KR0161142B1 - Production method for recombinant granulocyte colony stimulating factor by yeast - Google Patents

Abstract

The present invention relates to a method for producing granulocyte-colony stimulating factor (hereinafter referred to as G-CSF) protein using yeast, wherein the nucleotide sequence of the amino terminus is substituted without changing the amino acid, and from the amino terminus 17 Yeast cell transformed with an expression vector for expression type yeast comprising a gene encoding granulocyte-colonal stimulator (G-CSF), characterized in that the first amino acid cysteine is substituted with serine and is expressed in yeast By culturing the granulocyte-colon stimulating factor can be efficiently produced by the present invention comprising recovering the native G-CSF.

Description

Method for preparing granulocyte-colon stimulating factor (G-CSF) using yeast

Figure 1 shows the gene sequence and amino acid sequence of G-CSF derived from human U-937 cells, the nucleotide sequence of part of the amino terminal is substituted, and the cysteine, the 17th amino acid, is substituted with serine,

2 shows the substitution of serine for cysteine, the 17th amino acid, from the amino terminus of the G-CSF gene derived from human U-937 cells with serine and the cloning of the yeast expression vector pYLBC A / G αfG-CSF Cys17 → Ser. Shown,

Figure 3 shows the result of denatured polyacrylamide gel electrophoresis after culturing yeast transformed with the expression vector pYLBC A / G αfG-CSF Cys17 → Ser of the present invention,

Figure 4 is a Western blot using an antibody against G-CSF after culturing yeast cells transformed with the expression vector pYLBC A / G αfG-CSF Cys17 → Ser of the present invention and denatured polyacrylamide gel electrophoresis blotting) shows the results,

5 shows standard growth curves and activity calculations indicating the extent of cell growth upon treatment with the G-CSF international standard and G-CSF Cys17 → Ser protein of the present invention.

The present invention relates to a method for producing granulocyte-colony stimulating factor (hereinafter referred to as G-CSF) protein using yeast, and more specifically, to the 17th amino acid from the amino terminus of native G-CSF. The present invention relates to a method for preparing G-CSF having enhanced stability by modifying phosphorus cysteine to serine.

G-CSF is known to be involved in the growth and differentiation of neutrophil granulocytes among colony stimulatin factors that promote differentiation and proliferation of bone marrow cells (Nicola, et al., J. Biol. Chem. 252, 9017 ( 1983), G-CSF derived from human bladder cancer cells was purified (Welte, et al., Proc. Natl. Acad. Sci., 82, 1526 (1985)), and its cDNA sequence was also readily available (Shigekazu et al. et al., Nature, 319, 415 (1986)) and Sousa et al. (Souza et al., Science, 232, 61 (1986)).

The mature protein of G-CSF is a glycoprotein with a molecular weight of 19,600 Daltons, which acts in combination with neutrophil granulocytes, thereby increasing the number of neutrophil granulocytes in a short period of time, and thus can be applied to various therapeutic fields. For example, it can be used for the repair of destroyed immune system cells after chemotherapy chemotherapy and for the treatment of bone marrow transplantation, severe burns and leukemia.

Current methods for producing G-CSF include US Amgen's method (US Pat. No. 4,810,643) and Chinese Hamster Obari (CHO) animal cells using E. coli to make G-CSF protein with methionine at the amino terminus. Japan's method of making a natural glycosylated G-CSF protein is known using the method (J Korean Patent Publication Nos. 92-5752 and 91-5624). However, Amgen's method is capable of forming antibodies in vivo by expressing them in E. coli cells that express only 3 to 5% of the total protein and do not have the native protein, and that are not capable of glycosylation. In addition to this, there is a problem in that a purification step for removing pyrogenic substances generated using E. coli must be added separately. Moreover, although the natural protein can be obtained by the method of Jugai company using an animal cell, there exists a problem that it cannot compare with the method using E. coli, yeast, etc. in the cost. Therefore, G-CSF is expressed in a secretory form in microbial cells suitable for mass production, but it is necessary to avoid using E. coli.

The native G-CSF has a problem in that the cysteine residue, which is the 17th amino acid from the amino terminus in the protein primary structure, produces a reduced form of unstable protein and, as oxidized, forms protein duplexes, thereby reducing biological activity. According to Masaharu Ishikawa et al., When the cysteine, the 17th amino acid of G-CSF, is substituted with serine or another amino acid, the produced G-CSF not only has the same level of biochemical activity as that of the natural G-CSF, but also has a higher biochemical activity. It is known to have stability (Masaharu Ishikawa et al., Cell Structure and Function, 17, 61 (1992)).

The present inventors have studied a method for producing unglycosylated native G-CSF using yeast free of pyrogenic substances. As a result, the native G-CSF is substituted by replacing part of the amino terminal sequence of the G-CSF gene without changing the amino acid. It was found that can be obtained at high expression rate, and furthermore, the natural type with much higher stability when substituted with serine for cysteine, which can increase the purification efficiency of expressed natural G-CSF and affect the protein's pleating. The discovery that G-CSF proteins can be expressed has led to the completion of the present invention.

It is an object of the present invention to provide an improved method for producing native G-CSF protein which increases expression rate and stability using yeast cells.

Another object of the present invention is to provide a modified recombinant G-CSF gene used for producing a native G-CSF protein from yeast cells, an expression vector comprising the same, and a transformant transformed with the expression vector.

In accordance with the above object, in the present invention, the granulocyte-collecting stimulating factor, which is substituted without changing the nucleotide sequence of the amino terminus, and the cysteine, which is the 17th amino acid from the amino terminus, is substituted with serine and expressed in the yeast secreted form. Provided are a gene encoding (G-CSF), a G-CSF expression vector for secretory yeast comprising the gene at the 3′-end of an alpha factor, and a yeast cell line transformed with the expression vector.

In another aspect, the present invention provides a method for producing a G-CSF protein comprising culturing the yeast cells to recover the natural G-CSF.

When cloned into the yeast expression vector of the native G-CSF gene is transformed into yeast and expressed, the expression level of the G-CSF protein is very low, less than 5% of the total protein.

In the present invention, by replacing some of the nucleotide sequence of the cDNA amino terminal of the G-CSF with the amino acid sequence as follows, the expression rate of the G-CSF protein is greatly increased, and the cysteine, which is the 17th amino acid from the amino terminal, is replaced with serine. This greatly increased the biological stability of the G-CSF protein:

In addition, according to the present invention by linking the G-CSF gene modified as described above to the alpha factor 3'-end of the yeast to produce an expression vector for yeast, the alpha factor is cleaved while secreted out of the yeast cells after protein expression, natural G- Can be produced as CSF.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1

Isolation of G-CSF Gene from Human U-937 Cells]

RNA was extracted from human U-937 cells (ATCC CRL 1593) according to the RNAzolB method (Cat # CS-105, Biotecx Laboratories, INC). After adding 1 ml of RNAzolB solution to 5 × 10 ⁶ cells and disrupting the cell membrane using a 10 ml pipette, 0.1 ml of chloroform was added and mixed well for 15 seconds and left at 4 ° C. for 15 minutes, followed by 15 at 4 ° C. Centrifuged at 12000G for minutes. The aqueous solution of the upper layer was transferred to a new tube, the same volume of isopropanol was added and left at 4 ° C. for 15 minutes, followed by centrifugation at 12000 G for 15 minutes at 4 ° C. to obtain RNA precipitate. The precipitate was mixed well with 75% ethanol and then centrifuged at 7500G for 8 minutes at 4 ° C to obtain a clean RNA precipitate. It was dissolved in water treated with DEPC (diethylpyrocarbonate).

To synthesize cDNA single strands, 13.5 μl of the RNA solution obtained above was taken and heated at 65 ° C. for 10 minutes to release the secondary structure of RNA, followed by RNase inhibitors (Rnasin, IU / μl, Promega, USA) 1.0 ㎕ and 5.0 OD 260 oligo (dT) _12-18 primer _{(oligo (dT) 12-18 primer,} GibcoBRL, USA) was added and allowed to stand at 70 ℃ 2㎕ for 10 minutes, followed by 10-fold concentrated M-MULV reverse transcriptase reaction 2.0 μl of solution, 1 μl of 10 mM dNTP mixture (10 mM each of dGTP, dATP, dTTP and dCTP) and 1 μl of M-MULV reverse transcriptase (New England Biolabs) (IU / μl) were added and reacted at 37 ° C. for 1 hour to strand cDNA. Was synthesized.

Two oligonucleotide primers were synthesized using a nucleic acid synthesizer (DNA Synthesizer, Applied Biosystems INC. USA) to obtain G-CSF gene from cDNA using polymerase chain reaction. The synthesized XbaGCSF primer (38-mer: 5'-GTACTCTAGATAAGAGAACTCCTTTAGGTCCTGCTAG-3 ') comprises an Xbal recognition site and is part of the α factor precursor sequence up to the 18th nucleic acid and from the 19th nucleic acid to the G in the base sequence of the G-CSF gene. The base sequence of a part of the amino terminus of the G-CSF gene having a relatively high ratio of / C bases was replaced with no amino acid changes as follows:

In addition, the GCSFSal primer (37-mer: 5'-ATCATGTCGACTATTAGGGCTGGGCAAGGTGGCCTAG-3 ') contains a SalI recognition site (underlined) and two termination codons and is complementary to the carboxy terminal base sequence of the G-CSF gene from the 17th nucleic acid. It is designed to have.

Polymerase chain reaction was carried out using the cDNA strand as a template. 2 μl of the synthesized cDNA template, 10 μl of the 10-fold concentrated reaction buffer solution, 6 μl of 25 mM MgCl ₂ , ₂ μl of 10 mM dNTP, 0.75 μg XbaGCSF primer , 75 μl of distilled water was added to 3 μl of 0.75 μg GCSFSal primer and 0.4 μl of Taq polymerase (5 U / μl, Promega, USA) to make a reaction solution. 1 minute 30 seconds at 94 ° C; 1 minute at 50 ° C .; The reaction was repeated 40 times at 72 DEG C for 1 minute 30 seconds to amplify the 553 base pair G-CSF gene. DNA was extracted using conventional methods using phenol / chloroform and 100% ethanol and dissolved in 20 μl of TE buffer solution (10 mM Tris-HCl, pH 7.5, 1 mM EDTA).

Take 16 µl of the DNA solution obtained above, add 1 µl of restriction enzymes XbaI and SalI, and 2 µl of 10-fold restriction enzyme reaction solution for 1 hour at 37 ° C, and then use phenol / chloroform and 100% ethanol. In this manner, G-CSF DNA including XbaI and SalI recognition sites was obtained. To the M13mp18 vector digested with the restriction enzymes XbaI and SalI, the G-CSF DNA fragment, 1 μl of T4 DNA ligase, and 1 μl of 10-fold reaction solution were added and conjugated to react overnight at 16 ° C. (M13mp18-G-CSF). The conjugated reaction solution was added to JM105 (ATCC 47016) competent cells, transformed with the vector according to one method (J. Mol. Biol. 116, 557 (1983)), and plated in LB medium. E. coli transformants were selected.

According to Sanger et al. (Sanger, F. et al., Proc. Natl. Acad. Sci. USA 74, 5463 (1977)), the entire nucleic acid sequence of the G-CSF gene was identified to determine the base sequence of the known G-CSF. (Souza LM et al., Science, 232, 61 (1986)) was confirmed.

Example 2

Method for substituting cysteine, the amino acid at position 17, with serine and for producing the yeast expression vector pYLBC A / G αfG-CSF Cys17 → Ser

A strand of DNA of the M13mp18-G-CSF gene was purified according to Maniatis's method (Maniatis et al., Molecular cloning), adjusted to a concentration of 1 μg / μl, and used as a template for mutation.

Oligomer 5'-CTTCCTCACTTGCTCTAAGGACTTGAGCAGGAA-3 'was synthesized with a DNA synthesizer to replace cysteine, the seventeenth amino acid, from the G-CSF amino terminus with serine, and a mutation was performed according to the Sculptor method of Amersham, USA. It was confirmed that the substitution with the desired base sequence. FIG. 1 shows the gene base sequence and amino acid sequence of G-CSF derived from human U-937 cells with the nucleotide sequence of a part of the amino terminal substituted and the cysteine 17th amino acid substituted with serine.

To prepare a yeast expression vector of the modified G-CSF gene as described above, 2 μg of the yeast expression vector pYLBC A / G αf IGF-I in Korean Patent No. 60252 (expression vector of human insulin-like growth factor) was prepared. Fully digested with restriction enzymes PstI and XbaI, and 2 µg of the same plasmid were completely digested with restriction enzymes PstI and SalI, and then subjected to 1% agarose gel electrophoresis to obtain nucleic acid fragments of 4.5 kb and 9.8 kb, respectively. Hereinafter, these fragments are called fragments PX and PL, respectively. On the other hand, M13mp18-G-CSF Cys17 → Ser, whose nucleotide sequence was identified above, was digested with restriction enzymes XbaI and SalI and subjected to 1.5% agarose gel electrophoresis to separate G-CSF Cys17 → Ser gene nucleic acid fragments. G-CSF Cys17-> Ser nucleic acid fragments, fragment PX, and fragment PL were linked-reacted as follows: 0.1 μg fragment PX, 0.1 μg fragment PL, 0.1 μg G-CSF Cys17 → Ser gene fragment, 2 μl of 10 Pear concentration coupling reaction solution (500mM Tris-HCl, pH 7.8, 100mM DTT, 10mM ATP), 10 units of T4 DNA ligase was added and distilled water was added to a total volume of 20μL and reacted for 12 hours at 16 ℃. The reaction solution was added to E. coli HB101 (ATCC 33694) competent cells and transformed, and then, E. coli transformants were selected by plating on LB-Epicillin (LB medium containing 50 µg / ml Empicillin) plate.

2 is a process for substituting seri for the 17th amino acid cysteine from the amino terminus of a G-CSF gene derived from human U-937 cells and cloning it into the yeast expression vector pYLBC A / G αfG-CSF Cys17 → Ser. It is shown.

Example 3

Expression and Western Blotting of Secretory G-CSF Cys17 → Ser Protein]

Saccharomyces according to Hinnen et al. (Hinnen, et al., Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)) by mass extraction of the recombinant plasmid pYLBC A / G αfG-CSF Cys17 → Ser. Transformed into Saccharomyces Cerevesiae and applied to a plate lacking leucine (Leucine) and incubated for 5 days at 30 ℃ to obtain a transformant containing pYLBC A / G αfG-CSF Cys17 → Ser .

The transformed Saccharomyces cerevisiae pYLBC A / G αfG-CSF Cys17 → Ser was deposited on October 31, 1995 as a deposit No. KCTC 0208BP to the Gene Bank of Korea Institute of Science and Technology.

The transformed yeast strain was cultured without 3 ml of leucine (yeast nitrogen base without amino acids (Difco, USA) 6.7 g per 1 liter of culture, 0.25 g and 5% leucine deficient amino acid mixture) Glucose) was incubated for 24 hours at 30 ℃, and added to 50 ml of YEP medium (2% peptone, 1% yeast extract) containing 2% glucose and incubated for 48 hours. The culture was centrifuged at 120,000 G for 5 minutes to obtain 200 µl of the supernatant containing no yeast cells and mixed well with 200 µl of Ramley buffer solution (Laemli, et al., Nature 227, 680 (1970)) and 15%. After SDS-polyacrylamide gel electrophoresis, the protein was stained with Coomassie Brilliant blue R250.

Figure 3 shows the result of denatured polyacrylamide gel electrophoresis after culturing yeast transformed with the expression vector pYLBC A / G αfG-CSF Cys17 → Ser. In FIG. 3, columns 1, 3, and 4 are cultures in which yeast cells transformed with the expression vector pYLBC A / G αfG-CSF Cys17 → Ser of the present invention are cultured and centrifuged to remove the cells. Indicates pre-stained standard protein molecular weight (112000, 84000, 53200, 34900, 28700, 10500 Daltons; Bio-Rad) and column 5 indicates standard protein molecular weight (97400, 66200, 45000, 31000 , 21500, 14400 Daltons; Bio-Rad). As shown here, it was confirmed that human G-CSF Cys17-> Ser protein was expressed at a size of about 19,000 Daltons.

In addition, Western blotting was performed to confirm whether the secreted G-CSF Cys17 → Ser protein is a human granulocyte-colon stimulating factor protein (Manniatis, et al., Molecular Cloning). The G-CSF Cys17-Ser protein obtained above was subjected to 15% SDS-polyacrylamide gel electrophoresis using G-CSF antibody (Cat # BL-GCP) purchased from Genzyme (USA), followed by transfer solution ( 1x SDS Running solution, 20% methanol) was transferred to a Nitro cellulose membrane. To remove nonspecific binding, 50 ml of 1 × TBS solution (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5% bovine serum albumin) was added and left to shake for 1 hour at room temperature. Discard the solution, add the antibody against G-CSF to 50 ml of 1X TBS, leave for 1 hour with shaking at room temperature, discard the solution again and wash the mixture with 1X TBS and 0.1% Triton X-100 for 4 minutes for 10 minutes. Antibodies to -CSF were removed. Subsequently, goat-anti-rabbit-IgG, a second antibody against G-CSF antibody, was added to 50 ml of 1X TBS (5% BSA) and 0.1% Triton X-100 solution, and the mixture was left to shake at room temperature for 1 hour. The developing solution was used after switching to 1X TBS and 0.1% Triton X-100 solution four times for 10 minutes.

4 shows Western blotting results showing that the G-CSF Cys17 → Ser protein reacts with G-CSF antibody of Genzyme. Columns 1, 2, and 4 are the results for the culture medium in which the cells were removed by centrifugation after culturing yeast cells transformed with the expression vector pYLBC A / G αfG-CSF Cys17 → Ser of the present invention, and column 3 was preliminary. It represents the standard protein molecular weight stained (Bio-Rad). Here, it can be seen that the protein produced by the present invention is G-CSF.

Example 4

Activity Measurement of Secreted Cys17 → Ser Protein by In Vitro Analysis]

The titer of G-CSF can be measured to the extent that Granulocyte Progenitor Cells (NFS-60, hereinafter referred to as GPC) are grown by G-CSF in an in vitro bioassay, and after treatment with G-CSF in GPC Modification of titer (Proc. Natl. Acad. Sci. USA, 83, 7633 (1986)) by quantifying and measuring the growth of cells by adding ³ H, -thymidine (radioactive labeling substance, Amersham) will be.

Continuously dilute WHO NIBSC International Standard from 12.5 IU / mL in RPMI medium (GibcoBRL # 23400-062) containing 5% fetal calf serum (GibcoBRL # 16000-044) in a 96-well container, or add 50 mL to AD column. In addition, 50 μl of G-CSF protein purified by the same method was serially diluted twice and added to each corresponding hole of the EH column. M-NFS-60 (CRL-1838) purchased from ATCC was used as a cell for bioanalysis. Cell suspensions centrifuged at least three times with G-CSF-free medium were 5 × 10 ⁵ cells per ml. 50 ml of each was put in the empty container with the sample solution as much as possible, and then incubated for 24 hours in a 5% CO ₂ incubator. ³ H-thymidine was added to 0.25 μCi per well and incubated for 4 hours at 37 ° C. in a 5% CO ₂ incubator, and the contents of each ball were harvested on a glass fiber filter using a micro harvester. . The fibrous membrane from which cells were harvested was placed in a scintillation bottle, 5 ml of scintillation solution (Packcard # 6013329), and radioactivity was measured by a liquid scintillation counter (Packcard Tri-Carb 2500 TR). Experimental results showed that when treated with purified G-CSF Cys17 → Ser, cell growth similar to that when treated with WHO NiBSC International Standard was detected, and the CPM value of ³ H-thymidine was absorbed by cells. Estimating the titer showed 1.72 × 10 ⁷ IU / mg / ml activity.

5 (a) and (b) show standard growth curves indicating the degree of cell growth upon treatment with G-CSF international standard and G-CSF Cys17 → Ser protein of the present invention, and (c) shows G-CSF protein. It represents the activity calculation of

1 is the cpm value after 2-fold diluted G-CSF treatment;

2 is a sample cpm value converted into a Log ₂ index by substituting an international standard STD curve;

Log ₂ A = {sample cpm-STD Y intercept cpm} / STD slope;

3 is the dilution multiple (factor: D. F);

4 is the arithmetic conversion of the Log ₂ value in column ₂ , then multiplied by the dilution factor (BU / mL-2Log ₂ A * D. F / 1000).

As described above, the secreted G-CSF Cys17 → Ser protein expressed by culturing yeast cells transformed with the yeast expression vector pYLBC A / G αfG-CSF Cys17 → Ser of the present invention was treated with the international standard G-CSF. It is expected to be used in the treatment of various diseases because it is not only high activity equivalent to that given, but also suitable for mass production to be useful for commercialization.

Claims (7)

The nucleotide sequence of the amino terminus is substituted without changing the amino acid, and the cysteine, which is the 17th amino acid from the amino terminus, is substituted with serine, and is expressed in the yeast secretion form. Gene. The gene according to claim 1, wherein the nucleotide sequence of the amino terminal is substituted as follows: A G-CSF expression vector for secretory yeast comprising the gene of claim 1 at the 3'-terminus of an alpha factor. The method of claim 3, wherein pYLBC A / G αfG-CSF Cys 17 → Ser. A yeast cell line transformed with the expression vector of claim 3. Saccharomyces cerevisiae pYLBC A / G αfG-CSF Cys17 → Ser (KCTC 0208BP). A method for producing a G-CSF protein comprising culturing the yeast cells of claim 5 to recover native G-CSF.