance to a selection agent, or by compensating for metabolic or catabolic defects in the host cell. Conversely, cells that have received the selectable marker gene can be selectively cleared by a negative selectable marker. An example of this is the thymidine kinase gene of herpes simplex virus, whose expression in cells results in the clearance of cells by the simultaneous addition of acyclovir (acyclovir) and 9- [1, 3-dihydroxy-2-propoxymethyl ] guanine (ganciclovir). Selectable markers, including amplifiable selectable markers, including genetically modified mutants and variants, fragments, functional equivalents, derivatives, homologs, and fusions to other proteins or peptides, are used in the invention so long as the selectable marker retains its selectable properties. Such derivatives exhibit considerable homology in the amino acid sequence, in regions or domains which are considered to be selective. The literature describes a number of selectable marker genes, including bifunctional (positive/negative) markers (see, e.g., WO 92/08796 and WO 94/28143). Examples of selectable markers often used in eukaryotic cells include the genes for Aminoglycoside Phosphotransferase (APH), hygromycin phosphotransferase (HYG), dihydrofolate reductase (DHFR), Thymidine Kinase (TK), glutamine synthetase, asparagine synthetase, and genes that mediate resistance to neomycin (G418), puromycin, histidinol D, bleomycin, phleomycin, and lipoxin (zeocin).

It is also possible to select transformed cells by Fluorescence Activated Cell Sorting (FACS). In this regard, transformed cells may be selected, for example, using bacterial beta-galactosidase, cell surface markers, or fluorescent proteins, such as Green Fluorescent Protein (GFP) and variants thereof from Victoria and nephroleph or other species, red fluorescent protein and proteins that fluoresce in other colors, and variants thereof from non-bioluminescent organisms such as Corallocta shiitake, Helianthus rugosa, Corallocta yunnanensis, and Helichrysum nikosum.

Gene expression and selection of high-producing host cells

The term gene expression is used in relation to the transcription and/or translation of heterologous gene sequences in a host cell. The expression rate can generally be determined based on the amount of the corresponding mRNA present in the host cell or based on the amount of the gene product encoded by the gene of interest produced. The amount of mRNA prepared by transcription from a specific nucleotide sequence can be determined, for example, by northern blot hybridization, ribonuclease-RNA protection, in situ hybridization of cellular RNA, or by PCR (Sambrook et al, 1989; Ausubel et al, 1994). The protein encoded by a particular nucleotide sequence may also be determined by a number of methods, such as, for example, ELISA, Western blotting, radioimmunoassay, immunoprecipitation, detection of the biological activity of the protein, or by immunostaining of the protein followed by FACS analysis (Sambrook et al, 1989; Ausubel et al, 1994).

The terms "high expression level (or rate)", "high expression", "increased expression" or "high productivity" mean a sustained and sufficiently high expression or synthesis of a heterologous sequence (e.g., a gene encoding a therapeutic protein) introduced into a host cell. If the cells according to the invention are cultured by one of the methods described herein according to the invention, without gene amplification, and if the cells produce at least more than about 0.5pg of the desired gene product per day (0.5 pg/cell/day), an increased or high expression level (rate) or high productivity is exhibited. Cells according to the invention also exhibit increased or high expression levels (rates) or high productivity if they produce at least more than about 1.0pg of the desired gene product per day (1.0 pg/cell/day) without prior gene amplification. In particular, cells according to the invention exhibit increased or high expression levels (rates) or high productivity if they produce at least more than about 1.5pg of the desired gene product per day (1.5 pg/cell/day) without prior gene amplification. In particular, cells according to the invention exhibit increased or high expression levels (rates) or high productivity if they produce at least more than about 2.0pg of the desired gene product per day (2.0 pg/cell/day) without prior gene amplification. A cell according to the invention exhibits a particularly increased or high expression or a particularly high expression level (rate) or a particularly high productivity if it produces at least more than about 3.0pg of the desired gene product per day (3.0 pg/cell/day) without prior gene amplification. Productivity may be increased by at least 2 to 10 fold by a single gene amplification step, such as by using the DHFR/MTX amplification system described hereinafter, so the use of the words "high expression", "increased expression" or "high productivity" to refer to a cell that has been subjected to a gene amplification step if the cell produces at least more than about 5pg of the desired gene product per day (5 pg/cell/day), preferably at least more than about 10 pg/cell/day, more preferably at least more than about 15 pg/cell/day, even more preferably at least more than about 20 pg/cell/day or at least more than about 30 pg/cell/day.

High or increased expression, high productivity or high expression levels (rates) can be achieved by using one of the expression vectors according to the invention and by using one of the methods according to the invention.

For example, by co-expressing the gene of interest and the modified NPT gene, cells expressing a heterologous gene to a high degree can be selected and identified. Compared to wtNPT, the modified NPT allows for more efficient selection of stably transfected host cells with highly expressed heterologous genes of interest.

The invention therefore also relates to a method for expressing at least one gene of interest in recombinant mammalian cells, characterized in that (i) a collection of mammalian cells is transfected with at least one gene of interest and a modified neomycin phosphotransferase gene which has an activity of only 1 to 80%, preferably only 1 to 60%, more preferably only 1.5 to 30%, particularly preferably only 1.5 to 26%, compared to the wild-type neomycin phosphotransferase; (ii) culturing the cells under conditions that allow expression of the gene of interest and the modified neomycin phosphotransferase gene; (iii) culturing mammalian cells in the presence of at least one selective agent, preferably G418, which selectively acts on the growth of mammalian cells and confers a growth advantage on cells expressing the modified neomycin phosphotransferase gene; and (iv) obtaining the protein of interest from the mammalian cells or from the culture supernatant. It is preferred to use recombinant mammalian cells which have been transfected with an expression vector according to the invention.

The invention also relates to a method for selecting recombinant mammalian cells expressing at least one gene of interest, wherein (i) a collection of mammalian cells is transfected with at least one gene of interest and a modified neomycin phosphotransferase gene which has only 1 to 80% activity, preferably only 1 to 60%, more preferably only 1.5 to 30%, particularly preferably only 1.5 to 26% compared to the wild-type neomycin phosphotransferase; (ii) culturing mammalian cells under conditions that allow expression of the gene of interest and the modified neomycin phosphotransferase gene; (iii) mammalian cells are cultured in the presence of at least one selective agent, preferably G418, which selectively acts on the growth of mammalian cells and confers a growth advantage on cells expressing the modified neomycin phosphotransferase gene.

If the modified NPT genes described in detail in this application are used, a particularly preferred method for expressing at least one gene of interest and for selecting recombinant cells which express the corresponding gene of interest, in particular if the modified NPT gene which encodes glycine or aspartic acid at amino acid position 182, alanine at amino acid position 91, glycine at amino acid position 198, alanine, glycine or valine at amino acid position 227, glycine or asparagine at amino acid position 261 or isoleucine at amino acid position 240 is used in comparison with the wild-type gene. Particularly preferred is the use of Asp227Val, Asp227Gly, Asp261Asn, Phe240Ile or Trp91Ala mutants. In general, all modified neomycin phosphotransferase genes mentioned in the present patent specification according to the invention are suitable for use in this process. For a preferred neomycin phosphotransferase gene, see the section on modified neomycin phosphotransferase genes.

Selection of cells expressing the gene of interest and the modified NPT gene is performed, for example, by adding G418 as a selection agent. However, other aminoglycoside antibiotics such as neomycin or kanamycin can also be utilized. Cells according to the invention are preferably cultured and selected in 200 to 800. mu.g of G418 per ml of culture medium. It has proven particularly preferred to add between 300 and 700. mu.g G418 per ml of culture medium. The preferred embodiment is about 400 micrograms G418 added per milliliter of medium. Using this method, recombinant cells having particularly high expression rates can be selected. In comparison with the use of wtNPT, after selection at 400 μ G418 per ml medium as selectable marker, the cells showed a fold increase in productivity of 1.4-2.4 in the case of the Glu182Gly, Glu182Asp and Val198Gly mutants, 1.6-4.1 in the case of the Asp227Gly mutant, 2.2 or 4 in the case of the Asp227Ala or Trp91Ala mutants, 5.7 or 7.3 in the case of the Phe240Ile or Asp261Asn mutants and even 9.3 or 14.6 in the case of the Asp261Gly or Asp227Val mutants. The specific productivity of the different modified NPT genes is shown in figure 6.

Corresponding methods can be combined with FACS-assisted selection of recombinant host cells containing one or more fluorescent proteins (e.g., GFP) or cell surface markers as additional selectable markers. Other methods for obtaining increased expression, and combinations of different methods, can also be used, based on e.g.treating the cells with (artificial) transcription factors, with natural or synthetic agents to up-regulate endogenous or heterologous gene expression, to improve the stability (half-life) of mRNA or protein, to improve the initiation of mRNA translation, to increase gene dosage by using episomal plasmids (based on using viral sequences as replication origins, such as SV40, polyoma virus, adenovirus, EBV or BPV), to use amplification promoting sequences (Hemann et al, 1994) or in vitro amplification systems based on DNA concatemers (Monaco et al, 1996).

Coupled transcription of the gene of interest and the gene encoding the fluorescent protein has proven particularly effective in connection with the use of the modified NPT gene as a selectable marker. The resulting dicistronic mRNA expresses both the protein/product of interest and the fluorescent protein. Based on this coupling of the expressed gene of interest and the fluorescent protein, high-yielding recombinant host cells can be easily identified and isolated according to the present invention by selecting the expressed fluorescent protein, e.g., by using a fluorescence-activated cell sorter (FACS).

The selection of recombinant host cells that exhibit high viability and increased expression levels of the desired gene product is a multi-staged process. For example, host cells that have been transfected with an expression vector according to the invention, or optionally co-transfected with other vectors, are cultured under conditions that allow selection of cells expressing the modified NPT, e.g., by culturing in the presence of a selection agent such as G418 at a concentration of 100, 200, 400, 600, 800 micrograms or more of G418 per milliliter of medium. The corresponding cells are then studied for expression of at least the gene encoding the fluorescent protein and coupled to the gene of interest in order to identify and select the cells/cell populations exhibiting the highest expression rate of the fluorescent protein. Preferably, 10 to 20% of the cells belonging to the highest expression rate of the fluorescent protein are separated and further cultured. In practice, this means that 10% of the brightest fluorescent cells are separated and further cultured. Thus, the brightest 5%, preferably the brightest 3% or even the brightest 1% of the fluorescent cells of the cell mixture can also be sorted out and replicated. In a particularly preferred embodiment, only the brightest 0.5% or brightest 0.1% fluorescent cells are separated and replicated.

The selection step may be performed in a cell pool or using a pre-selected cell pool/cell clone. One or more, preferably two or more and especially three or more sorting steps may be performed, while the cells may be cultured and replicated for a certain period of time between individual sorting steps, e.g. about two weeks in the case of pools. For example, FIGS. 11 and 12 show the specific productivity of the mutant Asp227Gly after FACS-based selection with and without gene amplification step.

The present invention therefore relates to a method for obtaining and selecting recombinant mammalian cells expressing at least one heterologous gene of interest, characterized in that (i) recombinant mammalian cells are transfected with an expression vector according to the invention; (ii) culturing the transfected cells under conditions which allow expression of the gene of interest, the gene encoding the fluorescent protein and the modified neomycin phosphotransferase gene; (iii) culturing mammalian cells in the presence of at least one selective agent that selectively acts on the growth of the mammalian cells and confers a growth advantage on the cells expressing the modified neomycin phosphotransferase gene; and (iv) selecting mammalian cells exhibiting particularly high fluorescent gene expression by flow cytometry analysis. If desired, the cells obtained in step (iv) may be repeated one or several times in steps (ii) to (iv).

A corresponding method is preferred, characterized in that the mammalian cells are selected to have an average specific productivity (without additional gene amplification step) of more than 0.5Pg of the desired gene product per cell per day (0.5 Pg/cell/day), preferably more than 1 Pg/cell/day, particularly preferably more than 2 Pg/cell/day, even more preferably more than 3 Pg/cell/day, even more preferably more than 4 Pg/cell/day, e.g.more than 5, 6, 7, 8, 9, 10 etc., more than 15, 20, 25 Pg/cell/day etc. As described above, the productivity of such cells can be increased by at least 2 to 10 fold by a simple gene amplification step, such as using the DHFR/MTX system. This is shown, for example, in figure 12 to utilize NTP mutant Asp227Gly for selection. Specific productivity was between 20 and 25 pg/cell/day.

Also preferred according to the invention is a method wherein suitably selected cells are replicated and used to prepare the encoded gene product of interest. For this purpose, the selected high producing cells are preferably cultured in serum free medium and preferably in suspension culture under conditions allowing the expression of the gene of interest. Preferably the protein/product of interest is a gene product that is secreted from the cell culture medium. However, if the protein is expressed without a secretion signal, the gene product may also be isolated from the cell lysate. To obtain a pure homogeneous product substantially free of other recombinant proteins and host cell proteins, conventional purification procedures are performed. First, cells and cell debris are removed from the culture medium or lysate. The desired gene product can then be decontaminated from soluble proteins, polypeptides and nucleic acids, for example by separation on an immunoaffinity and ion exchange column, ethanol precipitation, reverse phase HPLC or chromatography on sephadex, silica gel or a cation exchange resin such as DEAE. Methods leading to the purification of heterologous proteins expressed by recombinant host cells are known to the person skilled in the art and are described in the literature, e.g.by Harr1s et al, 1995 and Scopes 1998.

Amplifiable selectable marker genes

Furthermore, it is also possible to subject the cells according to the invention to one or more gene amplification steps, in which they are cultured in the presence of a selection agent which leads to the amplification of an amplifiable selectable marker gene. This step can be performed both on cells expressing the fluorescent protein and which have been pre-selected, preferably one or several times (preferably in one of the ways described herein), by FACS, and on non-selected cells.

Provided that the host cell is additionally transfected with a gene encoding an amplifiable selectable marker. It is envisaged that the gene encoding the amplifiable selectable marker may be present on one of the expression vectors according to the present invention or introduced into the host cell by means of another vector.

Amplifiable selectable marker genes typically encode enzymes required for eukaryotic cells to grow under certain culture conditions. For example, the amplifiable selectable marker gene may encode dihydrofolate reductase (DHFR). In this case, if the host cell transfected therewith is cultured in the presence of the selection agent Methotrexate (MTX), the gene is amplified.

Table 2 below provides examples of other amplifiable selectable marker genes and associated selection agents that may be used in accordance with the present invention, and are described in Kaufman, "methods of enzymology", 185: 537-.

Table 2: amplifiable selectable marker gene

Amplifiable selectable marker gene	Deposit number	Selection agent
			Dihydrofolate reductase	M19869 (hamster) E00236 (mouse)	Methotrexate (MTX)
Metallothionein	D10551 (hamster) M13003 (human) M11794 (rat)	Cadmium (Cd)
			CAD (carbamyl phosphate synthase: aspartate transcarbamylase: dihydroorotase)	M23652 (hamster) D78586 (human)	N-phosphoric acid acetyl-L-aspartic acid
Adenosine deaminase	K02567 (human) M10319 (mouse)	Xyl-A-or adenosine, 2' -deoxysynemetic
			AMP (adenylate) -deaminase	D12775 (human) J02811 (rat)	Adenine, azaserine, synbiotics
UMP-synthetase	J03626 (human)	6-azauridine, pyrazole furan
			IMP 5' -dehydrogenase	J04209 (hamster) J04208 (human) M33934 (mouse)	Mycophenolic acid
Xanthine-guanine-phosphoribosyl transferase	X00221 (Escherichia coli)	Mycophenolic acid with limited xanthines
			Mutated HGPRT enzymes or mutated thymidine-kinases	J00060 (hamster) M13542 (hamster),	hypoxanthine, aminopterin and thymidine (HAT)

	K02581 (human) J00423, M68489 (mouse) M63983 (rat) M36160 (herpes virus)
			Thymidylate-synthetase	D00596 (human) M13019 (mouse) L12138 (rat)	5-Fluorodeoxyuridine
P-glycoprotein 170(MDR1)	AF016535 (human) J03398 (mouse)	Several drugs, e.g. adriamycin, vincristine, colchicine
			Ribonucleotide reductase	M124223, K02927 (mouse)	Aphidicolin
Glutamine-synthetase	AF150961 (hamster) U09114, M60803 (mouse) M29579 (rat)	Methionine sulfoxide amine (Methioninseulfoximin) (MSX)
			Asparagine-synthetase	M27838 (hamster) M27396 (human) U38940 (mouse) U07202 (rat)	Beta-aspartyl hydroxamic acid, Albizidine, 5' azacytidine
Argininosuccinate synthetase	X01630 (human) M31690 (mouse) M26198 (cattle)	Canavalia gladiata nitric acid
			Ornithine-decarboxylase	M34158 (human) J03733 (mouse) M16982 (rat)	Alpha-difluoromethyl ornithine
HMG-CoA-reductase	L00183, M12705 (hamster) M11058 (human)	Compactin
			N-acetyl glucosaminyl-transfer	M55621 (human)	Tunicamycin

Enzyme
			Sumoyl-tRNA-synthetases	M63180 (human)	Borrelidin
NaK-ATPase	J05096 (human) M14511 (rat)	Ouabain

The amplifiable selectable marker gene used according to the present invention is preferably a gene encoding a polypeptide having DHFR function, such as DHFR or a fusion protein derived from a fluorescent protein and DHFR. DHFR is essential for purine biosynthesis. Cells lacking the DHFR gene cannot grow in medium lacking purines. The DHFR gene is therefore a useful selectable marker for selecting and amplifying genes in cells cultured in purine-free medium. The selection medium used in conjunction with the DHFR gene was Methotrexate (MTX).

The invention therefore includes a method for preparing and selecting recombinant mammalian cells comprising the steps of: (i) transfecting a host cell with a gene encoding at least one protein of interest, a modified neomycin phosphotransferase and DHFR; (ii) culturing the cells under conditions that allow for expression of the different genes; and (iii) amplifying the co-integrated gene by culturing the cells in the presence of a selection agent, such as methotrexate, that allows amplification of at least the amplifiable selectable marker gene. Preferably, the transfected cells are cultured in media devoid of hypoxanthine/thymidine, devoid of serum, and added with increasing concentrations of MTX. Preferably, the concentration of MTX in the first amplification step is at least 5 nM. However, the concentration of MTX may also be at least 20nM or 100nM and may be increased stepwise up to 1. mu.M. In the independent case, concentrations greater than 1. mu.M, such as 2. mu.M, may be used.

If the corresponding cells are additionally transformed with the gene for the fluorescent protein, these cells can be identified and selected using a Fluorescence Activated Cell Sorter (FACS), then cultured in the presence of at least 20, preferably in the presence of 50 or 100nM MTX and amplified in a gene amplification step. In this way, productivity can be increased substantially to more than 20pg gene product per cell and day, preferably to more than 21, 22, 23, 24, 25 etc., 30, 35, 40 etc. The host cell may be subjected to one or more gene amplification steps in order to increase the copy number of at least the gene of interest and the amplifiable selectable marker gene. According to the present invention, the high productivity achievable is combined with efficient preselection by neomycin phosphotransferase-mediated resistance to aminoglycoside antibiotics such as neomycin, kanamycin and G418. It is possible to reduce the number of gene amplification steps required and, for example, to carry out only a single gene amplification.

Thus in another embodiment, the invention also relates to a method for obtaining and selecting recombinant mammalian cells expressing at least one heterologous gene of interest, characterized in that: (i) transfecting recombinant mammalian cells with an expression vector according to the present invention and a gene that amplifies a selectable marker gene; (ii) culturing mammalian cells under conditions that allow expression of the gene of interest, the modified neomycin phosphotransferase gene, and the gene encoding the fluorescent protein; (iii) culturing mammalian cells in the presence of at least one selective agent that selectively acts on the growth of the mammalian cells and confers a growth advantage on the cells expressing the modified neomycin phosphotransferase gene; (iv) selecting mammalian cells exhibiting particularly high fluorescent gene expression by flow cytometry analysis; (v) culturing the selected cells under conditions that express the amplifiable selectable marker gene; and (vi) adding a selection agent to the medium which results in amplification of the amplifiable selectable marker gene.

Particularly preferred is a corresponding method in which the modified neomycin phosphotransferase gene described in the present invention is used. Also preferred is a method wherein the amplification step is performed only once. Also preferred is a corresponding method which results in recombinant mammalian cells exhibiting an average specific productivity of greater than 20pg per cell per day, preferably greater than 21, 22, 23, 24, 25, etc., 30, 35, 40, etc., of the desired gene product.

Mammalian cells, preferably mouse myeloma and hamster cells, are preferred host cells for use of DHFR as an amplifiable selectable marker. The cell lines CHO-DUKX (ATCC CRL-9096) and CHO-DG44(Urlaub et al, 1983) are particularly preferred since they have no DHFR activity per se as a result of the mutation. In order to be able to use DHFR-dependent amplification in other cell types with their own endogenous DHFR activity, it is possible to use mutated DHFR genes encoding proteins with reduced sensitivity to methotrexate during transfection (Simonson et al, 1983; Wigler et al, 1980; Haber et al, 1982).

The DHFR markers are particularly useful for selection and subsequent expansion when using DHFR negative primary cells such as CHO-GD44 or CHO-DUKX, so these cells do not express endogenous DHFR and therefore do not grow in purine-free medium. Thus, the DHFR gene can be used herein as a dominant amplifiable selectable marker and transformed cells selected in hypoxanthine/thymidine free medium.

In the following, the invention is explained in more detail by means of non-limiting embodiments.

Examples of the invention

Abbreviations

Ala (═ A) alanine

AP: alkaline phosphatase

Asn (═ N) asparagine

Asp (═ D) aspartic acid

bp: base pairing

BSA: bovine serum albumin

CHO: chinese hamster ovary

dhfr: dihydrofolate reductase

DMSO, DMSO: dimethyl sulfoxide

ELISA: enzyme linked immunosorbent assay

FACS: fluorescence activated cell sorter

FITC: fluorescein isothiocyanate

GFP: green fluorescent protein

Glu (═ E): glutamic acid

Gly (═ G): glycine

HBSS: hanks Balanced salt solution

HT: hypoxanthine/thymidine

Ile (═ I): isoleucine

IRES: internal ribosome entry site

kb: kilobases

mAb: monoclonal antibodies

MCP-1: monocyte chemotactic protein-1

MTX: methotrexate (MTX)

MW: mean value of

NPT: neomycin phosphotransferase

And (3) PCR: polymerase chain reaction

PBS: phosphate buffer

Phe (═ F): phenylalanine

Trp (═ W): tryptophan

Val (═ V): valine

WT: wild type

Method

1. Cell culture and transfection

The cells CHO-DG44/dhfr^-/-(Urlaub et al, 1983) in CHO-S-SFMII medium without serum, supplemented with hypoxanthine and thymidine (InVitrogen GmbH, Karlsruhe, DE) in cell culture flasks and humidified atmosphere at 37 ℃ and 5% CO₂The medium is permanently cultured as suspension cells. Cell number and viability were determined by CASY1 cell counter (Schaerfe System, DE) or by trypan blue staining, and cells were then applied at a concentration of 1-3X 10⁵Inoculations were performed every 2-3 days.

Lipofectamine Plus reagent (Invitrogen Gm) was used for transfection of CHO-DG44bH). For each transfection mixture, a total of 1. mu.g of plasmid DNA, 4. mu.l of Lipofectamine and 6. mu.l of Plus reagent were mixed together according to the manufacturer' S instructions and added in a volume of 200. mu.l to 6X 10 in CHO-S-SFMII medium supplemented with 0.8 ml of HT⁵Exponentially growing CHO-DG44 cells. After 3 hours of incubation at 37 ℃ in a cell culture incubator, 2 ml of HT supplemented CHO-S-SFMII medium was added. For NPT-based selection, cells were transferred 2 days post transfection to HT-supplemented CHO-S-SFMII medium with G418(invitrogen), with medium changes every 3 to 4 days. Typically, 400. mu.g/ml G418 is added for selection, and in some experimental sequences the concentration is also reduced to 200. mu.g/ml or increased to 500, 600 or 800. mu.g/ml. In the case of co-transfection in DHFR-and NPT-based selection, where one expression vector contains DHFR and the other expression vector contains a neomycin phosphotransferase selectable marker, cells were transferred 2 days after transfection to CHO-S-SFMII medium without xanthine and thymidine while G418(Invitrogen) was added to the medium at a concentration of 400. mu.g/ml.

DHFR-based gene amplification with integration of heterologous genes can be obtained by adding MTX (Sigma, Deisenhofen, DE) at a concentration of 5-2000nM as a selection agent to HT-free CHO-S-SFMII medium.

2. Expression vector

For analytical presentation, eukaryotic expression vectors based on pAD-CMV vector (Werner et al, 1998) and constitutive expression of heterologous genes mediated by a combination of CMV enhancer/hamster ubiquitin/S27 a promoter (WO97/15664) were used. Although the basic vector pBID contains the dhfr minigene which serves as an amplifiable selectable marker (see, e.g., EP-0-393-438), the dhfr minigene has been replaced in the vector pBIN by a neomycin phosphotransferase resistance gene (FIG. 1). For this purpose, the selectable marker neomycin phosphotransferase (including the SV40 early promoter and the TK-polyadenylation signal) was isolated as a 1640bp Bsu36I fragment from a commercial column pBK-CMV (Stratagene, La Jolla, Calif., USA). After the reaction of filling the fragment ends with Klenow-DNA polymerase, the fragments were ligated with the 3750bp Bsu36I/StuI fragment of the vector pBID, which was also treated with Klenow-DNA polymerase.

In the bicistronic basic vector pBIDG (FIG. 1), the IRES-GFP gene region was isolated from the vector pIRES2-EGFP (Clontech, Palo Alto, Calif., USA) and introduced into the vector pBID in a manner under the control of the CMV enhancer/promoter to preserve the multiple cloning site between the promoter region and the IRES-element. The following procedure was used. In the PCR mutagenesis, in which the plasmid pIRES2-EGFP acts as template, it is eliminated, on the one hand, by converting the HindIII cleavage site AAGCTT in the IRES sequence into the sequence ATGCTT using mutagenic primers. On the other hand, the XbaI cleavage site was inserted by a primer complementary to the 5 'end of the IRES sequence or the SpeI cleavage site was introduced by a primer complementary to the 3' end of the GFP sequence. The resulting PCR fragment (which contained the complete IRES and GFP sequence) was digested with XbaI and SpeI and cloned into the vector pBID at a separate XbaI cleavage site 3' of the multiple cloning site. In the same way, the IRES-GFP gene region from the vector pIRES2-EGFP was placed under the control of the CMV enhancer/hamster ubiquitin/S27 a promoter in the vector pBIN. This resulted in the bicistronic basic vector pBING (FIG. 1).

Human MCP-1cDNA (Yoshimura et al, 1989) was cloned as a 0.3kb HindIII/EcoRI fragment into the corresponding cleavage site of vector pBIN, resulting in vector pBIN-MCP1 (FIG. 2A).

For the expression of monoclonal human IgG2 antibody, the heavy chain was cloned as a 1.5kb BamHI/HindIII fragment into the vector pBID or pBIDG digested with BamHI and HindIII to give the vector pBID-HC or pBIDG-HC (FIG. 2B). On the other hand, the light chain was cloned with a 0.7kb BamHI/HindIII fragment at the corresponding cleavage position of the vector pBIN or pBING, resulting in the vector pBIN-LC or pBING-LC (FIG. 2B).

3.FACS

Flow cytometry analysis and sorting were performed with a Coulter Epics Altra apparatus. The FACS was set up with a helium-argon laser with an excitation wavelength of 488 nm. The fluorescence intensity was absorbed at a wavelength suitable for the fluorescent protein and processed by the attached software Coulter Expo 32. Picking is typically performed at a rate of 8000-And (4) selecting. The suspended cells can be centrifuged (at 180 Xg for5 minutes) and the cell concentration adjusted to 1-1.5X 10 in HBSS⁷Per milliliter. Cells can then be selected based on their fluorescent protein signals. The cells are collected in tubes already containing the culture medium, centrifuged and, depending on the number of cells selected, either inoculated into suitable culture vessels or placed directly in microtiter plates.

4.ELISA

The titer of MCP-1 in the supernatant of stably transfected CHO-DG44 cells was quantified by ELISA using the OptEIA human MCP-1 kit according to the manufacturer's instructions (BDbiosciences Pharmingen, Heidelberg, DE).

The IgG2 monoclonal antibody from the supernatant of stably transfected CHO-DG44 was quantified by ELISA using goat anti-human IgG Fc fragment (Dianova, Hamburg, DE) on the one hand and AP-conjugated goat anti-human K light chain antibody (Sigma) on the other hand according to standard procedures (Current protocols in Molecular Biology, Ausubel et al, 1994, update). Purified IgG2 antibody was used as a standard.

Productivity (pg/cell/day) was calculated from the formula pg/((Ct-Co) t/ln (Ct-Co)), where Co and Ct are the number of cells at implantation and harvest, respectively, and t is the culture time.

5. Dot assay to determine NPT enzyme Activity

To prepare the cell extract, 6X 10 was prepared according to the method of Duch et al, 1990⁶The cells were washed twice with PBS and resuspended in 600. mu.l extraction buffer (0.135M Tris-HCl pH6.8, 20% glycerol, 4 mM dithiothreitol). After four cycles of freezing and thawing in dry ice or water bath, the cell debris was removed by centrifugation and the supernatant was used for subsequent enzyme analysis. The protein concentration in the cell extracts was determined by the (Bradford) assay using the BIO-RAD protein assay (Bio-Rad Laboratories GmbH, Munich, DE) using BSA as standard protein (Current Protocols in molecular Biology, Ausubel et al, 1994, update). To determine the NPT enzyme activity, a dot-blot was carried out (based on the method of Platt et al, 1987)Analytical method (DotAssay). For this purpose, 5. mu.g, 2.5. mu.g and 1.25. mu.g of protein were adjusted to a final volume of 20. mu.l with extraction buffer, and the total protein mass was increased to 5. mu.g with a cellular extract from non-transfected CHO-DG44 cells. After addition of 200. mu.l of assay buffer (67mM Tiis-HCl pH7.1, 42mM MgCl)₂，400mM NH₄Cl) plus/minus 40 μ G/ml G418 and plus/minus 5 microCurie [ gamma-³³p]After ATP/ml (NEN), the extract was incubated at 27 ℃ for 135 minutes. The extract was then placed in a 96-well vacuum manifold (Schleicher)&Schull, Dassel, DE) was made through a layer of Whatman 3MM filter paper, P81 phosphocellulose membrane (Whatman laboratory division, Maidstone, Great Britain) and nitrocellulose membrane (Schleicher)&Schull). Proteins phosphorylated by protein kinases and non-phosphorylated proteins bind to nitrocellulose, while phosphorylated G418 passes through nitrocellulose and binds to phosphocellulose. The membrane was removed from the apparatus after three washes with deionized water, washed with water and then air dried. The radiation signals were quantified using a phosphoimager (phosphor Imager) (Molecular Dynamics, Krefeld, DE).

Northern blot analysis

Total RNA was isolated from cells using TRIZOL reagent according to the manufacturer's instructions (Invitrogen GmbH, Karlsruhe, DE) and 30. mu.g of RNA was isolated by gel electrophoresis and transferred to Hybond N + nylon membrane (Amersham Biosciences, Freiburg, DE) according to the standard procedure for glyoxal/DMSO-denatured RNA (Current protocols in Molecular Biology, Ausubel et al, 1994, update). The probe used for subsequent nonradioactive hybridization with the GennImages CDP-Star detection kit (Amersham biosciences) was a PCR product containing the coding region of the NPT gene, labeled with the GeneImages random primer labeling kit (Amersham biosciences, Freiburg, DE) FITC-dUTP-according to the manufacturer's instructions.

7. Dot blot analysis

Genomic DNA was isolated from cells using a DNA isolation kit according to the manufacturer's instructions (DNA isolation kit for cells and tissues; Roche Diagnostics GmbH, Mannheim, DE). Different amounts of DNA (10. mu.g, 5. mu.g, 2.5. mu.g, 1.25. mu.g, 0.63. mu.g and 0.32. mu.g) were filtered onto Hybond N + nylon membranes (Amersham Biosciences, Freiburg, DE) in alkaline buffer using a 96-well vacuum manifold (Schleicher & Schull, Dassel, DE) by standard methods (Ausubel et al, 1994). As a negative control, untransfected CHO-DG44 cells were used. Plasmid pBIN-LC was used as standard (320pg, 160pg, 80pg, 40pg, 20pg, 10pg, 5pg, 2.5 pg). The probe used for subsequent nonradioactive hybridization with the GnenImages CDP-Star detection kit (Amersham Biosciences) was a PCR product containing the coding region of the NPT gene, labeled with the GeneImages random primer labeling kit (Amersham Biosciences, Freiburg, DE) FITC-dUTP-according to the manufacturer's instructions. Chemiluminescence signals were quantified using ImageMaster VDS-CL (Amersham biosciences). The copy number of the npt gene in the cells was then determined using a standard series of signal intensities obtained from calibrated plasmid DNA. The number of plasmid molecules was calculated using Afugardro's constant and the DNA content of CHO cells set to about 5 pg.

Example 1: mutagenesis of neomycin phosphotransferase

Base substitutions of the wild-type NPT gene required for the NPT mutant Glu182Gly (SEQ ID NO: 3), Trp91Ala (SEQ ID NO: 5), Val198Gly (SEQ ID NO: 7), Asp227Ala (SEQ ID NO: 9), Asp227Val (SEQ ID NO: 11), Asp261Gly (SEQ ID NO: 13), Asp261Asn (SEQ ID NO: 15), Phe240Ile (SEQ ID NO: 17), Glu182Asp (SEQ ID NO: 19), Asp227Gly (SEQ ID NO: 21), Asp190Gly (SEQ ID NO: 23) and Asp208Gly (SEQ ID NO: 25) were prepared by PCR using mutagenic primers (FIG. 3). The vector pBIN (FIG. 1) or pBK-CMV (Stratagene, La Jolla, USA) was used as a template for PCR mutagenesis. First, the 5 'or 3' portion of the mutant was prepared in a different PCR mix. To prepare mutants Glu182Gly, Glu182Asp, Trp91Ala, Asp190Gly, Val198Gly, Asp208Gly and Asp227Gly, amplification was performed using a primer combination consisting of neocor 5(SEQ ID NO: 27) and related mutagenic reverse (reverse) primer or neolev 5(SEQ ID NO: 28) and related mutagenic forward (forward) primer:

neocor 5(SEQ ID NO: 27) and E182Grev (SEQ ID NO: 32) or Neorev5(SEQ ID NO: 28) and E182Gfor (SEQ ID NO: 31) in the case of the NPT mutant Glu182Gly (SEQ ID NO: 3);

neocor 5(SEQ ID NO: 27) and E182Drev (SEQ ID NO: 48) or Neorev5(SEQ ID NO: 28) and E182Dfor (SEQ ID NO: 47) in the case of NPT mutant Glu182Asp (SEQ ID NO: 19);

neocor 5(SEQ ID NO: 27) and W91Arev (SEQ ID NO: 34) or Neorev5(SEQ ID NO: 28) and W91Afor (SEQ ID NO: 33) in the case of the NPT mutant Trp91Ala (SEQ ID NO: 5);

neocor 5(SEQ ID NO: 27) and V198Grev (SEQ ID NO: 36) or Neorev5(SEQ ID NO: 28) and V198Gfor (SEQ ID NO: 35) in the case of the NPT mutant Val198Gly (SEQ ID NO: 7);

neocor 5(SEQ ID NO: 27) and D190Grev (SEQ ID NO: 50) or Neorev5(SEQ ID NO: 28) and D190Gfor (SEQ ID NO: 49) in the case of the NPT mutant Asp190Gly (SEQ ID NO: 23);

neocor 5(SEQ ID NO: 27) and D208Grev (SEQ ID NO: 52) or Neoref 5(SEQ ID NO: 28) and D208Gfor (SEQ ID NO: 51) in the case of the NPT mutant Asp208Gly (SEQ ID NO: 25);

neocor 5(SEQ ID NO: 27) and D227Grev (SEQ ID NO: 54) or Neorev5(SEQ ID NO: 28) and D227Gfor (SEQ ID NO: 52) in the case of the NPT mutant Asp227Gly (SEQ ID NO: 21);

to prepare mutants Asp227Ala, Asp227Val, Asp261Gly, Asp261Asn and Phe240Ile, a primer combination was used for amplification consisting of Neocor 2(SEQ ID NO: 29) and the related mutagenic reverse (rev) primer or IC49(SEQ ID NO: 30) and the related mutagenic forward (for) primer:

neocor 2(SEQ ID NO: 29) and D227Arev (SEQ ID NO: 38) or IC49(SEQ ID NO: 30) and D227Afor (SEQ ID NO: 37) in the case of the NPT mutant Asp227Ala (SEQ ID NO: 9);

neocor 2(SEQ ID NO: 29) and D227Vrev (SEQ ID NO: 40) or IC49(SEQ ID NO: 30) and D227 Vcor (SEQ ID NO: 39) in the case of the NPT mutant Asp227Val (SEQ ID NO: 11);

neocor 2(SEQ ID NO: 29) and D261Grev (SEQ ID NO: 42) or IC49(SEQ ID NO: 30) and D261Gfor (SEQ ID NO: 41) in the case of the NPT mutant Asp261Gly (SEQ ID NO: 13);

neocor 2(SEQ ID NO: 29) and D261Nrev (SEQ ID NO: 44) or IC49(SEQ ID NO: 30) and D261Nfor (SEQ ID NO: 43) in the case of the NPT mutant Asp261Asn (SEQ ID NO: 15);

neocor 2(SEQ ID NO: 29) and F240Irev (SEQ ID NO: 46) or IC49(SEQ ID NO: 30) and F240Ifor (SEQ ID NO: 45) in the case of the NPT mutant Phe240Ile (SEQ ID NO: 17);

the coding strand of the 5 'part and the complementary strand of the 3' part of the mutant under investigation were then bound by hybridization to the overlapping region formed by the mutagenic primer sequences, the single-stranded region was filled and the total product was amplified again in a PCR with the primers Neocor 5(SEQ ID NO: 27) and Neorev5(SEQ ID NO: 28) or Neocor 2(SEQ ID NO: 29) and IC49(SEQ ID NO: 30). The PCR products were digested with StuI/RsrII (Neocor 5/Neorev5PCR products of mutants Glu182Gly, Trp91Ala and Val198 Gly), StuI/BstBI (Neocor 5/Neorev5PCR products of mutants Glu182Asp, Asp190Gly, Asp208Gly and Asp227Gly) or DraIII/RsrII (Neocor 2/IC49PCR products of mutants Asp227Ala, Asp227Val, Asp26lGly, Asp261Asn and Phe240 Ile). Then in the vector pBIN-LC (FIG. 2B) or pBK-CMV (Stratagene, La Jolla, USA), part of the wild-type NPT sequence was removed by StuI/RsrII digestion, DraIII/RsrII digestion or StuI/BstBI digestion and replaced with the corresponding fragment of the PCR product. By sequence analysis of both the complementary and coding strands, the required base substitutions in the different mutants were checked to ensure that the remaining DNA sequence corresponded to the wild type NPT sequence. In this way, expression vectors pBIN1-LC, pBIN2-LC, pBIN3-LC, pBIN4-LC, pBIN5-LC, pBIN6-LC, pBIN7-LC and pBIN8-LC were generated which contained NPT mutants Glu182Gly, Trp91Ala, Val198Gly, Asp227Ala, Asp227Val, Asp26lGly, Asp261Asn or Phe240Ile (FIG. 2B).

The remaining NTP mutants were isolated from the modified pBK-CMV as a 1640bp Bsu36I fragment, the ends of which were filled with Klenow-DNA polymerase and ligated with the 3750bp Bsu36I/StuI fragment of vector pBID, which was also treated with Klenow-DNA polymerase. In this way, expression vectors pKS-N5, pKS-N6, pKS-N7 and pKS-N8 were generated, containing NPT mutant Glu182As p, Asp190Gly, Asp208Gly or Asp227Gly, respectively. Human MCP-1cDNA was then cloned into these expression vectors as a 0.3kb HindIII/EocRI fragment (FIG. 2A) or the light chain of humanized IgG2 antibody was cloned into these expression vectors as a 0.7kb HindIII/BamHI fragment (FIG. 2B).

Mutations which insert neomycin phosphotransferase are, on the one hand, more (Val198Gly, Phe240Ile) or less (Trp9lAla, Glul82Gly, Glul82Asp, Asp227Ala, Asp227Val, Asp227Gly) flanked by conserved domains, such as conserved amino acids of motifs 1, 2 and 3(Shaw et al, 1993) (FIG. 4). On the other hand, the mutation is located within conserved motif 1(Asp190Gly), 2(Asp208Gly) or 3(Asp261Gly, Asp261Asn) and relates to conserved amino acids.

Example 2: effect of NPT mutations on selection of stably transfected MCP-1 expressing cells

MCP-1 was used as an example of expression of single-chain proteins in CHO cells. For this purpose, CHO-DG44 cells were transfected with pKS-N5-MCP1, pKS-N6-MCP1, pKS-N7-MCP1, pKS-N8-MCP1 or pBIN-MCP1 (FIG. 2A). Duplicate preparations were performed. Two days after transfection, cells were plated into 96-well plates (2000 cells/well) and selected at 400. mu.g/ml G418 in HT-supplemented CHO-S-SFMII medium. In the case of cells transfected with pBIN-MCP1, selection was performed simultaneously with 800. mu.g/ml G418. The resulting cell populations were transferred sequentially through 24-well plates into 6-well plates. Even if differences could be detected between different transfection mixtures during the selection phase. In contrast to the cell population selected with the NPT wild-type gene (SEQ ID NO: 1), fewer cells in the cell population that have been transfected with the mutated NPT are tolerant to the initial selection with G418. These cell populations could not be transferred into 24-well plates until about four days later. In the mixture transfected with pKS-N6-MCP1 and pKS-N7-MPCI, cells without stable transfection were selected at a concentration of 400. mu.g/ml G418. It is speculated that the enzyme function in NPT mutants with the mutation Asp190Gly or Asp208Gly is so severely impaired that not enough G418 molecules can be inactivated to grow stably transfected cells. Although some cells survive in the first selection phase when the G418 concentration is reduced to 200. mu.g/ml, they are severely hampered in their growth and survival and cannot be expanded with a few exceptions in the case of the mutant Asp208 Gly.

From cells transfected with the mutants Glu182Asp and Asp227Gly or with NPT wild type, 18 pools (9 pools of mixtures 1 and 2, respectively) were cultured for four passages in 6-well plates and the concentration of MCP-1 produced in the cell culture supernatants was determined by ELISA. Pools of cells that have used the NPT mutant as a selectable marker show on average higher productivity of 50-57% (Glu182Asp mutant) or 57-65% (Asp227Gly mutant) than pools of cells selected with the NPT wild type at a concentration of 400 or even 800 μ G/ml G418 (figure 5). Thus, by using NPT mutants as selectable markers, the proportion of high producers in the transfected cell population can be increased reliably.

Example 3: effect of NPT mutations on cell selection for expression of stably transfected monoclonal antibodies

In co-transfection, CHO-DG44 cells were first transfected with plasmid combinations pBIDG-HC/pBIN-LC (NTP wild type), pBIDG-HC/pKS-N5-LC (Glu182Asp NPT mutant) or pBIDG-HC/pKS-N8-LC (Asp227Gly NPT mutant) (FIG. 2B). In the vector configuration used, the two protein chains of the humanized IgG2 antibody were each expressed as their own vector, which additionally encodes a DHFR or neomycin selectable marker in a different transcription unit. Expression of the product gene is mediated by the CMV-enhancer/hamster ubiquitin/S27 a promoter combination. However, comparable data can also be obtained, for example, using the CMV enhancer/promoter, SV40 enhancer/hamster ubiquitin/S27 a promoter, or other promoter combinations.

In total, four transfection series were carried out, in each case 6 pools per plasmid combination. In contrast to the cell population selected with the NPT wild-type gene, fewer cells in the cell population that had been transfected with the mutant NPT tolerated the initial selection with G418. Antibody titers in cell culture supernatants were determined by ELISA several times (6-8 times) 2-3 weeks after selection of transfected cell pools in non-HT CHO-S-SFMII medium supplemented with 400. mu.g/ml G418. Cells selected with the Glu182Asp mutant showed 86% or 77% increase in average productivity and titer, respectively, and cells selected with the Asp227Gly mutant showed 126% and 107% increase in average productivity and titer, respectively, compared to using the NTP wild-type gene as selectable marker. Thus, by using NPT mutants with reduced enzymatic activity, cells with up to two times the basal productivity can be selectively enriched.

In another transfection series, the effect of different concentrations of G418 on selection was tested. Pools of transfected cells, in each case 3 pools, were selected using 400, 500 or 600. mu.g/ml G418. At higher concentrations, significantly fewer cells survived the initial selection in the cell population selected with the NPT wild-type gene, with the Asp227Gly mutant being the most effective. However, the resulting population of stably transfected cells showed no hindrance to growth and viability. However, no significant difference was detected between the productivity and the titer achieved within the plasmid combination used for transfection. However, even here, the cells selected by the NPT mutant again had the highest productivity on average, being preceded by the Asp227Gly mutant, which was four times higher than the NPT wild type, and then by the Glu182Asp mutant, which was 2.4 times higher (fig. 6A).

CHO-DG44 cells were then transfected with plasmid combinations pBIDG-HC/pBIN-LC (NTP wild type), pBIDG-HC/pBIN1-LC (Glu182Gly NPT mutant), pBIDG-HC/pBIN2-LC (Trp91Ala NPT mutant), pBIDG-HC/pBIN3-LC (Val198Gly NPT mutant), pBIDG-HC/pBIN4-LC (Asp227Ala), pBIDG-HC/pBIN5-LC (Asp227Val NPT mutant), pBIDG-HC/pBIN6-LC (Asp261Gly NPT mutant), pBIDG-HC/pBIN7-LC (Asp261Asn NPT mutant), or pBIDG-HC/pBIN8-LC (Phe240Ile NPT mutant) (FIG. 2B) in co-transfections. In the vector configuration used, the two protein chains of the monoclonal humanized IgG2 antibody were again each expressed in their own vector, which additionally also encodes a DHFR or neomycin selectable marker in a different transcription unit.

For each plasmid combination, 5 pools were transfected. In contrast to the cell population selected with the NPT wild-type gene, fewer cells in the cell population that had been transfected with the mutant NPT tolerated the initial selection with G418. Antibody titers in cell culture supernatants were determined by ELISA six times two to three weeks after selection of transfected cell pools in non-HT CHO-S-SFMII medium supplemented with 400. mu.g/ml G418. Figure 6B summarizes the average titer and productivity determined from the pools in the test. All pools of cells that had been selected with NPT mutants showed an average increase in productivity and titer of 1.4-14.6-fold or 1.4-10.8-fold, respectively, compared to using the NPT wild-type gene as selectable marker (fig. 6B). Thus, the optimum selectivity increase for cells with higher basal productivity was obtained with the NPT mutant Asp227Val or Asp261Gly, which increased the average productivity by 14.6 or 9.3 fold, respectively.

The vector pBIDG-HC contained the alternative marker GFP. GFP is transcriptionally linked to the heavy chain via an IRES element. The correlation formed between the expression of the target protein and the selectable marker GFP also enables rapid assessment of the magnitude and distribution of expression levels in transfected cell populations based on GFP fluorescence as determined in FACS analysis. GBP fluorescence was measured in FACS analysis after two to three weeks selection of transfected cell aggregates in G418-free CHO-S-SFMII medium (FIG. 7). In fact, the GFP fluorescent signal was correlated with the potency data obtained with the monoclonal antibody IgG2 antibody. Pools selected with NPT mutants Asp227Val, Asp261Gly, Asp161Asn and Phe240Ile also had a higher proportion of cells with high GFP fluorescence, followed by cells selected with NPT mutants Trp91Ala, Asp227Gly, Gly182Asp, Asp227Ala, Glu182Gly and Val198 Gly.

The productivity of cells can be further increased by inducing dhfr-mediated gene amplification by adding the selective agent Methotrexate (MTX, Methotrexate) to the culture medium. Thus, for example, after a single gene amplification step at 100 nMTX, the specific productivity in cell aggregates resulting from cotransfection with plasmid combinations pBIDG-HC/pBIN5-LC (NPT mutant Asp227Val), pBIDG-HC/pBIN6-LC (NPT mutant Asp261Gly), pBIDG-HC/pBIN7-LC (NPT mutant Asp261Asn) and pBIDG-HC/pBIN8-LC (NPT mutant Phe240Ile) can be increased by 2 to 4-fold, depending on the aggregate, productivities of 4 to 14/cell/day can be achieved. FIG. 8 shows, by way of example, that the cell pool resulting from co-transfection of pBIDG-HC/pBIN5-LC (NPT mutant Asp227Val) achieves an increase in productivity of 27 pg/cell/day by the addition of MTX (100nM MTX followed by 500nM MTX).

Example 4: determination and comparison of NPT enzyme Activity

To compare the enzymatic activity of the NPT mutant with that of the NPT wild type, a dot assay was performed to determine NPT activity in the cell extracts, based on the procedure of Platt et al 1987 and shown by way of example in FIG. 9A for the NPT mutants Glu182Asp and Asp227 Gly. Cell extracts were prepared from pools of different monoclonal antibody-expressing cells, which had been transfected and selected with the NPT wild-type gene (SEQ ID NO: 1) or with the NPT mutant Glu182Gly (SEQ ID NO: 3), Glu182Asp (SEQ ID NO: 19), Tr91Ala (SEQ ID NO: 5), Asp190Gly (SEQ ID NO: 23), Val198Gly (SEQ ID NO: 7), Asp208Gly (SEQ ID NO: 25), Asp227Ala (SEQ ID NO: 9), Asp227Val (SEQ ID NO: 11), Asp227Gly (SEQ ID NO: 21), Asp261Gly (SEQ ID NO: 13), Asp261Asn (SEQ ID NO: 15) or Phe240Ile (SEQ ID NO: 17). Cell extracts from untransfected CHO-DG44 cells were used as negative controls.

Compared with the NPT wild type, the enzymatic activity of the NPT mutant was significantly reduced. The average NPT mutants only had 1.5% to 62% of the wild-type enzyme activity, whereas the NPT mutants Asp261Gly and Asp261Asn had the lowest residual activity at 3.1% or 1.5% and the NPT mutants Val198Gly and Trp91Ala had the highest residual activity at 61.9% or 53.2% (fig. 9B). The signal obtained on phosphocellulose is directed against G418 phosphorylation by NPT enzyme activity. No NPT substrate G418 was added to the assay buffer and no activity was detectable on phosphocellulose. The signal obtained on the nitrocellulose membrane, which is formed by the protein phosphorylated by the protein kinase in the cell extract, serves as an internal control for an equal amount of sample used.

The reduced enzyme activity of the NPT mutant compared to the NPT wild type cannot be attributed to reduced gene expression. In contrast, northern blot analysis of total RNA showed that the pool of cells transfected with NPT wild type and exhibiting high NPT enzyme activity expressed less RNA than the pool of cells transfected with NPT mutant (fig. 10). The only exception was cells transfected with the NPT mutant Glu182Gly and expressing comparable amounts of NPT-mRNA. In dot blot analysis performed on genomic DNA from these transfected cell populations, it was shown that higher expression of NPT mutants resulted from gene dose effects and/or from integration of foreign DNA into transcriptionally active genomic regions (fig. 10). For example, in cells selected with NPT mutant Trp91Ala, gene dose effects were significant in pool 1 and integration effects were significant in pool 2. In this way, a marker with reduced enzymatic activity is used to select transfected cells that are capable of synthesizing enough marker protein under the same selection pressure to compensate for the reduced tolerance to the selection agent.

Example 5: isolation of cells with high expression of monoclonal antibodies by GFP-based FACS selection

In co-transfection, CHO-DG44 cells were transfected with a plasmid combination pBID-HC and pBING-LC encoding monoclonal humanized IgG2 antibody (FIG. 2B). In the vector configuration used, the two protein chains of the antibody are each expressed in their own vector, which also encodes in a different transcription unit in addition a DHFR or modified neomycin phosphotransferase selectable marker (Asp227Gly mutant; SEQ ID NO: 21). In addition, the vector pBING-LC contains another selectable marker, GFP. As a result of the transcriptional ligation of the expression of GFP and light chains by IRES elements, the co-transfection of CHO-DG44 with the vector pBID-HC/pBING-LC enabled selection of cells with high GFP content in a short time only by serial FACS selection. Isolating cells with high expression of monoclonal antibodies. In total, 8 different pools of cells were transfected, thus giving a population of stably transfected cells after the first two to three weeks of selection with the addition of 400. mu.g/ml G418 in non-HTCHO-S-SFMII medium. The titer and productivity of all 8 pool cells were determined by ELISA several times (7-8 times). The mean titer was about 1.4 mg/l, and the productivity was about 1.3 pg/cell/day. For subsequent serial FACS-based sorting, 5 th and 8 th pools were selected, the 5 th pool having the highest productivity and the 8 th pool having an average productivity equivalent to that of all pools. In each step, 5% of the cells with the highest GFP fluorescence were screened by FACS and further cultured in the pool. This selection was performed up to a total of six times with about two weeks of incubation between each selection. Surprisingly, a good correlation between monoclonal antibody productivity and GFP fluorescence was found (fig. 13), although both protein chains were each expressed from their own vector and only the expression of the light chain could be selected during GFP-based FACS selection, due to their co-transcriptional coupling to GFP. Selection based on FACS alone increased productivity to 9.5 pg/cell/day (fig. 11). Comparable data were also obtained when the hamster promoter was functionally linked to the SV40 enhancer instead of the CMV enhancer. By a single subsequent MTX amplification step, starting from pool 5 and pool 8 of the first selection step, the productivity of pools could be increased to an average of more than 20 pg/cell/day by adding 100nM MTX to the selection medium (figure 12). High expression levels of fluorescent protein did not adversely affect cell growth and viability. During gene amplification, cell growth properties are also severely negatively affected by the addition of MTX, especially when it is added at higher concentrations. However, pre-selected cell aggregates showed significantly more robust properties in response to a fairly high initial dose of 100nM MTX. It is more resistant to the selection phase, i.e. only after 2 weeks a population of cells with high viability and good growth rate is obtained.

Furthermore, the development time for selecting highly productive cells is reduced to about 6 weeks compared to the traditional stepwise gene amplification strategy, which typically comprises 4 amplification stages with increasing MTX addition. This is achieved by the combined use of an increase in transfected cells with increased expression of the gene of interest by the use of a modified NPT selectable marker with reduced enzymatic activity, followed by GFP-based FACS selection and subsequent gene amplification steps.

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sequence listing

<110> Beilinger Engelham Frama Bihe (BOEHRINGER INGELHEIM PHARMA GMBH & CO KG)

<120> novel neomycin phosphotransferase gene and method for selecting high-producing recombinant cells

<130>Case 1-1411

<140>

<141>

<160>55

<170>PatentIn Ver.3.1

<210>1

<211>795

<212>DNA

<213> Escherichia coli (Escherichia coli)

<400>1

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>2

<211>264

<212>PRT

<213> Escherichia coli (Escherichia coli)

<400>2

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>3

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant E182G

<400>3

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcggggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>4

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant E182G

<400>4

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu ValAsp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Gly Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>5

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant W91A

<400>5

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac gcgctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>6

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant W91A

<400>6

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Ala Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>7

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant V198G

<400>7

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat gggggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>8

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant V198G

<400>8

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Gly Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>9

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D227A

<400>9

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg teactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtgc tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>10

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D227A

<400>10

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Ala Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>11

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D227V

<400>11

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtgt tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>12

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D227V

<400>12

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Val Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>13

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D261G

<400>13

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

ggcgagttct tctga 795

<210>14

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D261G

<400>14

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Gly Glu Phe Phe

260

<210>15

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D261N

<400>15

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

aacgagttct tctga 795

<210>16

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D261N

<400>16

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asn Glu Phe Phe

260

<210>17

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant F240I

<400>17

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcatc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>18

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant F240I

<400>18

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Ile

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>19

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant E182D

<400>19

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgatgatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>20

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant E182D

<400>20

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Asp Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>21

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D227G

<400>21

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtgg tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>22

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D227G

<400>22

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Gly Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<0210>23

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D190G

<400>23

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcggt gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>24

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D190G

<400>24

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Gly Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Asp

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>25

<211>795

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

neomycin mutant D208G

<400>25

atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc 60

ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca 120

gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg 180

caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg 240

ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag 300

gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg 360

cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc 420

atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa 480

gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac 540

ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat 600

ggccgctttt ctggattcat cggctgtggc cggctgggtg tggcggaccg ctatcaggac 660

atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc 720

ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt 780

gacgagttct tctga 795

<210>26

<211>264

<212>PRT

<213> Artificial sequence

<220>

<223> description of Artificial sequences

Neomycin mutant D208G

<400>26

Met Ile Glu Gln Asp Gly Leu His Ala Gly Ser Pro Ala Ala Trp Val

1 5 10 15

Glu Arg Leu Phe Gly Tyr Asp Trp Ala Gln Gln Thr Ile Gly Cys Ser

20 25 30

Asp Ala Ala Val Phe Arg Leu Ser Ala Gln Gly Arg Pro Val Leu Phe

35 40 45

Val Lys Thr Asp Leu Ser Gly Ala Leu Asn Glu Leu Gln Asp Glu Ala

50 55 60

Ala Arg Leu Ser Trp Leu Ala Thr Thr Gly Val Pro Cys Ala Ala Val

65 70 75 80

Leu Asp Val Val Thr Glu Ala Gly Arg Asp Trp Leu Leu Leu Gly Glu

85 90 95

Val Pro Gly Gln Asp Leu Leu Ser Ser His Leu Ala Pro Ala Glu Lys

100 105 110

Val Ser Ile Met Ala Asp Ala Met Arg Arg Leu His Thr Leu Asp Pro

115 120 125

Ala Thr Cys Pro Phe Asp His Gln Ala Lys His Arg Ile Glu Arg Ala

130 135 140

Arg Thr Arg Met Glu Ala Gly Leu Val Asp Gln Asp Asp Leu Asp Glu

145 150 155 160

Glu His Gln Gly Leu Ala Pro Ala Glu Leu Phe Ala Arg Leu Lys Ala

165 170 175

Ser Met Pro Asp Gly Glu Asp Leu Val Val Thr His Gly Asp Ala Cys

180 185 190

Leu Pro Asn Ile Met Val Glu Asn Gly Arg Phe Ser Gly Phe Ile Gly

195 200 205

Cys Gly Arg Leu Gly Val Ala Asp Arg Tyr Gln Asp Ile Ala Leu Ala

210 215 220

Thr Arg Asp Ile Ala Glu Glu Leu Gly Gly Glu Trp Ala Asp Arg Phe

225 230 235 240

Leu Val Leu Tyr Gly Ile Ala Ala Pro Asp Ser Gln Arg Ile Ala Phe

245 250 255

Tyr Arg Leu Leu Asp Glu Phe Phe

260

<210>27

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences

Oligonucleotide Neocor 5

<400>27

ttccagaagt agtgaggagg c 21

<210>28

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide Neorev5

<400>28

atggcaggtt gggcgtcgc 19

<210>29

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide Neocor 2

<400>29

gaactgttcg ccaggctcaa g 21

<210>30

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide IC49

<400>30

cggcaaaatc ccttataaat ca 22

<210>31

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide E182Gfor

<400>31

gacggcgggg atctcgtcgt 20

<210>32

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide E182Grev

<400>32

acgacgagat ccccgccgtc 20

<210>33

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide W91Afor

<400>33

gggaagggac gcgctgctat tgg 23

<210>34

<211>23

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide W91Arev

<400>34

ccaatagcag cgcgtccctt ccc 23

<210>35

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide V198Gfor

<400>35

ccgaatatca tgggggaaaa tggc 24

<210>36

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide V198Grev

<400>36

gccattttcc cccatgatat tcgg 24

<210>37

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences

Oligonucleotide D227Afor

<400>37

ctacccgtgc tattgctgaa g 21

<210>38

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D227Arev

<400>38

cttcagcaat agcacgggta g 21

<210>39

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D227 Vpor

<400>39

ctacccgtgt tattgctgaa g 21

<210>40

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D227Vrev

<400>40

cttcagcaat aacacgggta g 21

<210>41

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences

Oligonucleotide D261Gfor

<400>41

gccttcttgg cgagttcttc tgag 24

<210>42

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D261Grev

<400>42

ctcagaagaa ctcgccaaga aggc 24

<210>43

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D261Nfor

<400>43

gccttcttaa cgagttcttc tgag 24

<210>44

<211>24

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D261Nrev

<400>44

ctcagaagaa ctcgttaaga aggc 24

<210>45

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide F240Ifor

<400>45

ggctgaccgc atcctcgtgc tt 22

<210>46

<211>22

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide F240Irev

<400>46

aagcacgagg atgcggtcag cc 22

<210>47

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide E182Dfor

<400>47

gacggcgatg atctcgtcgt 20

<210>48

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide E182Drev

<400>48

acgacgagat catcgccgtc 20

<210>49

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D190Gfor

<400>49

catggcggtg cctgct tgc 19

<210>50

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of Artificial sequences

Oligonucleotide D190Grev

<400>50

gcaagcaggc accgccatg 19

<210>51

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D208Gfor

<400>51

gattcatcgg ctgtggccg 19

<210>52

<211>19

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D208Grev

<400>52

cggccacagc cgatgaatc 19

<210>53

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D227Gfor

<400>53

ctacccgtgg tattgctgaa g 21

<210>54

<211>21

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences:

oligonucleotide D227Grev

<400>54

cttcagcaat accacgggta g 21

<210>55

<211>2406

<212>DNA

<213> Chinese hamster (Cricetulus griseus)

<300>

<310>PCT/EP/96/04631

<311>1996-10-24

<312>1997-05-01

<400>55

gatctccagg acagccatgg ctattacaca gagaaaccct gtctggaaaa acaaaaaatt 60

agtgtccatg tgtaaatgtg tggagtatgc ttgtcatgcc acatacagag gtagagggca 120

gtttatggga gtcagttcct attcttcctt tatgggggac ctggggactg aactcaggtc 180

atcaggcttg gcagaaagtg cattagctca cggagcctta tcattggcga aagctctctc 240

aagtagaaaa tcaatgtgtt tgctcatagt gcaatcatta tgtttcgaga ggggaagggt 300

acaatcgttg gggcatgtgt ggtcacatct gaatagcagt agctccctag gagaattcca 360

agttctttgg tggtgtatca atgcccttaa aggggtcaac aacttttttt ccctctgaca 420

aaactatctt cttatgtcct tgtccctcat atttgaagta ttttattctt tgcagtgttg 480

aatatcaatt ctagcacctc agacatgtta ggtaagtacc ctacaactca ggttaactaa 540

tttaatttaa ctaatttaac cccaacactt tttctttgtt tatccacatt tgtggagtgt 600

gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gtgtgtgtgc 660

gcgcgcgcgc gcgctcggat cattctacct tttgtttaaa aaatgttagt ccaggggtgg 720

ggtgcactgt gaaagtctga gggtaacttg ctggggtcag ttctttccac tataggacag 780

aactccaggt gtcaactctt tactgacaga accatccaaa tagccctatc taattttagt 840

tttttattta tttatttttt gtttttcgag acagggtttc tctgtggctt tggaggctgt 900

cctggaacta gctcttgtag accaggctgg tctcgaactc agagatccac ctgcctctgc 960

ctcctgagtg ctgggattaa aggcatgcgc caccaacgct tggctctacc taattttaaa 1020

agagattgtg tgtcacaagg gtgtcatgtc gccctgcaac cacccccccc ccaaaaaaaa 1080

aaaaaaaaaa acttcactga agctgaagca cgatgatttg gttactctgg ctggccaatg 1140

agctctaggg agtctcctgt caaacagaat ctcaacaggc gcagcagtct tttttaaagt 1200

ggggttacaa cacaggtttt tgcatatcag gcattttatc taagctattt cccagccaaa 1260

aatgtgtatt ttggaggcag cagagctaat agattaaaat gagggaagag cccacacagg 1320

ttattaggaa gataagcatc ttctttatat aaaacaaaac caaaccaaac tggaggaggt 1380

ctacctttag ggatggaaga aaagacattt agagggtgca atagaaaggg cactgagttt 1440

gtgaggtgga ggactgggag agggcgcaac cgctttaact gtcctgtttt gcctattttt 1500

tggggacagc acatgttcct atttttccca ggatgggcaa tctccacgtc caaacttgcg 1560

gtcgaggact acagtcattt tgcaggtttc cttactgtat ggcttttaaa acgtgcaaag 1620

gtgaccatta accgtttcac gctgggaggg cacgtgcggc tcagatgctt cctctgactg 1680

agggccagga gggggctaca cggaagaggc cacacccgca cttgggaaga ctcgatttgg 1740

gcttcagctg gctgagacgc cccagcaggc tcctcggcta caccttcagc cccgaatgcc 1800

ttccggccca taacccttcc cttctaggca tttccggcga ggacccaccc tcgcgccaaa 1860

cattcggccc catcccccgg tcctcacctg aatctctaac tctgactcca gagtttagag 1920

actataacca gatagcccgg atgtgtggaa ctgcatcttg ggacgagtag ttttagcaaa 1980

aagaaagcga cgaaaaacta caattcccag acagacttgt gttacctctc ttctcatgct 2040

aaacaagccc cctttaaagg aaagcccctc ttagtcgcat cgactgtgta agaaaggcgt 2100

ttgaaacatt ttaatgttgg gcacaccgtt tcgaggaccg aaatgagaaa gagcataggg 2160

aaacggagcg cccgagctag tctggcactg cgttagacag ccgcggtcgt tgcagcgggc 2220

aggcacttgc gtggacgcct aaggggcggg tctttcggcc gggaagcccc gttggtccgc 2280

gcggctcttc ctttccgatc cgccatccgt ggtgagtgtg tgctgcgggc tgccgctccg 2340

gcttggggct tcccgcgtcg ctctcaccct ggtcggcggc tctaatccgt ctcttttcga 2400

atgtag 2406

Claims (47)

1. A modified neomycin phosphotransferase gene, characterized in that it encodes an amino acid different from the wild-type neomycin phosphotransferase at amino acid positions 91, 198 and/or 240 relative to the wild-type gene and is used for the generation and selection of high-yielding recombinant cells.

2. The modified neomycin phosphotransferase gene according to claim 1, characterized in that the modified neomycin phosphotransferase encoded by the neomycin phosphotransferase gene has a lower enzymatic activity than the wild-type neomycin phosphotransferase.

3. The modified neomycin phosphotransferase gene according to claim 1 or2, characterized in that it encodes an alanine at amino acid position 91, a glycine at amino acid position 198 and/or an isoleucine at amino acid position 240 relative to the wild-type gene, compared to the wild-type gene.

4. A modified neomycin phosphotransferase gene according to any one of claims 1 to 3, characterized in that it encodes a polypeptide having the sequence given in SEQ ID NO: 6. SEQ ID NO: 8 or SEQ ID NO: 18.

5. The modified neomycin phosphotransferase gene according to any one of claims 1-4, comprising the amino acid sequence of SEQ ID NO: 5. SEQ ID NO: 7 or SEQ ID NO: 17 or consists of these sequences.

6. A modified neomycin phosphotransferase gene, characterized in that it encodes a glycine at amino acid position 182, an alanine or valine at amino acid position 227 and/or a glycine at amino acid position 261, relative to the wild-type gene, and is used for the production and selection of high-yielding recombinant cells, in comparison with the wild-type gene.

7. The modified neomycin phosphotransferase gene according to claim 6, characterized in that it encodes a polypeptide having the sequence of SEQ ID NO: 4. SEQ ID NO: 10. SEQ ID NO: 12 or SEQ ID NO: 14.

8. The modified neomycin phosphotransferase gene according to any one of claims 6 or 7, comprising the amino acid sequence of SEQ ID NO: 3. SEQ ID NO: 9. SEQ ID NO: 11 or SEQ ID NO: 13 or consists of these sequences.

9. A modified neomycin phosphotransferase encoded by the modified neomycin phosphotransferase gene according to any one of claims 1 to 8.

10. A eukaryotic expression vector comprising the modified neomycin phosphotransferase gene according to any one of claims 1 to 8.

11. A eukaryotic expression vector comprising a heterologous gene of interest functionally linked to a heterologous promoter and a modified neomycin phosphotransferase gene according to any one of claims 1 to 8 encoding a neomycin phosphotransferase having a lower enzymatic activity compared to the wild-type neomycin phosphotransferase.

12. The expression vector according to claim 10 or 11, characterized in that the modified neomycin phosphotransferase gene encodes a polypeptide having the sequence given in SEQ ID NO: 4. SEQ ID NO: 6. SEQ ID NO: 8. SEQ ID NO: 10. SEQ ID NO: 12. SEQ ID NO: 14. or SEQ ID NO: 18.

13. The expression vector according to any one of claims 10 to 12, characterized in that the expression vector comprises one or more enhancers functionally linked to one or more promoters.

14. The expression vector according to claim 13, characterized in that the enhancer is the CMV or SV40 enhancer.

15. Expression vector according to any one of claims 10 to 14, characterized in that it comprises the hamster ubiquitin/S27 a promoter.

16. The expression vector according to claim 15, characterized in that the heterologous gene of interest is under the control of the ubiquitin/S27 a promoter.

17. The expression vector according to any one of claims 10 to 16, characterized in that the expression vector additionally comprises a gene for a fluorescent protein.

18. The expression vector according to claim 17, characterized in that the gene of the fluorescent protein is functionally linked or will be linked to a gene of interest and a heterologous promoter.

19. The expression vector according to claim 17 or 18, characterized in that said expression vector additionally comprises an Internal Ribosome Entry Site (IRES) allowing the gene encoding the fluorescent protein and the gene encoding the protein/product of interest to be expressed bicistronically under the control of a heterologous promoter.

20. The expression vector according to any one of claims 17 to 19, characterized in that the gene encoding the fluorescent protein and the modified neomycin phosphotransferase gene are located in one or two separate transcription units.

21. A mammalian cell comprising a modified neomycin phosphotransferase gene according to any one of claims 1 to 8.

22. A mammalian cell which has been transfected with an expression vector according to any one of claims 10 to 16.

23. A mammalian cell which has been transfected with an expression vector according to any one of claims 17-20.

24. Mammalian cell according to any of claims 21 to 23, characterized in that the mammalian cell is further transfected with an amplifiable selectable marker gene.

25. Mammalian cell according to claim 24, characterized in that the gene amplifiable selectable marker is the dihydrofolate reductase (DHFR) gene.

26. Mammalian cell according to any of claims 21 to 25, characterised in that the mammalian cell is a rodent cell.

27. Mammalian cell according to claim 26, characterized in that the rodent cell is a CHO or BHK cell.

28. A method of enriching mammalian cells, comprising:

(i) transfecting a collection of mammalian cells with a modified neomycin phosphotransferase gene according to any one of claims 1 to 8;

(ii) culturing a mammalian cell under conditions that allow expression of the modified neomycin phosphotransferase gene; and

(iii) mammalian cells are cultured in the presence of at least one selective agent that selectively acts on the growth of mammalian cells and confers a growth advantage on the cells expressing the modified neomycin phosphotransferase gene.

29. A method for obtaining and selecting mammalian cells expressing at least one heterologous gene of interest, comprising:

(i) transfecting a collection of mammalian cells with at least one gene of interest and the modified neomycin phosphotransferase gene according to any one of claims 1 to 8;

(ii) culturing mammalian cells under conditions that allow expression of one or more genes of interest and the modified neomycin phosphotransferase gene; and

(iii) culturing mammalian cells in the presence of at least one selective agent that selectively acts on the growth of mammalian cells and confers a growth advantage on cells expressing the modified neomycin phosphotransferase gene.

30. Method according to claim 29, characterized in that the mammalian cells are additionally transfected with a gene amplifying a selectable marker.

31. The method according to claim 30, characterized in that the selected mammalian cells are subjected to at least one gene amplification step.

32. The method according to claim 30 or 31, wherein the gene amplifying the selectable marker is DHFR.

33. Method according to any one of claims 30 to 32, characterized in that the gene amplification step is carried out by adding methotrexate.

34. A method for obtaining and selecting mammalian cells expressing at least one heterologous gene of interest, characterized in that

(i) Transforming a recombinant mammalian cell with an expression vector according to any one of claims 17-20;

(ii) culturing the cell under conditions that allow expression of one or more genes of interest, a gene encoding a fluorescent protein, and a modified neomycin phosphotransferase gene;

(iii) culturing mammalian cells in the presence of at least one selective agent that selectively acts on the growth of mammalian cells and confers a growth advantage on cells expressing the modified neomycin phosphotransferase gene; and

(iv) mammalian cells were selected by flow cytometry analysis.

35. Method according to claim 34, characterized in that the mammalian cells are additionally transfected with a gene for an amplifiable selectable marker and the cells sorted by flow cytometry analysis are subjected to at least one gene amplification step.

36. The method of claim 35 wherein the gene that amplifies the selectable marker is DHFR.

37. The method according to claim 35, characterized in that the gene amplification is carried out by adding methotrexate.

38. A method for producing at least one protein of interest in a recombinant mammalian cell, comprising:

(i) transfecting a collection of mammalian cells with at least one gene of interest and the modified neomycin phosphotransferase gene according to any one of claims 1 to 8;

(ii) culturing the cells under conditions that allow expression of the one or more genes of interest and the modified neomycin phosphotransferase gene;

(iii) culturing mammalian cells in the presence of at least one selective agent that selectively acts on the growth of mammalian cells and confers a growth advantage on cells expressing the modified neomycin phosphotransferase gene; and

(iv) obtaining one or more proteins of interest from the mammalian cells or from the culture supernatant.

39. A method for producing at least one protein of interest in recombinant mammalian cells, characterized in that

(i) Transfecting a recombinant mammalian cell with an expression vector according to any one of claims 17-20;

(ii) culturing the cells under conditions that allow expression of one or more genes of interest, a gene encoding a fluorescent protein, and a modified neomycin phosphotransferase gene;

(iii) culturing mammalian cells in the presence of at least one selective agent that selectively acts on the growth of mammalian cells and confers a growth advantage on cells expressing the modified neomycin phosphotransferase gene; and

(iv) sorting mammalian cells by flow cytometry analysis;

(v) obtaining one or more proteins of interest from the mammalian cells or from the culture supernatant.

40. A method of preparing at least one protein of interest, characterized by:

(i) culturing a mammalian cell according to any of claims 24 or 25 under conditions which allow expression of the gene of interest, the modified neomycin phosphotransferase gene and the gene for the amplifiable selectable marker;

(ii) culturing and selecting mammalian cells in the presence of at least one selective agent that selectively acts on the growth of mammalian cells and confers a growth advantage on cells expressing the modified neomycin phosphotransferase gene;

(iii) subjecting the selected mammalian cells to at least one gene amplification step; and

(iv) one or more proteins of interest are subsequently obtained from the mammalian cells or from the culture supernatant.

41. Method according to any one of claims 38 to 40, characterized in that mammalian cells are transfected with at least two genes of interest encoding proteins/products in heteromeric form, and

(i) culturing the cells under conditions that allow expression of the subunit of protein/product in heteromeric form; and

(ii) isolating the protein/product in heteromeric form from the culture or culture medium.

42. The method according to any one of claims 38 to 41, characterized in that the sorted mammalian cells exhibit an average specific productivity of more than 5pg of one or more desired gene products per cell per day.

43. The method according to claim 40 or 41, characterized in that the sorted host cells exhibit an average specific productivity of more than 20pg of one or more desired gene products per cell per day.

44. The method according to any one of claims 28 to 43, characterized in that the mammalian cell is a rodent cell.

45. The method according to claim 44, characterized in that the rodent cell is a CHO or BHK cell.

46. The method according to any one of claims 28 to 45, characterized in that the mammalian cells are cultured in suspension culture.

47. The method according to any one of claims 28 to 46, characterized in that the mammalian cells are cultured serum-free.