KR0145802B1 - Modified erythropoietin gene and expression vectors thereof - Google Patents

Abstract

The present invention provides a synthetic human erythrocyte growth promoter (EPO) gene using a human preferred codon and an expression vector comprising the same, and provides a method of mass-producing EPO in animal cells using the expression vector.

Description

Modified human erythrocyte growth promoter gene and expression vector thereof

Figure 1 shows the nucleotide sequence of the EPO 59 gene designed with codons that are frequently found in humans and the CD5 leader sequence linked to its 5 'end,

Figure 2 shows the nucleotide sequence of the EPO 60 gene and the EPO leader sequence linked to its 5 'end,

Figure 3 shows the nucleotide sequences of the primer DNA used to make the EPO 59 gene and EPO 60 gene,

Figure 4 shows the manufacturing process and structure of the expression vector pM1-EPO 59,

Figure 5 shows the manufacturing process and structure of the expression vector pM1-EPO 60,

6 shows the results of Western blotting using a single antibody against EPO after introducing pM1-EPO 59 and pM1-EPO 60 expression vectors into COS-7 cells to express EPO.

7 shows the nucleotide sequence of the EPO gene designed with the yeast preference codon described in the prior patent application 93-16865

8 is a result of Western blotting comparing the expression rate of the EPO 60 gene designed with the human line codon of the present invention and the EPO gene designed with the yeast preference codon of the prior patent application 93-165865.

The present invention relates to a large amount of human erythrocyte growth promoting factor (eryhtropoietin; hereinafter abbreviated as EPO) having excellent biological activity, and more particularly, an EPO gene using a human preferred codon, an expression vector including the same, and the expression thereof. A host cell transformed with a vector and a method for mass production of EPO using the host cell.

EPO is a glycoprotein hormone that promotes red blood cell growth in higher organisms (see Carnot et al., Compt. Ren. 143, 384 (1906)). EPO is produced in the liver of infants and in the kidneys and livers of adults, and in bone marrow and spleen of adults. , Naughton, BA et al, Science 196, 301-302 (1979); Krantz, SB, J. Biol. Chem. 238.4058-4090 (1963): Krstal, G., Expl. Hemat. 11, 649-660 (1983). EPO is maintained at about 15-30 milliunits (mU) or 0.01 mM per milliliter of blood (Garcia, J. F., Lab. Clin. Med. 99, 624-635 (1982)). Patients suffering from diseases such as aplastic anemia produce higher concentrations of EPO than normal individuals, and their blood or urine can be used for EPO production (White et al., Rec. Progr. Horm. Res. 16.219 ( 1960): Espada et al., Biochem. Med. 3. 475 (1970): Fisher Pharmacol. Rev. 24.459 (1972)).

EPO products can generally be used separately from the urine of patients with aplastic anemia, but mass production of EPO is very difficult due to limitations in the supply and demand of raw materials. In the urine of normal people, the concentration of EPO is low and there is an EPO activity inhibitor, there is a disadvantage that high purity purification is required to remove it. (See US Pat. Nos. 4,397,840; 4,303,650; 3,865,810). EPO can be extracted from sheep plasma, but sheep EPO has the disadvantage of acting as an antigen to humans (Goldwasser, Control Cellular Dif. Divelop., Part A; pp 487-494, Alan R. Liss. INC., NY (1981). EPO can be produced by animal in vivo proliferation of human lymphocytes, including Namalva, BALL-1, NALL-1, TALL-1 and JBL (Sugimoto et al., US Pat. No. 4,377,513). Since this method uses human tumor cells as a material, more intensive investigation is required to study stability. Recently, a method of cloning human EPO genes and expressing them in animal cells or insect cells has been developed (Domestic Patent Publication No. 93-5917, Domestic Patent Application No. 93-16865). However, these methods also have a disadvantage of low expression.,

After studying for mass production of EPO, the present inventors designed a gene that induces high EPO expression by using a human preferred codon, and by using an EPO leader sequence and an extracellular secretory protein leader sequence other than EPO before the EPO gene, It was found that the mass expression can be completed the present invention. It is therefore an object of the present invention to provide synthetic EPO genes using human preferred codons. Another object of the present invention is to provide an expression vector comprising the gene and a method for mass production of EPO using the same.

In the present invention, based on the known EPO amino acid sequence (Kennech Jacobs et al., Nature 313, 806 (1985)) high in animal cells prepared by employing codons (human preference codons) most frequently appear in humans An EPO gene showing the expression rate is provided. Human preferred codon refers to the codon that occurs most frequently in the human gene for a particular aminoline codon, as shown below;

The design principle of the synthetic EPO gene of the present invention codes for the same amino acid sequence as the amino acid sequence of natural EPO, wherein at least 70% of the total codons are the human preferred codons, more preferably at least 90%, most preferably at least 95% The base sequence. Also, optionally, one or two amino acid codons of N-terminal and C-terminal which are known to be ineffective may be lacking, and some codons such as N-terminal amino acid codons may have low G / C base content sequences. It may be substituted with a codon having a.

Representative genes of the present invention include the EPO59 gene and EPO 60 gene. The EPO 59 gene is designed to make human preferred codons 97.6% of the total codons, encoding proteins with one non-functional N- and C-terminal amino acids removed one by one. Based on the designed nucleotide sequence, the oligonucleotides were chemically synthesized to synthesize the EPO gene using a polymerase chain reaction (PCR, Saiki et al., Science 230,1350 (1985)), and then cloned into the M13 phage vector. Check it. This gene is cloned into CD5-IgGl (Aruffo et al., Cell, 61,1303 (1990)), which is an expression vector containing a CD5 leader sequence. The expression vector thus created is named pM1-EPO59. pM1-EPO59 includes cytomegalovirus (CMV) promoter, CD5 leader sequence, EPO59 gene, SV40 polyadenylation sequence, and the like. The characteristic of pM1-EPO59 vector is that the EPO gene is made from only the codons most frequently found in humans, and the CD leader sequence is used as a leader sequence to let EPO out into the cell (culture solution). There is an advantage. The leader sequence located before the gene of the present invention, such as EPO59, in the EPO gene expression vector may include, in addition to the CD5 leader sequence, an original EPO leader sequence or a cell isolation protein or a type-I cell membrane labeling protein (eg, TNF receptor (Smith et al. , Science, 248, 1019 (1990); IL-13 (Minty et al., Nature, 362, 248 (1993); Ita et al., Cell, 66,233 (1991); and CD 40 (Stamenkovic et al. , EMBO J., 8, 1403 (1989), etc.) can be used.

The EPO 60 gene is a further improvement of the EPO 59 gene, which is further enhanced by replacing the base sequence encoding the 5 amino acids of the 5 'terminal of the EPO 59 gene with a sequence having a low G / C base content. Lowering the G / C base content of the 5 'end of the EPO gene promotes transcription of the mRNA of the EPO, leading to an improvement in expression. The pMI-EPO 60 vector includes the cytomegalovirus (CMV) promoter, the EPO leader sequence and the newly designed EPO 60 gene, the SV 40 polyadenylation sequence, and the like. The expression vector pMI-EPO60 is characterized by adopting a human preferred codon, designing the 5 'end of the gene to promote mRNA transcription, and adopting the EPO leader sequence. E. coli MC1061 / P3 / PMI-EPO 60 transformed with the expression vector pMI-EPO60 was deposited with the accession number KCTC 0110BP dated May 17, 1994 to the Bank of Biotechnology.

Expression vectors of the present invention comprising the two expression vectors can be expressed in animal cells, such as COS, CHO, BHK.

For example, 96 hours after infection of the expression vector with COS-7 (ATCC CRL 1651, derived from African Green Monkey Kidney Cell), the supernatant was subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by EPO. The degree of expression can be confirmed by performing a Western blot using a monoclonal antibody against. The supernatant with EPO expressed in animal cells such as COS-7 can be measured by the ³ H thymidine method (Gerald Krystal Exp, Hematol., 7, 649 (1983)). As a result, when pMI-EPO 59 and pMI-EPO60 were expressed in COS-7, EPO was 37.0 U / ml for the pMI-EPO59 vector per milliliter of cell culture supernatant, and 540.0 U / ml for the pMI-EPO60 vector. It can be seen that this value is much higher than the expression rate in any animal cells published so far (expression in the COS cells of the invention of the domestic patent publication 93-5917 is 3.1 + 1.8 U / ml) . The invention of Korean Patent Application No. 93-16865 is characterized by expressing insect cell EPO. The expression level of the present invention was such that EPO was detected by Western blotting by precipitating 1 ml of cell culture with trichloroacetic acid. Outstanding expression rate is detected.

Hereinafter, the present invention will be described in detail with reference to Examples. The following examples are only intended to illustrate the invention specifically, it is clear that the present invention is not limited by the following examples.

All DNA manipulations in the examples below were followed by methods described in Frederckck et al., Short Protocols in Molecular Biology 2nd ED., 1992, Greene Publishing Associates and John Wiely Sons.

Example 1 Preparation of EPO59 Gene

To make the N-terminal half of EPO59, add 1 μl of primers 1, 2, 3, and 4 in FIG. 3 into one tube in the appropriate conditions (10 mM Tris-HCl, 1.5 mM MgCl ₂ , 50 mM KCl, 30 reactions (94 ° C, 55 ° C, 72 ° C) at 20 ° C in pH 8.3, 0.2mM dATP, 0.2mM dGTP, 0.2mM dCTP, 0.2mM dTTP, 10% DMSO, 1U Taq DNA Polymerase, final 50 μl volume , Each for 1 minute). The C-terminal was made by chain reaction under the same conditions using primers 5, 6, 7, and 8. A total of 520 bp of the EPO 59 gene (FIG. 3) was generated by a chain polymerization (PCR) reaction using primers 9 and 10 using the N-terminal and C-terminal EPO59 fragments prepared as templates.

Example 2 Preparation of Expression Vector pMI-EPO59

The EPO59 gene produced by the chain polymerization in Example 1 was cut with restriction enzymes NheI and NotI, electrophoresed on agarose gel, cut out, and then cut with CD5-IgGl (Aruffo et al, Cell, 61, 1303 (1990)) and the ligation of the (kb) fragment (ligation), E. coli MC1061 / P3 was transformed to obtain an E. coli transformant containing the desired vector. Expression vectors were isolated from the transformants and named pMI-EPO59.

Example 3 Formation of EPO60 Gene and Expression Vector pMI-EPO60

The vector pMI-EPO59 prepared in Example 2 was cut with XbaI, 0.7kb DNA fragments were isolated by agarose gel electrophoresis, and cut with Sau3A to obtain 0.5kb EPO DNA fragments without N-terminus. The primers 11 and 12 were used to react under the chain polymerization conditions of Example 1 to obtain a 0.1 kb DNA fragment, which was digested with Sau3A and XbaI to prepare fragment 2. pCDM8 vector (Invitrogen, USA) was cut with NheI and SacII, treated with Klenow enzyme to make blunt ends, and then conjugated to remove pDMBS (587-1179 nucleotide sequence) of pCDM8 vector. Is made). This was digested with Xbal to obtain a fragment of 3.8 kb, conjugated with fragment 1 and fragment 2 prepared above, and transformed with E. coli MC1061 / P3 to obtain an E. coli transformant containing the desired vector. From this, the vector pMI-EPO 60 Was separated. Escherichia coli MC1061 / P3 / PMI-EPO60 transformed with the expression vector pMI-EPO60 was deposited with KCTC0110BP dated May 17, 1994 to the Bank of Biotechnology.

Example 4 Transformation and Expression of EPO Gene in COS-7 Cells

Two 6 cm cell culture dishes containing 5 ml of DMEM (Gibco BRL) medium containing 10% FBS (fetal bovine serum) were grown to 50-80% COS cells and 2 µg of purified pMI-EPO59 and pMI-EPO60. Vectors were incubated with COS cells with 2 ml of infectious solution (10% Nuserum (Collaborative Biomedical Products, USA), 0.04% DEAE Dextran (Sigma), 0.05% Chloroquine (Sigma), 90% DMEM (Gibco BRL)) Each plate was incubated at 37 ° C. and 5% CO ₂ for 4.5 hours. At the end of the incubation, the infection solution was removed, treated with PBS (Gibco BRL, Catalog No: 14040-034) containing 2 ml of 10% DMSO for 2 minutes, and then in 5 ml of fresh DMEM (containing 10% fetal bovine serum). The cells were incubated for 12 hours at 37 ° C. and 5% CO ₂ . After incubation, cells were detached with trypsin for cell detachment (Gibro BRL, Catalog No .: 25300-013), and then in a new dish containing DMEM (containing 10% fetal bovine serum (Gibco BRL, Catalog No .: 16140-014)). 96 hours incubation at 37 ℃, 5% CO ₂ conditions. After incubation, the supernatant was used for expression analysis by centrifugation at 200 g for 5 minutes.

10 μl of the supernatant was subjected to SDS-PAGE on a 15% gel, followed by Western blotting according to the method of Ed Harlow and David Lane, Antibodees, A Laborative Manual 1988, Cold Spring Harbor Laboratory. Protein bands separated on gels after SDS-PAGE were transferred electrically to nitro cellulose paper. Nitro cellulose paper was treated for 1 hour in PBS solution containing 1% casein and treated for 2 hours in casein / PBS solution containing 500-fold dilution of monoclonal antibody (Genzyme cat.No.AE7A5, USA) against EPO. After treatment, the cells were washed with PBS and treated with alkaline phosphatase (alkaline phosphatase-conjugated goat affinity purified antibody to mouse IgG, Cappel, Cat.No 59296, USA; diluted 500-fold in casein / PBS solution) for 1 hour. Washed off. Nitro cellulose paper was developed with BCIP / NBT (5-brmo-4-chloro-3'-indolylphosphate-p-toluidine salt / nitro-blue tetrazolium chloride; PIERCE), an alkaline phosphatase color developing reagent. Color development revealed that the vectors pMI-EPO59 and pMI-EPO60 both expressed EPO in the range of approximately 32-39 kD on polyacrylamide gel (see FIG. 6). Compared to the EPO of the type suggests the same in terms of molecular weight of glycosylated protein.

Example 5 Measurement of Bioactivity of Expressed EPO

Bioactivity analysis was performed according to the method described in Gerald Krystal, Exp. Hematol., 7, 649-660 (1983). 20 g of C57 / BL6 mice (obtained from the Korea Research Institute of Chemical Technology) were injected intraperitoneally with phenyl hydrazine chloride (Sigma inoculation: 60 mg / kg) once a day for two consecutive days to obtain spleens. The spleen was struck with a syringe to obtain the cells, followed by one assay of 50 μl of activity assay mixture (2.5 X 105 cells in DMEM, 20% fetal bovine serum, 0.1 mM 2-mercaptoethanol, 1X Antibiotic-Antimycotic (Gibco). BRL) was prepared by diluting (200-20000 times) the expression supernatant with EPO to be analyzed with DMEM with 20% fetal bovine serum, and adding 50 µl to the previous assay mixture. Incubated for 20 hours at% CO ₂ condition After adding 20 μl of ³ H thymidine ([methyl- ³ H] -thymidine, Amersham) containing 0.5 μCi per assay to the culture medium, and incubated for 2.5 hours, the glass fiber filter After drying, the filter was placed in a vial, 3 ml hydrofluor (National Diagnostics, USA) was added, and the cpm value was measured on a liquid scintillation counter (Pharmacia). Products (Erythropoietin, human, recom binant, 250 U / ml, Cat.No:1120 166), and the bioactivity assay revealed that 37.0 U / ml for the pMI-EPO59 vector and 540.0 U / ml for the pMI-EPO60 vector. This result is higher than 3.1 U / ml, which is a conventional result (Temp. 93-5197: Jacobs et al., Nature 313, 28 (1985), etc.), which was temporarily expressed in COS cells. Excellent level.

Example 6 Expression Comparison with EPO Gene of Korean Patent Application No. 93-16865

The recombinant virus BVLUC-EPO (ATCC dated July 23, 1993) was used to compare the expression rate of the EPO gene (see FIG. 7) and EPO60 gene in animal cells of Korean patent application No. 93-16865. Deposition: Accession No. ATCC VR2421) DNA was obtained and cleaved with XbaI to have a cleavage site of XbaI at the 5 'and 3' ends, and then conjugated to the pCDM8 vector cleaved with XbaI. Thus made vector (pCDM-L-EPO) contains the cytomegalovirus promoter, EPO leader sequence, EPO gene in the same structure as pMI-EPO60 of the present invention. The difference between the two vectors in EPO expression is that they only differ in the nucleotide sequence of the EPO gene (the amino acid sequence is the same). These two vectors were infected with COS-7 cells as in Example 4 and their expression was fertilized by Western blotting analysis using monoclonal antibody against EPO. (See FIG. 8) After Western blotting, the filter was When comparing the expression levels of the two vectors using an image scanner (Digital Imaging Scanner, IS1000, Innotech Scientific System), pMI-EPO60 vector of the present invention showed 12.2 times higher expression level than pCDM-L-EPO. In the same manner as in Example 5, the expression level was confirmed by measuring the bioactivity by the ³ H thymidine method, and as a result, the EPO60 gene showed 38.6 times higher bioactivity than the EPO gene of the yeast preference codon. This result shows that EPO60 gene designed with human preference codon shows better expression rate than EPO user of Korean Patent Application No. 93-16865.

Claims (5)

Synthetic EPO gene encoding human erythrocyte growth promoting factor (EPO) consisting of a base sequence in which the following human preferred codons for each of the following amino acids range from 90 to 100% of the total codons: The synthetic EPO gene according to claim 1, having the following nucleotide sequence: The synthetic EPO gene according to claim 1, having the following nucleotide sequence: Expression vector pMI-EPO 60 for animal cells comprising the gene of claim 3. A method for producing human erythrocyte growth promoting factor comprising culturing animal cells transformed with the expression vector for animal cells of claim 4.