RU2763990C2 - Cell that produces active protein aryl sulfatase b with high efficiency, and method for obtaining this cell - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: transformed CHO-K1 cell is proposed, which is not defective in endosomal acidification, producing aryl sulfatase B with high yield achieved due to its co-expression with recombinant sulfatase-modifying factor 1 as part of an expression vector under EF1a promoter. A vector expression structure for obtaining a transformed cell, which essentially contains sequences: CMVe/EF1a pro, BGH pA, a regulatory element that increases gene expression, and a gene encoding the expressed protein selected from ARSB and SUMF1 are also disclosed.

EFFECT: invention can be used for the production of active lysosomal aryl sulfatases for the treatment of diseases of lysosomal accumulation by enzyme replacement therapy.

7 cl, 8 dwg, 1 tbl, 5 ex

Description

The basis of the invention was the discovery that a derivative of the CHO-K1 cell line expressing human arylsulfatase B, adapted to suspension cultivation, significantly increases its productivity when constructs expressing recombinant sulfatase-modifying factor 1 are introduced into it. The result of such modifications are cells that produce with high yield of active recombinant lysosomal sulfatase enzymes (10 times higher than similar cells without SUMF1). The invention also relates to the use of any derivative of the CHO-K1 cell line co-expressing recombinant human SUMF1 and recombinant lysosomal sulfatase enzyme, and may be suitable for the manufacture of active lysosomal sulfatases for the treatment of lysosomal storage diseases by enzyme replacement therapy.

Brief description of the drawings.

Fig. 1. Cell productivity during screening of 96 well plates of each group. ARSB+SUMF1 - cells transfected with two plasmids carrying the ARSB and SUMF1 genes; ARSB - cells transfected with a single plasmid carrying the ARSB gene. For statistical calculation of data, Unpaired t test (GraphPad program) P _value <0.0001 was used.

Fig. 2. Cell productivity on the 6th day of the batch cultivation process. ARSB+SUMF1 - cells transfected with two plasmids carrying the ARSB and SUMF1 genes; ARSB - cells transfected with a single plasmid carrying the ARSB gene. Unpaired t test (GraphPad program) was used for statistical calculation of data.

_Pvalue <0.0001.

Fig. 3. Specific productivity of cells. ARSB+SUMF1 - cells transfected with two plasmids carrying the ARSB and SUMF1 genes; ARSB - cells transfected with a single plasmid carrying the ARSB gene. For statistical calculation of data, Unpaired t test (GraphPad program) P _value =0.0001 was used.

Fig. 4. Electrophoregram (left) and Western blot (right) of cell QOL. Cell QOL samples were applied in 20 µl per track. Ladder - Protein Ladder marker (Thermo Fisher Scientific, USA). Standard - recombinant galsulfase protein, preparation "Naglazyme" (BIOMARIN, England). ARSB+SUMF1 - cells transfected with two plasmids carrying the ARSB and SUMF1 genes; ARSB - cells transfected with a single plasmid carrying the ARSB gene.

Fig. 5. Electropherogram (left) and Western blot (right) of cell lysates. The samples were applied in 8×10 ⁴ cells per track. Ladder - Protein Ladder marker, 10 µl. ASB - recombinant galsulfase protein, drug "Naglazyme" (BIOMARIN, England). ARSB+SUMF1 - cells transfected with two plasmids carrying the ARSB and SUMF1 genes; ARSB - cells transfected with a single plasmid carrying the ARSB gene.

Fig. Fig. 6. Productivity of CHO-K1-V and CHO-K1-GEN cells after super-transfection with a plasmid carrying the SUMF1 gene on day 6 of batch cultivation. ARSB+SUMF1 - cells transfected with two plasmids carrying the ARSB and SUMF1 genes; ARSB - cells transfected with a single plasmid carrying the ARSB gene.

Unpaired t test (GraphPad program) was used for statistical calculation of data. Unpaired t test for CHO-K1-V: P _value <0.0001, Unpaired t test for CHO-K1-GEN: P _value < 0.0001.

Fig. 7. Electropherogram (left) and Western blot (right) of cell lysates after super-transfection with the SUMF1 gene. The samples were applied in 8×10 ⁴ cells per track. Ladder - Protein Ladder marker, 10 µl per track. ARSB+SUMF1 - cells transfected with two plasmids carrying the ARSB and SUMF1 genes; ARSB - cells transfected with a single plasmid carrying the ARSB gene.

Fig. Fig. 8. Electrophoregram (left) and Western blot (right) of cell QOL samples after super-transfection with the SUMF1 gene on day 6 of batch cultivation. Samples were applied at 25 µl per track. Ladder - Protein Ladder marker, 10 µl. The standard is "Goddamn". ARSB+SUMF1 - cells transfected with two plasmids carrying the ARSB and SUMF1 genes; ARSB - cells transfected with a single plasmid carrying the ARSB gene.

List of used abbreviations

The present invention relates to a new highly effective modified producer strain of CHO-K1 cells or its derivatives for obtaining active recombinant lysosomal human sulfatase enzymes or their biologically active fragments, mutants, variants in amounts that provide their therapeutic use. In general, the proposed producer cell contains (a) a first expression vector for recombinant human sulfatase modifying factor 1, containing the sequences CMVe/EF1a pro, SUMF1 gene, BGH pA, and a regulatory element that increase gene expression, and (b) a second expressing vector for recombinant lysosomal arylsulfatase B enzyme containing sequences CMVe/EF1a pro, ARSB gene, BGH pA, and a regulatory element that increase gene expression. Obtaining a producer strain includes the steps of: (a) cultivating a CHO-K1 cell or a derivative thereof; (b) obtaining a first mammalian expression vector capable of expressing an active recombinant human lysosomal sulfatase enzyme or a biologically active fragment, mutant, variant or derivative thereof in a CHO-K1-derived cell or derivative thereof; (c) obtaining a second mammalian expression vector capable of expressing recombinant human sulfatase modifying factor 1 (SUMF1) or a biologically active fragment, mutant, variant or derivative thereof in a CHO-K1 cell or its derivative line, non-defective in endosomal acidification; (d) transfection of the CHO-K1 cell or derivative thereof with the first and second expression vectors; (e) selecting and cloning a CHO-K1 cell transfectant or derivative thereof that expresses an active recombinant human lysosomal sulfatase enzyme or a biologically active fragment, mutant, variant or derivative thereof; (e) optimizing a cell culture method for making a recombinant human lysosomal sulfatase enzyme or a biologically active fragment, mutant, variant or derivative thereof.

Preferably, the recombinant human lysosomal sulfatase is arylsulfatase B (ARSB gene). In other embodiments, arylsulfatase A (ARSA gene), iduronate-2-sulfatase (IDS gene), sulfamidase/heparin-N-sulfatase (SGSH gene), N-acetylglucosamine sulfatase (GNS gene ) and N-acetylgalactosamine-6-sulfatase (GALNS gene). When obtaining a cell, the steps of transfection of cDNA encoding all or part of the lysosomal sulfatase enzyme and cDNA encoding all or part of human SUMF1 into a CHO-K1 cell or its derivative not defective in endosomal acidification are used. In some embodiments, first and second expression vectors that are capable of expressing a cDNA encoding an active recombinant human lysosomal sulfatase enzyme and human SUMF1, respectively, are simultaneously transfected into a cell. In some embodiments, the first and second expression vectors are transfected into the cell sequentially. In some embodiments, a cDNA encoding a full-length human lysosomal sulfatase enzyme is used, while in other embodiments, a cDNA encoding a biologically active fragment, mutant, variant, or derivative thereof is used. In some embodiments, a cDNA encoding full-length human SUMF1 is used, while in other embodiments, a cDNA encoding a biologically active fragment, mutant, variant, or derivative thereof is used. In some embodiments, multiple expression vectors are used to transfer the human lysosomal sulfatase enzyme cDNA and SUMF1 simultaneously or sequentially into a CHO-K1 cell or derivative thereof. In some embodiments, a single expression vector is used to simultaneously transfer the cDNA of the lysosomal sulfatase enzyme and human SUMF1 into a CHO-K1 cell or derivative thereof. In a preferred embodiment, the CHO-K1 cell or derivative thereof is the CHO-K1 cell line. In a preferred embodiment, the method comprises the production of an active recombinant lysosomal arylsulfatase B (ASB) enzyme by the CHO-K1 cell line or a derivative thereof. The invention also relates to a modified CHO-K1 cell line characterized by its ability to produce active recombinant human lysosomal sulfatase enzymes in amounts that allow therapeutic use of the lysosomal sulfatase enzyme. In various embodiments, the invention also relates to CHO-K1 derived cell lines, or derivatives thereof, that are capable of producing active recombinant human lysosomal sulfatase enzymes in high yield, thereby allowing large scale production of such therapeutic lysosomal sulfatase enzymes. In one embodiment, the CHO-K1 cell line comprises (a) an expression vector for human recombinant sulfatase modifying factor 1 (SUMF1) whose nucleotide sequence includes the entire sequence of SEQ ID#1 and (b) an expression vector for recombinant lysosomal arylsulfatase enzyme B (ARSB), the nucleotide sequence of which includes the entire sequence of SEQ ID#2.

A known method for producing various sulfatases, incl. arylsulfatase B, during their co-expression with the SUMF1 gene (patents RU 2510820 and RU 2607376). The inventions relate to a G71 cell line adapted for growth in a serum-free suspension culture, referred to as G71S. The endosomal acidification-deficient G71S cell line expressing human sulfatase modifying factor 1 (SUMF1) produces lysosomal sulfatase enzymes with higher levels of activity (i.e., conversion of the cysteine residue in the arylsulfatase B active site to Cα-formylglycine (FGly)). The known method includes the steps of cultivating G71S cells of the END3 complementation group or its derivative; obtaining a vector containing a recombinant lysosomal human sulfatase enzyme; obtaining a second expression vector containing a recombinant sulfatase modifying factor 1 (SUMF1); then transfecting said G71S cell line with the first and second expression vectors. The selectable marker was the neomycin phosphotransferase gene, which carries a point mutation that reduces the efficiency of the enzyme. This marker was expressed under a weak HSV-tk promoter. The productivity was 0.6-1.2 pg/cell/day, with an enzyme activity of 1000-1400 ng/ml. For this cell line, a positive effect of SUMF1 on both productivity and activity of the resulting enzyme was shown.

There is also known a method for producing arylsulfatase B in a CHO cell line that is not defective in the END3 complementation system, while showing the effect of SUMF1 co-expression on sulfatase activity, but not on cell productivity (WO 2004072275, this method is closest to the claimed invention).

Thus, there is currently no method known in the art that allows the production of arylsulfatase B with high productivity and activity in any cell line other than the defective G71S line.

In the claimed invention, as in the prototype of WO 2004072275, for the production of sulfatase, the CHO-K1 line, which was not defective in endosomal acidification, was used, while, due to the introduction into the cell of its own expression constructs having the sequences of SEQ ID#1 and SEQ ID#2, was achieved a higher productivity of the enzyme compared with the prototype and other known methods, amounting to 1-3.5 pg/cell/day (Fig. 3). Arylsulfatase B contains a cysteine residue in the catalytic center; This cysteine residue is modified post-translationally in C _α -formilglitsin (FGly) to activate the enzyme. Post-translational activation of the enzyme, which is catalyzed by the FGE accessory protein (SUMF1 gene), occurs immediately after translation on unfolded sulfatases in the endoplasmic reticulum, after which the arylsulfatase B molecule is excreted from the cell using a signal peptide. Since the cell overexpresses arylsulfatase B, there is not enough endogenous FGE in the endoplasmic reticulum to convert each arylsulfatase B molecule. ), which contributes to the correct folding of arylsulfatase B, it is easier for the cellular apparatus to carry out the biosynthesis of excess arylsulfatase B, as a result, protein yield increases. The use of DNA combinations containing protein coding sequences in the transfection of CHO-K1 cells in the proposed method for producing sulfatases led to an unexpected significant increase in the level of expression of the target sulfatase, not previously observed in the prior art. Although different ways of obtaining sulfatases in various cell lines are known, it has not been previously shown that the introduction of the SUMF1 expression construct into the cell increases the expression of the target sulfatase by an order of magnitude. This technical result is new and not obvious from the prior art. In our opinion, the key factor was the use of the CHO-K1 cell line, which is not defective in endosomal acidification, in combination with the expression of the FGE and ASB proteins in the original expression constructs that ensure the optimal level of production of both proteins, which is always a non-trivial task when co-expressing two and more proteins (see example 2, Fig. 2, 6). The expression plasmid essentially contains the following elements, in order: a gene conferring bacterial resistance to ampicillin (selective marker, SV40 pro - sv40 virus promoter, puro - a gene conferring bacterial resistance to puromycin or neomycin (selective marker), CMVe/EF1a pro - enhancer of CMV virus and transcription promoter of elongation factor alpha gene, expressed protein gene - ARSB or SUMF1, BGH рА - signal of bovine growth hormone polyadenylation, UCOE - universal chromatin unfolding element can be additionally introduced. the cell requires the presence of two coding sequences, one of which contains CMVe/EF1a pro, the gene encoding ARSB, BGH pA, and the other which contains CMVe/EF1a pro, the gene encoding FGE, BGH pA.

An additional contribution to productivity can be obtained by introducing into the composition of the plasmid a nucleotide sequence that specifically binds to the isolated nuclear matrix under in vitro conditions. The use of such sequences to enhance transgene expression has been known since the 1990s. BODE et al. BIOLOGICAL SIGNIFICANCE OF UNWINDING CAPABILITY OF NUCLEAR MATRIX-ASSOCIATING DNAs SCIENCE. - 1992, p. 195-197. J Biol Chem. 1995 Oct 13; 270(41):24010-8. Mutually exclusive interaction of a novel matrix attachment region binding protein and the NF-muNR enhancer repressor. Implications for regulation of immunoglobulin heavy chain expression. Zong R et al. J Biol Chem. 1995 Oct 13; 270(41):24010-8. J Immunol. 2002 Sep 1; 169(5):2477-87. High frequency of matrix attachment regions and cut-like protein x/CCAAT-displacement protein and B cell regulator of IgH transcription binding sites flanking Ig V region genes. Goebel Pat al., J Biol. Chem. 1997 Jul 18; 272(29):18440-52. Interaction of the nuclear matrix-associated region (MAR)-binding proteins, SATB1 and CDP/Cux., with a MAR element (L2a) in an upstream regulatory region of the mouse CD8a gene. Banan et al., Crit Rev Eukaryot Gene Expr. 1999; 9(3-4):311-8. Origin and roles of nuclear matrix proteins. Specific functions of the MAR-binding protein MeCP2/ARBP. Strätling WH, Yu F. Construction of a chromosome specific library of human MARs and mapping of matrix regions attachment on human chromosome 19 JOURNAL Nucleic Acids Res. 24 (7), 1330-1336 (1996) In the patent RU 2235786 (C2), publ. 01/22/1999, the use of the so-called "matrix attachment region" is also disclosed, which is a higher-order regulatory element of the structural organization of chromatin, with the help of which chromatin is divided into topologically independent "loops", which allows for independent spatial and temporal regulation of gene expression and DNA replication. In addition to S/MAR and MAR, other known regulatory elements that increase gene expression are, for example, UCOE, STAR elements (Regulatory elements in vectors for the efficient production of cell lines producing target proteins. Acta Naturae (Russian version), 2015 Maksimenko O. G. Gasanov N.B. Georgiev P.G.).

Implementation of the invention

1. Obtaining Expression Vectors

Full-length human sulfatase-modifying factor 1 (SUMF1) cDNA encoding a 374 amino acid polypeptide was cloned into the pGNR2 expression vector, which contains the CMV virus enhancer and the elongation factor alpha gene transcription promoter, the bovine growth hormone polyadenylation signal, and the universal chromatin opening element, UCOE. The selectable marker was a neomycin resistance gene under the control of the sv40 promoter. The resulting plasmid was designated pGNR-071-007.

Full-length human N-acetylgalactosamine-4-sulfatase (Arylsulfatase B) cDNA encoding a 515 amino acid polypeptide including a 20 amino acid signal peptide was cloned into the pGNR1 expression vector, which contains the CMV enhancer and elongation factor alpha gene transcription promoter, bovine polyadenylation signal growth hormone and the universal chromatin opening element - UCOE. The selectable marker was a puromycin resistance gene under the control of the sv40 promoter. The resulting plasmid was designated pGNR-071-006.

2. Generation of cell lines co-expressing ASB and FGE

CHO-K1 cells were adapted for suspension cultivation in a medium that does not contain serum or other components of animal origin. Cells were adapted to serum-free medium by gradually decreasing the volume of serum-containing medium (100%, 50%, 20% and 0%) and increasing the volume of serum-free medium (0%, 50% 80% and 100%) BalanCD (Irvine Scientific, USA). Cells were cultured at 37°C, 5% CO ₂ and standard humidification conditions.

Host cells were transfected using the Neon Transfection System (Thermo Fisher Scientific, USA) according to the standard protocol for CHO cells. For this, CHO cells were mixed with paired combinations of plasmids encoding the SUMF1 and ARSB genes. After transfection, transfectant pools were placed in BalanCD medium. Control electroporation was performed without adding the plasmid. Viability and viable cell density were counted 24 and 48 hours after transfection. Then, transfectant pools were cultured in BalanCD medium with selective antibiotics: puromycin 5 µg/mL (Sigma, Japan) and/or neomycin 400 µg/mL (Gibco, USA). The presence of the target enzyme expressed in CL was measured by ELISA.

The maximum cell productivity during the screening of 96-well plates with the SUMF1 accessory gene was 3.5 mg/l, the maximum cell productivity without the accessory protein was 1.5 mg/l (Fig. 1)

3. Conducting enzyme immunoassay

For sorption on the solid phase, purified anti-galsulfase polyclonal antibodies (MBC Generium) were diluted in carbonate-bicarbonate buffer pH 9.6 to a concentration of 5 µg/ml and added 50 µl per well of a 96-well sorption plate (Nunc MaxiSorp, ThermoFisher ). Diluted CL samples in PBS-Ta buffer were added to the wells, 50 μl each. The standard (galsulfase, Naglazyme preparation 1.0 mg/ml) was diluted in PBS-Ta buffer to a concentration of 40, 20, 10, 5, 2.5 1.25, 0.625 ng/ml and added 50 µl per well. The diluted QOL and standard samples were incubated for 1 hour at +37°C, 300 rpm. Washing was carried out with PBS-T buffer in 3 repetitions, 200 μl per well. Then added conjugated antibodies (anti-galsulfaze HRP) diluted in PBS-Ta. Washing was carried out with PBS-T buffer in 3 repetitions, 200 μl per well. Then, the TMB solution was added to the wells in 100 μL. To stop the reaction, STOP solution (0.5 M H ₂ SO ₄ ) was added at 50 μl per well. The measurements were carried out on a Benchmark Plus spectrophotometer (Bio-Rad, USA) at OD 450 nm.

Evaluation of the specific productivity of cell lines expressing arylsulfatase B with FGE and without an auxiliary protein showed that the maximum productivity of cells with SUMF1 during batch cultivation was about 90 mg/l. The productivity of arylsulfatase B in cells with the additional SUMF1 gene is on average 10 times higher (Fig. 2). Cell lines without the FGE accessory protein have the lowest productivity of about 5 mg/L (FIG. 2). The specific productivity of cell lines expressing only ASB and ABS+FGE is also shown in FIG. 3.

4. Evaluation of target protein activity

The target protein can be isolated and purified from the CL of the producers by any standard method. The specific activity of the arylsulfatase B obtained according to the invention is comparable to that of the Naglazyme standard preparation.

5. Super-transfection with the SUMF1 genome of low-yielding arylsulfatase B cell lines

For super-transfection, cell lines CHO-K1-GEN and CHO-K1-V were used, which expressed arylsulfatase B in very low yield (about 1 mg/l). Cell lines CHO-K1-GEN and CHO-K1-V were mixed with plasmids carrying SUMF1 genes. Transfection was performed on the Neon Transfection System (Thermo Fisher Scientific, USA) according to the standard protocol for CHO. Next, the cells were placed in a medium with a selective antibiotic, and the transfectant pool was stabilized.

Examples

Example 1 Construction of an expression plasmid carrying the ARSB gene.

To create target constructs, ready-made minipreps of plasmids with target genes pGNR-071-002 (ARSB gene), pGNR-071-001 (SUMF1 gene) and pGNR2 and pGNR1 vectors, respectively, with restriction at the HindIII+XbaI sites, were used. The desired fragments of backbones and inserts were purified from agarose gel on microcolumns according to a standard protocol. The ligation mixtures were transformed into competent E. coli XL10 Gold cells. After overnight incubation on Petri dishes, 2 colonies for each construct were cleaved and transferred to individual tubes with 3 mL of 2-YT+Amp medium. After overnight incubation in a shaker, 2 ml of each culture was used for DNA purification according to the standard procedure on silicate columns . The resulting preparations were validated by analytical restriction. In all clones, the sizes of the obtained fragments corresponded to the theoretical ones. Plasmids were generated from 100 ml overnight culture, rechecked by analytical restriction for PstI sites, and linearized for PvuI sites.

Example 2. Evaluation of the productivity of cell lines under conditions of batch cultivation.

To assess the effect of the auxiliary protein on arylsulfatase B productivity, 26 cell lines co-expressing the target protein (ASB) and auxiliary protein (FGE) and 4 cell lines expressing only ASB were obtained. Analysis of cell lines was carried out in batch mode: cell seed density 0.2×10 ⁶ cells/ml; temperature regime + 37 ° С; stirring speed 200 rpm; the content of CO ₂ 5%. Analysis of the results obtained during batch cultivation showed that co-transfection of two genes positively affects the yield of the target ASB protein (Fig. 2).

Example 3 Reducing electrophoresis and western blot of CL of cell lines expressing arylsulfatase B with FGE and no accessory protein.

Analysis of cell lines co-expressing the target protein (ASB) and helper protein (FGE) was also performed by reducing electrophoresis and Western blot.

To obtain samples of cell culture fluid, CL was mixed 1X with a denaturing buffer containing 2-mercaptoethanol, after which the samples were heated for 10 min at +95°C. Trichloroethanol was added for protein visualization in SDS-PAGE.

To obtain samples of cell lysates, a cell suspension (1×10 ⁶ cells) was besieged by centrifugation at 1000 rpm for 5 minutes, the supernatant was decanted, the precipitate was washed in PBS in a volume equal to the removed supernatant. After washing, the cells were pelleted by centrifugation at 1000 rpm for 5 minutes, the supernatant was removed and the cells were resuspended in 100 μl of 1X denaturing buffer with 2-mercaptoethanol, after which the samples were heated for 5 minutes at +95°C.

For Western blot analysis, proteins from the gel were transferred to a nitrocellulose membrane (Thermo Scientific, USA). The membrane was then transferred to a blocking solution (5% skimmed milk powder for microbiology (Merck, Germany) in PBS-Ta buffer). The membrane was incubated for 1 hour; the membrane was transferred into a solution of primary antibodies for 1 hour; the membrane was washed in PBS-T 3 times for 5 minutes; the membrane was transferred into a solution of secondary antibodies for 1 hour; the membrane was washed in PBS-T 4 times for 5 minutes; a Western blotting detection Kit (Advanstal, USA) developing solution was applied to the membrane; used the ChemDoc detection system (Bio-Rad, USA) to visualize specific protein binding sites. For the detection of arylsulfatase B, rat polyclonal antibodies against galsulfase (MBC Generium) were used as primary antibodies, and rabbit antibodies against rat immunoglobulins (p-RAQ-Iss, IMTEK, Russia) were used as staining antibodies. To detect the product of the SUMF1 gene, rabbit polyclonal antibodies Anti-SUMF1 (ab91479, Abeam, England) were used; rabbit antibodies to goat immunoglobulins (P-RAG Iss, IMTEK, Russia) were used as staining antibodies.

The electrophoretic mobility of the target ASB protein is similar to the standard used - the Naglazyme preparation, however, a more intense signal can be observed in the QOL of cells that were co-transfected with the ARSB+SUMF1 genes (Fig. 4, tracks 1-2), which confirms the ELISA data batch cultivation (Fig. 2). Cells that were transfected with a single plasmid containing the ARSB gene had very little specific signal (FIG. 4, tracks 4-7).

Example 4 Production of the SUMF1 gene product, FGE accessory protein. Expression of FGE in cells with high productivity of arylsulfatase B.

The FGE protein is found in the endoplasmic reticulum. The molecular weight according to the amino acid sequence of FGE is 37-42 kD. Denaturing reducing electrophoresis and Western blot of cell lysates were performed to test the expression of the FGE accessory protein. In FIG. 5 on the right, one can observe the signal obtained due to the interaction of specific antibodies to FGE. All cell lines that have a high productivity of arylsulfatase B (mean 50 mg/l) were co-transfected with the SUMF1 and ARSB genes and have the FGE accessory protein (Fig. 5, tracks 1-3). For comparison, cell lines without ancillary protein were shown (FIG. 5, track 5-8), for which no specific FGE signal was observed. The data obtained confirm the contribution of FGE to the increase in the yield of arylsulfatase B during biosynthesis.

Example 5 Super-transfection with the SUMF1 gene of low productive cell lines expressing arylsulfatases B.

After stabilization of the transfectant pools in the presence of two selective antibiotics, the cell lines were seeded for batch cultivation. The productivity of cell lines was measured by ELISA. As a result of super-transfection of low productive cell lines CHO-K1-V and CHO-K1-GEN with a plasmid carrying the SUMF1 gene, it was possible to increase the yield of the target product by 10 times (Fig. 6).

The increase in the yield of the target enzyme of the low productive CHO-K1-GEN-2G2 cell line by super-transfection with the SUMF1 helper gene is also confirmed by the results (Fig. 8). On tracks 1-2, CL samples of the 2G2+SUMF1 cell line were applied; the specific signal is clearly visible and comparable with the standard in the 5th track. A specific signal to galsulfase was not observed in the CL of the 2G2 cell line (4th track, Fig. 8), without the auxiliary SUMF1 gene, which is associated with a very low productivity of the cell line without SUMF1. To confirm the presence of FGE cells with additional super-transfection with SUMF1, in cells with higher productivity, electrophoresis and Western blot of lysates of the 2G2 cell line were performed (Fig. 7). A specific signal to SUMF1 can be clearly observed on the membrane, in the cell lysate, from producers with additional transfection with the SUMF1 gene (Fig. 7, tracks 2-3). This correlates with the results of electrophoresis and Western blot of CL cells (Fig. 7, tracks 1-2). Thus, the productivity of arylsulfatase B increased after super-transfection with SUMF1.

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List of nucleotide sequences

SEQ ID#1 CMVe/EF1a pro - CMV enhancer and promoter

transcription of the gene elongation factor alpha

CTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAG

TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGC

CCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGA

CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCAT

ATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC

CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCT

ATTACCATGGGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCC

CCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGG

TAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAAC

CGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAA

CACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCCTCTTTACGGGTTATGGCCCTT

GCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGG

TTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGTCTT

GAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCG

CCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGA

CGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTT

CGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGA

GGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGC

CTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGG

CCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGA

GCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGA

AAAGGGCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGT

CCAGGCACCCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGG

GGTTTTATGCGATGGAGTTTCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTT

GGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTC

TCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTTCCATTTCAGGTGTCGTGAGGAATTA

GCTTGGTACTAATACGACTC

SEQ ID#2 Galsulfase - arylsulfatase B gene with stop codon

ATGGTGAGCCAGGCCCTGAGACT

GCTGTGCCTGCTGCTGGGCCTGCAGGGCTGCCTGGCCGCTGGCGCCAGCAGACCTCCCCA

CCTGGTGTTCCTGCTGGCCGACGACCTGGGCTGGAACGACGTGGGCTTCCACGGCAGCAG

AATCAGAACCCCCCACCTGGACGCCCTGGCCTGCTGGAGGCGTGCTGCTGGACAACTACTA

CACCCAGCCCCTGTGCACCCCAGCAGAAGCCAGCTCCTGACAGGAAGATACCAGATCAG

AACCGGCCTGCAGCACCAGATCATCTGGCCCTGCCAGCCCAGCTGCGTGCCTCTGGACGA

GAAGCTGCTGCCCCAGCTGCTGAAGGAGGCCGGCTACACCACCCACATGGTGGGCAAGTG

GCACCTGGGCATGTACAGAAAGGAGTGCCTGCCCACCAGAAGAGGCTTCGACACCTACTT

CGGCTACCTGCTGGGCAGCGAGGACTACTACAGCCACGAGAGATGCACCCTGATCGACGC

CCTGAACGTGACCAGATGTGCCCTGGACTTCAGAGATGGCGAGGAGGTGGCCACCGGCTA

CAAGAACATGTACAGCACCAACATCTTCACCAAGAGAGCCATCGCCCTGATCACCAACCA

CCCCCCCGAGAAGCCCCTGTTCCTGTACCTGGCCCTGCAGAGCGTGCACGAGCCCCTGCA

GGTGCCCGAGGAGTACCTGAAGCCCTACGACTTCATCCAGGACAAGAACAGACACCACTA

CGCCGGCATGGTGAGCCTGATGGACGAGGCCGTGGGCAACGTGACCGCCGCCCTGAAGTC

CAGCGGCCTGTGGAACAACACCGTGTTCATCTTCAGCACCGACAATGGCGGCCAGACCCT

GGCTGGAGGCAACAATTGGCCCCTGAGAGGCAGAAAGTGGAGCCTGTGGGAGGGCGGCGT

GAGAGGCGTGGGCTTCGTGGCCAGCCCCCTGCTGAAGCAGAAGGGCGTGAAGAACAGAGA

GCTGATCCACATCAGCGACTGGCCTGCCCACCCTGGTGAAGCTGGCCAGAGGCCACACCAA

TGGCACCAAGCCCCTGGACGGCTTCGACGTGTGTGGAAGACCATCAGCGAGGGCAGCCCCAG

CCCCAGAATCGAGCTGCTGCACAACATCGACCCCAACTTCGTGGACAGCAGCCCCTGCCC

CAGAAACAGCATGGCCCCCGCCAAGGACGACAGCAGCCTGCCCGAGTACAGCGCCTTCAA

CACCAGCGTGCACGCCGCCATCAGACACGGCAACTGGAAGCTGCTGACCGGCTACCCCGG

CTGCGGCTACTGGTTCCCTCCTCCCAGCCAGTACAACGTGAGCGAGATCCCCAGCAGCGA

TCCCCCTACCAAGACCCTGTGGCTGTTCGACATCGACAGAGACCCCGAGGAGAGACACGA

CCTGAGCAGAGAGTACCCCCACATCGTGACCAAGCTGCTGAGCAGACTGCAGTTCTACCA

CAAGCACAGCGTGCCCGTGTACTTCCCCCGCCCAGGACCCCAGATGCGACCCCCAAGGCCAC

CGGCGTGTGGGGCCCTTGGATGTGA

SEQ ID#3 BGH pA - bovine polyadenylation signal

growth hormone

GCTCGCTGAT

CAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTT

CCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCAT

CGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGG

GGGAGGATTGGGAAGACAATAGCAGGCATGCTGGG

SEQ ID#4 Amp - β-lactamase gene

TTACCAATGCTTAATCAGTGAGGCACCTATCTCA

GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG

ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCA

CCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGT

CCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGT

AGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCA

CGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACA

TGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGA

AGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACT

GTCATGCCATCCGTAAGATGCTTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGA

GAATAGTGTATGCGGCGACCGAGTTGCCTTTGCCCGGCGTCAATACGGGATAATACCGCG

CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTC

TCAAGGATCTTACCGCTGTTGAGATTCCAGTTCGATGTAACCCACTCGTGCACCCAACTGA

TCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT

GCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT

SEQ ID#5 sv40 pro - sv40 virus promoter

TGCATCTCAATTAGTCA

GCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCC

CATTCTCCGCCCCATCGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCG

GCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA

AAGCT

SEQ ID#6 puro - puromycin resistance gene

ATGACCGAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTC

CCCAGGGCCGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACC

GTCGATCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCTCACGCGC

GTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGG

ACCAGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCC

GAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCAC

SEQ ID#7 UCOE - Universal Chromatin Opening Element

GGGCGGCCGCACGCGTGGCCCTCCGCGCCTACAGCTCAAGCCACATCCGAA

GGGGGAGGGAGCCGGGAGCTGCGCGCGGGGCCGCCGGGGGGAGGGGTGGCACCGCCCACG

CCGGGCGGCCAGAAGGGCGGGGCAGCGGGCGCGCGCGCGGCGGGGGGAGGGGCCGGCGC

CGCGCCCGCTGGGAATTGGGGCCCTAGGGGGAGGGCGGAGGCGCCGACGACCGCGGCACT

TACCGTTCGCGGCGTGGCGCCCGGTGGTCCCCAAGGGGAGGGAAGGGGGAGGCGGGGCGA

GGACAGTGACCGGAGTCTCTCAGCGGTGGCTTTTCTGCTTGGCAGCCTCAGCGGCTGGC

GCCAAAACCGGACTCCGCCCACTTCCTCGCCCGCCGGTGCGAGGGTGTGGAATCCTCAG

ACGCTGGGGGAGGGGGAGTTGGGAGCTTAAAAACTAGTACCCCTTTGGGACCACTTTCAG

CAGCGAACTCTCCTGTACACCAGGGGTCAGTTCCACAGACGCGGGCCAGGGGTGGGTCAT

TGCGGCGTGAACAATAATTTGACTAGAAGTTGATTCGGGTGTTTCCGGAAGGGGCCGAGT

CAATCCGCCGAGTTGGGGCACGGAAAACAAAAAGGGAAGGCTACTAAGATTTTTCTGGCG

GGGGTTATCATTGGCGTAACTGCAGGGACCACCTCCCGGGTTGAGGGGGCTGGATCTCCA

GGCTGCGGATTAAGCCCCTCCCGTCGGCGTTAATTTCAAACTGCGCGACGTTTCTCACCT

GCCTTCGCCAAGGCAGGGGCCGGGACCCTATTCCAAGAGGTAGTAACTAGCAGGACTCTA

GCCTTCCGCAATTCATTGAGCGCATTTACGGAAGTAACGTCGGGTACTGTCTCTGGCCGC

AAGGGTGGGAGGAGTACGCATTTGGCGTAAGGTGGGGCGTAGAGCCTTCCCGCCATTGGC

GGCGGATAGGGCGTTTACGCGACGGCCTGACGTAGCGGAAGACGCCTTAGTGGGGGGGAA

GGTTTCTAGAAAAGCGGCGGCAGCGGCCTCTAGCGGCAGTAGCAGCAGCGCCGGGTCCCGTG

CGGAGGTGCTCCTCGCAGAGTTGTTTCTCCAGCAGCGGCAGTTCTCACTACAGCGCCAGG

ACGAGTCCGGTTCGTGTTCGTCCGCGGAGATCTCTCTCATCTCGCTCGGCTGCGGGAAAT

CGGGCTGAAGCGACTGAGTCCGCGATGGAGGTAACGGGTTTGAAATCAATGAGTTATTGA

AAAGGGCATGGCGAGGCCGTTGGCGCCTCAGTGGAAGTCGGCCAGCCGCCTCCGTGGGAG

AGAGGCAGGAAATCGGACCAATTCAGTAGCAGTGGGGCTTAAGGTTTATGAACGGGGTCT

TGAGCGGAGGCCTGAGCGTACAAACAGCTTCCCCACCCTCAGCCTCCCGGCGCCATTTCC

CTTCACTGGGGGTGGGGGATGGGGAGCTTTCACATGGCGGACGCTGCCCCGCTGGGGTGA

AAGTGGGGCGCGGAGGCGGGACTTCTTATTCCCTTTCTAAAGCACGCTGCTTCGGGGGCC

ACGGCGTCTCCTCGGACGGCCGGGCGCGCCG

SEQ ID#8 SUMF - SUMF gene with stop codon

ATGGCCGCCCCTGCCCTGGGCCT

GGTGTGTGTGGCAGATGCCCTGAGCTGGGACTGGTGCTGCTCCTGCTGTTGCTCAGCCTGCT

GTGTGGCCGCCGCCGGCAGCCAGGAGGCCGGCACCGGCGCTGGAGCCGGCAGCCTGGCCGG

CAGCTGCGGCTGTGGCACCCCCCAGAGACCTGGCGCCCACGGCAGCTCTGCCGCTGCCCA

CAGATACAGCAGAGAGGCCAATGCCCCTGGCCCTGTGCCTGGCGAGAGACAGCTGGCCCA

CAGCAAGATGGTGCCCATTCCTGCCGGCGTGTTCACCATGGGCACCGACGATCCTCAGAT

CAAGCAGGATGGCGAAGCCCCTGCCAGAAGAGTGACCATCGACGCCTTCTACATGGACGC

CTATGAGGTGAGCAACACCGAGTTCGAGAAGTTCGTGAATAGCACCGGCTACCTGACCGA

AGCCGAGAAGTTTGGCGACAGCTTCGTGTTCGAGGGCATGCTGAGCGAGCAGGTGAAGAC

CAACATCCAGCAGGCCGTGGCCGCTGCCCCTTGGTGGCTGCCCGTGAAAGGCGCCAATTG

GAGACACCCCGAGGGCCCCGACAGCACCATCCCTGCACAGACCCGACCACCCTGTGCTGCA

CGTGAGCTGGAACGACGCCGTGGCCTACTGCACCTGGGCCGGCAAGAGACTGCCCCACCGA

GGCCGAGTGGGAGTACAGCTGCAGAGGCGGCCTGCACAATAGACTGTTTCCCTGGGGCAA

CAAGCTGCAGCCCAAAGGCCAGCACTATGCCAATATCTGGCAGGGCGAGTTTCCCGTGAC

CAATACCGGCGAGGACGGCTTCCAGGGCACCGCCCCTGTGGACGCCTTCCCTCCCAATGG

CTATGGCCTGTACAACATTGTGGGCAATGCCTGGGAGTGGACCAGCGACTGGTGGACCGT

GCACCACAGCGTGGAGGAGACCCTGAATCCCAAAGGCCCTCCCAGCGGCAAGGACAGAGT

GAAGAAAGGCGGCAGCTACATGTGCCACAGAAGCTACTGCTACAGATACAGATGTGCCGC

CAGAAGCCAGAACACCCCCGACAGCAGCGCCAGCAATCTGGGCTTTTAGATGCGCTGCCGA

CAGACTGCCCACCATGGACTGA

SEQ ID#9 Neo - neomycin resistance gene

ATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAG

AGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTC

CGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTG

AATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGC

GCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTG

CCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCT

GATGCAATCGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCG

AAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGAT

CTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGC

ATGCCCGACGGCGAGGATCTCGTCGTGACACATGGCGATGCCTGCTTGCCGAATATCATG

GTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGC

TATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCT

GACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTAT

CGCCTTCTTGACGAGTTTCTTCTGA

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Claims (7)

1. CHO-K1 cell co-expressing arylsulfatase B and FGE proteins, characterized in that it contains (a) the first expression vector for recombinant human sulfatase-modifying factor 1 containing the sequences CMVe/EF1a pro, SUMF1 gene, BGH pA, and a regulatory element, an expression-enhancing gene; and (b) a second expression vector for a recombinant lysosomal arylsulfatase B enzyme containing the sequences CMVe/EF1a pro, the ARSB gene, BGH pA, and a gene expression-enhancing regulatory element. 2. A cell according to claim 1 that is not defective in endosomal acidification. 3. The cell according to claim 1, where (a) the first expression vector includes the entire sequence of SEQ ID No. 2, and (b) the second expression vector includes the entire sequence of SEQ ID No. eight. 4. A vector expression construct for obtaining a cell according to claim 1, which essentially contains the sequences: CMVe/EF1a pro, BGH pA, a gene expression enhancing regulatory element, and a gene encoding a protein selected from ARSB and SUMF1. 5. The construction according to claim 4, which contains at least one selective marker. 6. The construction of claim. 4, which includes the entire sequence of SEQ ID No. 2. 7. The construction of claim. 4, which includes the entire sequence of SEQ ID No. eight.

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