KR0180081B1 - Preparation process of gcsf from yeast - Google Patents

Abstract

The present invention relates to a method for producing granulocyte colony stimulating factor (hereinafter, abbreviated as GCSF) in yeast using a DNA synthesis technique.

More specifically, the present invention uses a yeast expression vector having a secreted signal consisting of a complex promoter made of two yeast-derived promoters, a killing toxin of yeast and an amino terminus of interleukin 1β (IL-1β). It relates to a method for producing granulocyte colony stimulating factor.

The present invention also relates to a method for producing granulocyte colony stimulating factor in yeast by using an expression vector having a promoter and secretion signal sequence of a heat shock protein 150 of yeast.

Description

How to prepare GCSF in yeast

1 shows the manufacturing process of YEp2-k.

Figure 2 shows the manufacturing process of YEp2KIL20GC.

Figure 3 shows the manufacturing process of pIL20GC.

Figure 4 shows the manufacturing process of YEpHSPGC.

Figure 5 shows the amino acid sequence of the killing toxin leader sequence-IL 1β 24AA-GCSF and the site cleaved by the leader sequence peptidase and KEX2 peptidase.

Figure 6 shows the expression of GCS in yeast and analyzed by SDS-PAGE.

7 is a western blot after expressing GCSF using the yeast-expressed vector YEpHSPGC.

8 shows the purification of crude GCSF from yeast culture.

9 shows the purification of GCS by Sephacryl S-200 column chromatography.

10 shows the final purification of G-CSF.

The present invention relates to a method for producing granulocyte clony stimulating factor (hereinafter abbreviated as GCSF) in yeast using DNA recombination technology.

More specifically, the present invention uses a yeast expression vector having a secreted signal consisting of a complex promoter made of two yeast-derived promoters, a killing toxin of yeast and an amino terminus of interleukin 1β (IL-1β). It relates to a method for producing granulocyte colony stimulating factor.

The present invention also relates to a method for producing granulocyte colony stimulating factor in yeast by using an expression vector having a promoter and secretion signal sequence of a heat shock protein 150 of yeast.

Bone marrow differentiation and proliferation experiments have shown that colonies of neutrophil granulocytes or monocytic macrophages are formed in the human body, and that there is a factor that stimulates colonization in live animals [J. Cell. Comp. Physiol 66: 319 (1965); Aust. J. Exp. Biol. Med.Sci. 44: 287) (1996).

These factors, collectively referred to as Colony Stimulating Factor (hereinafter referred to as CSF), are classified as follows according to specificity.

(i) GM-CSF (granulocyte and monocytic macrophage stimulator) proliferates and differentiates granulocytes or monocytic macrophages to form colonies, and (ii) M-CSF (monocytic macrophages). Stimulating factors) mainly form colonies of monocytic macrophages, (iii) multi-CSF (pluripotent cell stimulating factor) acts on hepatocytes of undifferentiated pluripotent cells to form colonies of pluripotent cells, and (iv G-CSF (granulocyte leukocyte stimulator) forms granular leukocyte colony [JBC 252: 1998-2033 (1977). J.B.C. 252: 4045-4052 (1977), Biochem. J. 185: 341-343 (1980), J.B.C 258: 9017-9021 (1983).

GSCF is a glycoprotein of about 20 KDa and is produced in cells such as monocytes, monocytic macrophages, epithelial cells and fibroblasts. The human GCSF gene is present on chromosome 17.

Indeed, GCSF stimulates neutrophil colonization in vitro, works with IL-3 to stimulate colonization of blasts, eukaryotic and macrophages, and some leukemia myeloma lines are matured by GCSF. It is known to become. It also increases the number of neutrophils in vivo and slightly increases the number of monocytes.

GCSF has clinical uses as follows.

First, the number of neutrophils is dose-dependently increased in neutropenia in patients with solid tumors and hematologic malignancies.

Second, neutropenia is rapidly recovered by chemotherapy for malignant, lymphoma, lung cancer, ovarian cancer, testicular tumor, urethral epithelial cancer, and acute leukemia.

Third, the number of neutrophils is increased during bone marrow transplantation of acute non-lymphatic leukemia and chronic myeloid leukemia.

Fourthly, neutropenia caused by bone marrow dysplasia syndrome is rapidly recovered.

Fifth, the neutropenia associated with aplastic anemia is restored.

Sixth, it is useful for congenital and idiopathic neutropenia.

Seventh, antitumor chemotherapy prevents or reduces the occurrence of mucositis and febrile neutropenia (Drug Evaluations Annual 1993, American Medical Associations p2232-2333).

Various genetic engineering methods have been attempted to obtain such a large amount of GCSF. First, a method of cloning the GCSF gene and expressing it in E. coli was attempted (Science 232: 61-64 (1986)).

However, when E. coli is used as a host cell, there are the following problems.

Proteins such as GCSF are first made into precursors in the human body, and these precursors are matured by proteases to GCSF having the N-terminal amino acid sequence of Thr-Pro-Leu. However, when expressed in Escherichia coli, a methionine corresponding to the start codon is present at the N-terminus according to the amino peptidase enzyme titer. The mixture is very difficult to remove during the purification process, and when expressed as an inactive insoluble protein, the process must be made into a soluble form that has an active activity.

In addition, E. coli should pay attention to the contamination of endotoxin during purification.

In addition, glycoproteins such as GCSF do not have glycosylation because E. coli has no post-translational modification.

The GCSF gene was then cloned and expressed in animal cells such as CHU-2 (human GCSF producing cancer cell line; hunam GCSF-producing tumor cell line) or Chinese hamster ovary cell [EMBO J. 5: 871-. 876 (1980)] (Korean Patent, Publication No. 91-5624).

However, when animal cells are used as host cells, expensive serums must be used for cell culture, so that the culture conditions are difficult, and the yield is too small because a small amount of protein must be purified in a large amount of culture medium.

In order to solve the problems of the existing techniques as described above, a method of expressing GCS using a yeast host cell has been attempted.

How to obtain desired polytide or protein by mass culture using yeast as a host is well known by Loison et al. [Bio / Technol. 4: 433-437 (1986); Burrow, Baker's yeast, p 349-420, in The yeast vol. 3 Rose and Harrison, eds. Academic Oress, London (1970)), the use of yeast as a host has a number of advantages that can not be obtained when using animal cells or E. coli.

In this regard, the present inventors studied a method for preparing GCSF using yeast.

Yeast has been recognized for safety in humans by the US FDA, and most of the principles of gene expression regulation have been revealed (Strathern et al., The Molecular Biology of the Yeast Saccharomyces, Metabolism and Gene Expression, Cold Spring Harbor Laboratory, N.Y. (1982)).

When yeast is used as a host cell, it is safe for human body, it can be mass-produced because it can be cultured in high concentration, and its purification method is easy because it can secrete the soluble form protein out of the cell by using a signal peptide. Do.

Recently, a method for expressing foreign proteins such as hepatitis B virus interfeton, calf chymosin, and epidermal growth factor in yeast has been reported [Valensuela et al., Nature 298: 347-350. (1982); Hitzeman et al. NAR 11: 2745-2763 (1983); McAleer et al., Nature 307: 178-180 (1984); Tuite et al., EMBO, J. 1: 603-608 (1982); Mellor et al. Gene 24: 1-14 (1983); Urdea et al., PNAS 80: 7561-7465 (1983).

However, there is a problem that the expression of heterologous proteins in yeast produces very small amounts compared to the production of normal homologous gene expressions, and many researchers are trying to increase the expression of heterologous proteins in yeast by developing various expression vectors. Chen et al. NAR 12: 8951-8970 (1984).

For example, a method for producing a galactokinase-bovine prochymosin fusion protein in yeast is disclosed using a cloning vector having an external gene and a yeast GAL1 promoter. It is.

In addition, when a yeast gene GAL4 is inserted into a vector including an external gene and a GAL1 promoter, a method of increasing the expression of GAL4 by controlling the level of transcription by galactose is known [Laughon et al., PNAS is known. 79: 6827-6831 (1982).

European Patent 84302723 discloses human interferonγ, human serum albumin, bovine interferonα-1, α-2, tissue plasminogen activity using a promoter and secretion signal of the α-mating factor of yeast. Tissue plasminogen activator, rennin, and human insulin-like growth factor are described in yeast.

The inventors of the present invention have studied the following production method, focusing on the fact that in order to prepare GCSF in yeast using DNA recombination technology, not only a large amount of GCSF should be expressed but also the expressed GCSF should be secreted out of cells.

First, in order to secrete the mature GCSF, various secretion signals (signal peptides) were conjugated to the amino terminal region of the GCSF to try to secrete the cells. However, in this case, even though the secretion signal is present, it was not easy to secrete extracellularly.

On the other hand, interleukin IL-1β has been reported to be secreted out of the cell very efficiently by secretion signals in yeast (EMBO J. 6: 229-234 (1987)). The present inventors have found that the amino acid sequence of the killing toxin secretion signal and about 20 IL-1β in front of the GCSF gene is focused on the fact that it will help to secrete GCSF expressed in the amino acid amino terminal of IL-1β in addition to the secretion signal. By arranging the expression of GCSF and the secretion of cells as a result of successful expression and secretion was found to complete the present invention.

Since 20 amino acids of IL-1β are present in front of the GCSF protein, the cleavage site of KEX2, a dibasic endopeptidase of yeast, is induced to induce cleavage of 20 amino acids of IL-1β during secretion. Placed between IL-1β and mature GCSF gene.

The vector thus obtained has an arrangement of about 20 amino acids-KEX2 cleavage site-mature GCSF of toxic toxin secretion signal-IL 1β. The protein expressed by such expression vector is first secreted by the signal peptidase during secretion and extracellular secretion after protein synthesis, and then the IL-1β site is cut by the KEX2 peptidase. Mature GCSF with amino termini is finally secreted.

The GCSF expression vector used in the present invention will be described in more detail as follows.

GCSF expression vector is yeast expression vector YEpsec1-hI1 [C. Baldari et al., EMBO J. 6: 229-234 (1987)] substituted MFα-factor (Mating factor α1) promoters in place of the CYC-1 promoter, and the leading sequence of toxic toxins optimized by codon use (leader sequence) and a complex secretion signal consisting of 24 amino termini of IL-1β, GCSF gene, and GAL gene activator (GAL4). Saccharomyces cerevisiae host cells were transformed with this vector, and the correct transformants were selected from minimal medium without uracil and used as GCSF producing strains.

In addition, the present invention also attempted a vector that can control the expression of the desired GCSF protein by using only a heat shock protein (culture temperature difference). Unlike other known inducible yeast expression vectors such as the GAL promoter induced by galactose, the Pho5 promoter induced by phosphorus depletion, and the ADH II promoter induced by glucose depletion, thermal shock proteins are thermoregulated. (37-42 ℃) can easily control the expression of mRNA and protein, and is secreted out of the cell by the leader (leader sequence) (PNAS, 89: 3671-3675). Therefore, the present inventors invented a method of preparing GCSF using an expression vector including a promoter of the heat shock protein HSP 150 and a leader sequence of the heat shock protein.

Through the following examples will be described in more detail the contents of the present invention.

The examples are merely illustrative of the present invention and do not limit the content of the present invention.

1. YEp2-k manufacture

Originally, yeast expression vector YEpsec1-hI1 is the upstream activation sequence of Gal 1, 10 gene and the CYC-1 promoter, Kluyveromyces lactis. J. R. Stark et al., NAR 12: 6011-6031, (1984)], and the interleukin 1β) gene, and IL-1β is expressed by the inducer galactose.

To make YEpsec1-hI1 a more efficient vector, the toxic toxin sequence was optimized with codons of proteins expressed in yeast and the CYC-1 promoter was replaced with a more efficient MFα1 promoter.

In addition, the transcriptional terminator of GAPDH was put on the 3 'side of GCSF to clone the RNA transcription, and the Gal4 gene, the activator of the Gal gene, was cloned and included behind the GCSF.

One). Optimization of killer toxin leader codons

Preparation of (YEpsec-ok)

Example 1

Synthesis of Killer Toxin Lead Sequence Oligopeptides To replace the leader sequence codons of the killer toxin of yeast expression vector YEpsec1-hI1 with codons of proteins expressed in large quantities in Saccharomyces cerevisiae Oligo peptides were synthesized by a synthesizer (ABI, 392 DNA / RNA synthesizer) (J. Bennetzen, B. Hall. J. Biol. Chem. 257: 3026-3031).

In order to insert the synthesized oligo block into the site where the killer toxin leader sequence of YEpsec1-hI1 was cut out, it was processed as follows.

Each oligonucleotide reaction solution comprising ATP in order to phosphorylate peptide 5 '[70mM Tris-HCl ( pH 7.6), 10mM MgCl 2, 5mM DTT (dithiothreitol)] 30㎕ from T ₄ polynucleotide kinase (T ₄ polynucleotide kinase; NEB ) Was reacted at 37 ° C. for 1 hour.

The two reaction mixtures were mixed and left at 65 ° C. for 20 minutes, followed by annealing while slowly cooling to 30 ° C.

Example 2 YEpsecl-hI1 cleavage

1 μg of YEpsecl-hl was reacted with 40 μl of the reaction solution [20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT (pH7.9)] at 37 ° C. for 1 hour at 37 ° C. After electrophoresis separation with 1% plate agarose gel (electrophoresis separation), 8.4kb of bands were cut out and DNA was eluted from the bands cut using Jetsorb (GENOMED, cat # 110300).

Example 3 DNA-Linked Light Transformation

Oligoblocks of the killer toxic toxin sequence annealed in Example 1 and YEpsecl-hI1 digested with restriction enzymes SacI, KpnI in Example 2 were 50 mM Tris-hcl (pH7.6), 10 mM MgCl ₂ , 10 mM DTT, and 1 mM ATP. It was dissolved in 30 µl of the reaction solution consisting of, 100 units of T ₄ DNA ligase was added and reacted overnight at 16 ° C.

The reaction solution was prepared using E. coli XL-1 Blue (supE44 hsdR17 recA1 endA1 gyrA46 thi relA1 Iac ^- F '[proAB ⁺ lacI ^q lac ^Z ) by CaCl ₂ method according to Molecular Cloning A Laboratory Manual (Sambrook, Fritch, Maniatis. 2nd edition.CSH ^). M15 Tn10 (tet ^r )]), and then transformed into LB-Amp agar solid medium (10 g / l tryptone, 5 g / l yeast extract, 10 g / l NaCl, 1.5% Agar, 100 μl / ml) ampicillin). The plate was incubated at 37 ° C. for 50 hours at 50 μl / ml. Colonies of ampicillin resistant transformants (Amp ^R ) were incubated in 1.5 ml of liquid LB-Amp medium and dissolved by alkaline elution, and the plasmids were purified using an RPM rotation filter (BIO 101). Since codon optimization eliminated the restriction enzyme SmaI site present on the DNA of the toxic toxin sequence, a plasmid that was not cleaved by restriction element SmaI was selected and named YEpsec-ok.

Example 4 Short-Chain DNA

Sequencing was performed to identify the nucleotide sequence of the killer toxin codon substituted for codon optimization. In order to make a single-strand plasmid required for sequencing was carried out as follows.

The YEpsecl-ok plasmid obtained in Example 3 was further digested with restriction enzymes BamHI, SacI, isolated from 1.5% agarose gel, and the DNA fragment of 0.66 kb was cut out. DNA was eluted from the gel using GENE CLEAN KIT II.

1 μg of the vector M13mp19 was digested with restriction enzymes BamHI, SacI and purified using GENE CLEAN KIT II. In connection with the DNA of M13mp19 in the 0.66kb (ligation) reaction solution was added to T ₄ DNA ligase was reacted overnight at 16 ℃. After the reaction solution was transformed into transformant E. coli XL-1Blue, single-chain DNA was isolated according to the Molecular Cloning A Laboratory Manual (ibid). More specifically, ligation agar, 200 µl XL-1 Blue culture overnight, 40 µl X-Gal (20 mg / ml in dimethylformamide), 4 µl IPTG (200 mg / ml), LB agar (0.7 ml agar 3 ml) ) And spread on LB solid agar medium. After overnight incubation at 37 ° C., one white plaque formed on agar medium was inoculated into 200 ml of LB with 200 μl of XL-1Blue, shaken at 37 ° C. and 250 rpm for 5 hours, followed by centrifugation. Into the supernatant, add 1/5 volume of PEG (20% PEG8000 in 2.5 M NaCl) and leave on ice for 15 minutes. After centrifugation again, the supernatant was discarded and the M13 virus pellet was suspended in 200 [mu] l TE buffer (10 mM Tris-HCl (pH 7.6), 1 mM EDTA), followed by phenol / chloroform / isoamyl alcohol (25: 24: 1 Deproteinized by treatment with). After centrifugation, twice the volume of ethanol was added to the supernatant to precipitate the DNA, and the DNA pellet was washed with 70% ethanol. The pellet was vacuum dried and then dissolved in 20 µl of distilled water.

Example 5 Sequencing

The base sequence of the single-chain plasmid purified in Example 4 by the dideoxy chain termination DNA sequencing method was analyzed using Sequenase (USB). The primers required for sequencing were synthesized oligo DNA of Biolab by a synthesizer (ABI).

Oligo DNA for Sequencing

As a result of sequencing, it was found that the base was substituted in an optimized state as follows.

2) GAPDH (glyceraldehyde 3-phosphatase dehydrogenase) transcription terminator (preparation of yepsec-term)

Example 6 Oligopeptide Synthesis

Gall, 10 UAS (upstream activation sequence)-GAPDH [J. Biol. Chem. 245: 839-845. (1979)].

After synthesis, the oligopeptide was purified by OPC (oligo purification column). The oligopeptide was phosphorylated and annealed according to the method of Example 1.

Example 7 YEpsec1-hI1 cleavage

YEpsec1-hI1 was treated with restriction enzymes BamHI, SalI for 1 hour at 37 ° C. in order to insert GAPDH transcription terminator downstream of the gene to be expressed. The cleaved plasma was separated with a 1% plate agarose gel to cut a band of about 9 kb, and DNA was eluted from the cut band using Jetsorb.

Example 8 DNA Linkage and Transformation

In Example 6, the transcription terminator oligopeptide of phosphorylated and annealed YEpsec1-hI1 fmf T ₄ DNA ligase digested with restriction enzymes BamHI, SalI was linked at 16 ° C. Colonies of ampicillin-resistant transformants obtained by transforming E. coli XL-1 Blue according to the CaCl ₂ method were incubated in 1.5 ml of LB-Amp medium and plasmids were purified by RPM filters. The plasmid was digested with restriction enzymes BamHI, SalI, and analyzed by 8% polyacrylamide gelelectrophoresis (PAGE).

3) Preparation of YEpsec-kt

Example 9 YEpsec-ok Cutting

1 μg of YEpsec-ok was digested with restriction enzymes KpnI, SalI for 1 hour at 37 ° C., separated with 1% plate agarose gel, and cut out a band of about 8.3 kb to elute DNA using Jetsorb.

Example 10 YEpsec-term Cleavage

1 μg of YEpsec-term was digested with restriction enzyme KpnI, SalI for 1 hour at 37 ° C., and then separated with 1% plate agarose gel to cut a band of about 0.6 kb to elute DNA.

Example 11 DNA Linkage and Transformation

The 8.3 kb vector eluted in Example 9 and the 0.6 kb insert eluted in section B were dissolved in 30 µl of the ligation reaction solution and reacted by adding a T ₄ DNA ligase.

The reaction solution was transformed into E. coli XL-1 Blue according to CaCl ₂ method, colonies of ampicillin-resistant hemoglobin were incubated in 1.5 ml of LB-Amp, and the plasmid was purified using an RPM rotation filter. The plasmid was digested and analyzed with restriction enzymes, and YEpsec-kt was named as the electron terminator of GAPDH inserted into YEpsec-ok.

4) Substitution of CYC-1 promoter with MFα1 promoter (Preparation of Yepαkt)

Originally, YEpsec1-hI1 consists of a complex promoter of a GAL1-10 UAS regulatory site and a CYC-1 promoter, which binds to Gal ₄ protein, an activator of genes involved in galactose metabolism, but has a more efficient transcription initiation point in this patent. MFα1 promoter [Kurjan et al., Cell. 30: 933-943 (1982)] to replace the CYC-1 promoter.

Example 12 PCR (polymerase chain reaction) of MF α1 promoter

For convenience of cloning, PCR was performed using a primer having an appropriate cleavage site to obtain an MFα1 promoter. The 5 'end of each primer has a cleavage site of restriction enzymes SalI and SalI as shown below.

Primer for amplification of Transcription Initiation Sequence of MFα1 (mating factorα1)

The template used for PCR was the vector p70αT with the MFα1 promoter.

To 50 μmol of each primer, 10 μl of reaction solution containing 200 μg dNTP [10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-HCl (pH 8.8), 2 mM MgSO ₄ 0.1% Triton X-100] Two units of Vent DNA polymerase were added and reacted 35 times using PCR ROBOT (Fine) under the following conditions.

Pretreatment 94 ° C., 300 seconds; Annealing 53 ° C., 40 seconds; Polymerization 72 ℃, 40 seconds, heat denaturation 94 ℃, 40 seconds, post-treatment 53 ℃, 300 seconds

The amplified MFα1 promoter was electrophoresed on a 1.5% plate agarose gel to confirm a DNA band of about 105 bp and eluted from the gel. Purification was performed by digestion with restriction enzymes SalI, SacI.

Example 13 YEpsec-kt Cutting

1 μg of YEpsec-kt was digested with restriction enzymes XhoI, SacI at 37 ° C. for 1 hour. The cleaved plasmid was separated with a 1% plate agarose gel to cut a band of about 8.8 kb, and DNA was eluted using Jetsorb.

Example 14 Linking and Transformation

The α 1 promoter cleaved with SalI and SacI obtained in Example 12 and the 8.3 kb vector cleaved with XhoI and SacI obtained in Example 13 were reacted by adding T ₄ DNA ligase in 30 µl of the ligation reaction solution. After transforming the reaction solution into E. coli XL-1 Blue, colonies of ampicillin-resistant transformants were cultured in 1.5 ml LB-Amp, and then plasmids were purified. When the XhoI site and the SalI site are correctly linked, the plasmid cannot be cleaved by the two restriction enzymes. Therefore, the ones not selected by the restriction enzymes XhoI and SalI were selected and named as YEpαkt.

5) Gal4 gene (preparation of YEPαktGAL4)

Genes involved in galactose metabolism (Gal7, 10, 1, Gal2, Mel1) when galactose is a carbon source are GAL4 protein [Johnstone et al., Proc. Natl. Acad. Sci. USA. 79: 6971-6975 (1982).

This induction occurs at the transcription level of each gene, which acts as a transcriptional activator.

Gal1 (kinase) -Gal10 (epimerase) of YEpsec1-hI1 has a binding site of GAL4 UAS [Cirton et al., J. Bacteriol. 158: 269-278 (1984). YEpsec1-hI1 plasmid is a high-copy number plasmid of the initiation of replication of yeast 2μcircle, and since GAL4 protein is synthesized from chromosomal DNA, it lacks GAL4 protein that can bind to GAL1-10 UAS during galactose induction. It's hard to be.

Therefore, to maintain sufficient GAL4 protein, the Gal4 gene was inserted into the YEpsec1-hI1 plasmid.

Example 15 GAL4 Gene PCR

To PCR the GAL4 gene, the following primers were synthesized.

As a template, the genomic DNA of Saccharomyces cerevisiae 2805 was used and 50 μl of each primer, 100 μl of reaction solution containing 200 μm dNTP [10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-HCl (pH 8.8), 6mM MgSO ₄ , 0.1% Triton X-100] and 2 residues of Vent DNA polymerizing element were added and PCR ROBOT (Fine) was used for 35 reactions under the following conditions.

Pretreatment 94 ° C., 300 seconds; Annealing 53 ° C., 50 seconds; Polymerization 72 ° C., 260 seconds; Heat denaturation 94 ° C., 50 seconds; Post-treatment 53 ° C., 300 seconds.

The amplified GAL4 gene was electrophoresed on a 1% plate agarose gel to confirm a DNA band of about 3.5 kb, eluted from the gel, and purified by restriction enzyme EcoRI.

Example 16 pUCGAL4 Construction

pUC18 was digested with restriction enzyme EcoRI for 1 hour at 37 ° C, and then dephosphorylated by treatment with CIP (calf intestinal phosphatase, NEB).

Plasmids digested with EcoRI were purified using Jetsorb.

In a connection GAL4 gene and pUC18 cut with the EcoRI reaction mixture 30㎕, Lee T ₄ DNA was added to the reaction was carried out overnight at 16 ℃. The reaction solution was transformed into E. coli XL-1 Blue according to the CaCl2 method, colonies were cultured, and the plasmid was purified using an RPM rotary filter. When the plasmid was digested with the restriction enzyme EcoRI, a band of 3.5 kb was visible and named pUCGAL4.

Example 17 YEpαktGAL4

In order to insert the GAL4 gene into YEpαkt was performed as follows.

pUCGAL4 was digested with restriction enzyme EcoRI and electrophoresed on a 1% plate agarose gel to cut a DNA band of about 3.5 kb and eluted from the gel to purify.

1 μg of YEpαkt was digested with restriction enzyme EcoRI for 1 hour at 37 ° C., and then dephosphorylated by CIP (calf intestinal phosphatase, NEB), and the digested vector was purified using Jetsorb.

The GAL4 gene and YEpαkt digested with EcoRI were added with T ₄ DNA ligase in the ligation reaction and reacted overnight at 16 ° C. The reaction solution was transformed into Escherichia coli XL-1 Blue according to the CaCl2 method, and the plasmids were purified by culturing colonies. When the plasmid was digested with the restriction enzyme EcoRI, a band of 3.5 kb was selected and named YEpαktGAL4.

6) Preparation of YEp2-k

Example 18 Preparation of YEp2-k

YEpαktGAL4 has two restriction enzyme KpnI cleavage sites.

One is at the end of the toxic toxin leader sequence and the other is in the leu2-d gene, which is a selectable marker. In this patent, since URA3 is used as a selection marker of yeast, the KpnI cleavage site in the leu2-d gene was removed for the convenience of cloning.

After partial cleavage of YEpαktGAL, 10 units of T ₄ DNA polymerase were added in 50 µl of the reaction solution (10 mM Tris-HCl pH 8.0, 5 mM MgCl ₂ , 5 mM DTT, 100 µm dNTP, 50 µg / ml BSA), and The reaction was carried out for 1 hour to make KpnI cross section end, and ATP and T ₄ DNA ligase were added and reacted overnight at 16 ° C.

E. coli XL-1 Blue was transformed according to the CaCl ₂ method, and plasmids were purified by culturing colonies in 15 ml of LB-Amp. The plasmid was digested and analyzed with restriction enzymes to remove KpnI in the leu2-d gene and named YEp2-K.

II. Cloning of GCSF (Preparation of YEP3KGC)

Example 19 Preparation of GCSF

Oligonucleotides were synthesized as follows for PCR (polymerase chain reaction) of GCSF.

50 pmol of each primer using a macrophage cDNA library (Clontech) as a template to PCR GCSF.

2 units of Vent DNA in 100 μl of reaction solution containing 200 μm dNTP (10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 20 mM Tris-HCl (pH 8.8), 2 mM MgSO ₄ , 0.1% Triton X-100) The polymerase was added and reacted 35 times using PCR ROBOT (Fine) under the following conditions.

Pretreatment 94 DEG C, 300; Annealing 53 ° C., 30 seconds; Polymerization 72 ° C., 30 seconds; Heat denaturation at 94 ° C. for 30 seconds; Post-treatment 53 ℃, 300 sec

The amplified GCSF gene was electrophoresed on a 1% plate agarose gel to confirm and purify the DNA band of about 0.5 bp, and then digested with restriction enzymes KpnI and BamHI.

Example 20 YEpsec-kt Cutting

1 mg of YEp2-K was digested with restriction enzymes KpnI, BamHI for 1 hour at 37 ° C, separated with 1% plate agarose gel, and the IL-1β fragment was discarded. The remaining vector was taken out and eluted.

Example 21 DNA ligation and transformation

The above KpnI, GCSF gene and YEp2-k is cut with BamHI in the ligation reaction mixture 30㎕ T ₄ DNA Lee reacted to claim. After transforming the reaction solution with E. coli XL-1Blue, colonies were cultured to purify the plasmid.

When the plasmid was digested with the restriction enzymes KpnI and BamHI, the GCSF fragment was inserted and named YEp2KGC.

III. Construction of YEpGalMF

General cloning vector YEp352 [J. E. Hill et al. Yease. 2: 163-167 (1986)] to express the GCSF was carried out as follows.

An expression vector YEpGalMF was constructed by obtaining a portion necessary for GCSF expression in YpGX265Gal and used for GCSF expression.

Example 22 PCR of GAL4-GAL1, 10 UAS-MFα1

YpGX265GAL4 [US patents No. 2, pp. 6, 2015] 5,013,652] was performed using primers complementary to the GAL4 and MFα1 promoters to obtain UAS-MFα1 in GAL4-GAL1, 10.

Two units of Vent DNA polymerase were added to 100 μl of the reaction solution containing 500 pmol of each primer and 200 μm dNTP, and the reaction was performed 35 times using PCR ROBOT (Fine) under the following conditions.

Pretreatment 94 ° C., 300 seconds; Annealing 53 ° C., 30 seconds; Polymerization 72 ° C., 30 seconds; Heat denaturation at 94 ° C. for 30 seconds; Post-treatment 53 ℃, 300 sec

The amplified GAL4-GAL1 and 10 UAS MFα1 genes were electrophoresed on a 1% plate agarose gel to confirm DNA bands of about 4 bp, eluted from the gel, and purified by restriction enzymes SacI and EcoRI.

Example 23 YEp352 Cutting

1 μg of YEp352 was digested with restriction enzyme SacI, EcoRI for 1 hour at 37 ° C., and then separated with 1% plate agarose gel to cut a band of about kb and eluted DNA using Jetsorb.

Pretreatment 94%, 300 sec; Annealing 50 ° C., 30 seconds; Polymerization 72 ° C., 30 seconds; Heat denaturation at 94 ° C. for 30 seconds; Post-treatment 53 ℃, 300 sec

[Example 24] DNA ligation and transformation

GAL4-GAL1, 10 UAS-MFα1 and YEp352 of YpGX265GAL4 digested with SacI and EcoRI were reacted by adding T ₄ DNA ligase in 30 µl of the ligation reaction solution. After transforming the reaction solution with E. coli XL-1 Blue, colonies were cultured to purify the plasmid.

When the plasmid was digested with restriction enzymes SacI and EcoRI, 3.5 kb bands were selected and named YEp2GalMF.

IV. YEp2kIL20GC Implementation

Example 25 Preparation of IL20GCSF

1) The following primers were synthesized for PCR of the toxic toxin sequence, the 24 amino acids of the amino terminus of IL-1β, and the endotheptidase KEX2 cleavage site.

As a template, 2 units of Vent DNA polymerase were added to 100 μl of PCR reaction solution containing 50 pmol of each primer and 200 μM dNTP using YEp2-k, and the reaction was performed 35 times under the following conditions.

Pretreatment 94 ° C., 300 seconds; Annealing 50 ° C., 30 seconds; Polymerization 72 ° C., 30 seconds; Heat denaturation at 94 ° C. for 30 seconds; Post-treatment 53 ℃, 300 sec

The amplified killing toxin leader sequence ILβ24AA was electrophoresed on a 1.5% plate agarose gel to identify a DNA band of about 80 bp, eluted from the gel, and purified. The PCR DNA has the following nucleotide sequence.

2) GCSF was PCR using the PCR product thus synthesized and oligopeptides complementary to the carboxy terminus of GCSF.

Oligopeptides complementary to the carboxy terminus of GCSF were synthesized as follows.

As a template, 2 units of Vent DNA polymerase were added to 100 μl of a reaction solution containing 50 pmol of the primer and 24 AA of the toxic toxin sequence IL-1β and 20 μm dNTP using YEp2KGC obtained in Example 21. Reaction was carried out 35 times under.

Pretreatment 94 ° C., 300 seconds; Annealing 50 ° C., 30 seconds; Polymerization 72 ° C., 30 seconds; Heat denaturation at 94 ° C. for 30 seconds; Post-treatment 53 ℃, 300 sec

The elongated IL20GCSF was electrophoresed on a 1% plate agarose gel to identify a DNA band of about 0.66 kb, which was eluted from the gel and purified. IL20GCSF was digested with restriction enzymes SacI, BamHI and purified.

Example 26 YEp2-k Cleavage

1 μg of YEp2-k was digested with restriction enzymes SacI, BamHI for 1 hour at 37 ° C. The cleaved plasmid was separated with 1% agarose gel to cut a band of about 12 kb, and DNA was eluted using Jetsorb.

Example 27 DNA Linkage and Transformation

IL20GCSF cut with SacI and BamHI obtained in Example 26 and YEp2-K were reacted by adding T ₄ DNA ligase in 30 µl of the ligation reaction solution. After transforming the reaction solution with E. coli XL-1Blue, colonies were cultured to purify the plasmid. The plasmid was digested and analyzed with restriction enzymes, and IL20GCSF was inserted and named YEp2kIL20GC.

V. pIL20GC Implementation

Example 28 YEp2kIL20GC cleavage

In order to obtain IL20GCSF-GAPDH transcription terminator in YEp2kIL20GC, the enzyme was digested with restriction enzyme KpnI, SalI at 37 ° C. for 1 hour. The cleaved plasmid was separated by the above method to purify a band of about 0.66 kb.

Example 29 YEpGalMF Cleavage

1 μg of YEpGalMF was digested with restriction enzyme KpnI, SalI at 37 ° C. for 1 hour. The cleaved plasmid was isolated and purified by the above method.

[Example 30] DNA ligation and transformation

The IL20GCSF-GAPDH transcription terminator cut with KpnI and SalI and YEpGgalMF were linked with T ₄ DNA ligase in 30 µl of the ligation reaction solution, followed by transformation. Colonies were incubated to purify the plasmids. The plasmid was digested and analyzed with restriction enzymes, and the IL20GCSF-GAPDH transcription terminator was inserted and named pIL20GC.

VI. Manufacture of YEpHSPGC

HSP 150 (heat shock protein) is a type of glycoprotein secreted when yeast is incubated at 37 ℃ ~ 42 ℃. This advantage can be used for GCSF expression, making it easy to control the expression of GCSF. First, the secretion signal of the promoter of HSP150 was cloned and the GCSF gene was inserted to prepare YEpHSPGC.

Example 31 HSP 150 promoter and leader sequence PCR

The following primers were synthesized to PCR the HSP 150 promoter and leader sequence.

Using the above primers, PCR was performed for the HSP150 promoter and leader sequence of about 0.4 kb in S. cerevisiae genomic DNA. After purification, the mixture was digested with KpnI and SalI.

Example 32 Preparation of YEpHSPGC

The MFα1 (mating factor) promoter and killer toxin leader sequence of YEp2KGC was cut out, and the HSP 150 promoter and leader sequence were substituted and named as YEpHSPGC.

VII. GCSF Expression by pIL20GC and YEp2kIL20GC

Example 33 Yeast Transformation

GCSF was transformed with pIL20GC and YEp2kIL20GC to express in yeast, respectively.

S. cervisidae 2805 (a, pep4 :: HIS3, pro 1-δ, can1, GAL1, his3δ, ura3-52) was inoculated in 3 ml YEPD (1% yeast extract, 2% peptone, 2% glucose) medium at 30 ° C. Incubated overnight at 250 rpm. When re-inoculated with 15 ml YEPD, the OD ₆₀₀ was about 1, and after centrifugation, it was transformable according to the Alkali Cation-Yeast transfrom kit (Bio 101) protocol. competent) made yeast. Yeast pellets were washed with TE buffer, and then suspended in lithium-acetate and incubated at 30 ° C. and 120 rpm.

The suspension was centrifuged and the pellet suspended in TE buffer and mixed in an eppendorf tube containing the plasmid, carrier DNA, and histamine to be transformed. Thermal shock was applied at 42 ° C. for 5 minutes, followed by centrifugation, and then suspended in 200 μl of SOS medium and plated on SD agar solid medium [0.8 g / l Yeast Nitrogen Base without Amino Acid (DIFCO), 2% glucose 1.5% agar]. URA ⁺ colonies were selected by incubation at about 3 days. Yeast transformed with pIL20GC was named Saccharomyces cerevisiae GC1 and yeast transformed with YEp2kIL20GC, respectively, and it was deposited on September 27, 1995 to the Gene Bank of Korea Institute of Science and Technology. 0193BP, KCTC1095BP).

Example 34 GCSF Expression

Colonies were inoculated in SD medium and incubated overnight at 30 ° C. and 250 rpm.

After centrifugation of the culture solution, the pellet was suspended in 1 ml YEPGal (1% yeast extract, 2% peptone, 2% galactose) medium and incubated at 30 ° C. and 250 rpm for about 15 hours to induce GCSF. After the induction culture was centrifuged, 0.5 ml of the supernatant was left on ice for 10 minutes with 10% TCA in the presence of 100 µg / ml BSA, followed by centrifugation at 13000 rpm and 4 ° C for 10 minutes to precipitate GCSF. 20 µl of distilled water and 20 µl 2X SDS dye [125mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 10% 2-mercaptoethanol] was dissolved in the pellet, followed by electrophoresis. Stained with Coomassie. The band of GCSF 17.3kDa was shown.

VIII. Expression of GCSG by YEpHSPGC

Example 35 Expression of Yeast Disease and GCSF

YEpHSPGC was transformed into S. cerevisiae 2805 using Alkali-Cation yeast transform kit (Bio 101) to perform Ura ⁺ selection, and yeast transformed with YEpHSPGC was Saccharomyces cerevisiaeHGCA. It was named and deposited on September 27, 1995 to Gene Bank of Korea Institute of Science and Technology, Biotechnology Research Institute (Accession Number KCTC 0193BP).

Yeast colonies grown in SD medium without uracil were inoculated in 3 ml liquid medium and incubated overnight at 36 ° C. and 250 rpm.

The pellet was centrifuged and the pellet was suspended in 1 ml YEP Gal [1% yeast extract, 2% peptone, 2% galactose] and expressed at 37 ° C. and 250 rpm for 18 hours.

After centrifugation, the pellets and the supernatant were divided, and SDS dye was added to each of them to perform 16% SDS-PAGE to analyze proteins. The supernatant did not show protein bands induced by induction, but in the pellet, a new major band appeared at 20.1 KDa. Western blot was performed to confirm that this new band was GCSF. After SDS-PAGE, the cells were transferred to a nitrocellulose filter and detected using GCSF Ab (RD system) and anti-mouse (IgG-alkaline phosphatase).

IX. Purification of Expressed GCSF Protein

Example 36 Ammonium Sulfate Precipitation

Yeast cells were centrifuged at 10,000 × g for 10 minutes to remove yeast cells. After obtaining the supernatant from which the yeast cells were removed, (NH ₄ ) ₂ SO ₄ was saturated to 85% and left at 4 ° C. for 24 hours. The pellet was dissolved in 50mM Tris (pH 7.8), 0.1mM EDTA, 1mM DTT complete solution and insoluble material was removed by centrifugation at 10,000xg for 10 minutes. All of the above process was carried out at 4 ℃ (Fig. 7).

The crude GCSF thus obtained was used as a sample for the next purification gel-permeation chromatography.

Example 37 Gel-permeation Chromatography

Sepacryl-S-200 (from Pharmacia) was prepared as a medium for gel permeation chromatography by washing with 50 mM Tris (pH 7.8), 1 mM DTT, 0.1 mM EDTA buffer and filling into column (1.6 x 100 cm). (NH ₄ ) ₂ SO4 precipitate was sampled and the concentrated 20 ml culture broth was injected into the column, eluted with 50 mM Tris (pH 7.8) 1 mM DTT, 0.1 mM EDTA buffer, and each peak was collected and frozen based on absorbance at 280 nm. Dry and concentrate. Analysis of each peak by SDS-PAGE revealed GCSF bands and contaminated proteins in the first peak and media protein in the second largest peak (Fig. 8). Separcryl S-200 gel permeation chromatography was able to remove most of the media protein. C4 reversed phase HPLC was performed using GCSF, partially separated by gel permeation chromatography as a sample.

Example 38 C4 Reversed Phase HPLC

Partially purified GCSF via gel permeation chromatography was purified purely by reversed phase HPLC method using C4 column. The C4 column used is from Vydac and the column thickness is 1.0 x 25 cm. The flow rate was 2 ml / min. About 100 μg of the sample was injected into the column, 0.1% TFA (in water) was eluted for 5 minutes, and 0.1% later (in acetonitrile) was eluted with a linear gradient from 0 to 100%. GCSF eluted at 90% 0.1% TFA (in acetonitrile). As a result of analysis by SDS-PAGE, it was confirmed that GCSF was purely separated (Fig. 9).

The GCSF yield was 8% for purification from induction culture broth to C4 reversed phase HPLC. Approximately 1.6 mg of purified GCSF was obtained per 1 L of induction culture broth.

X. N-terminal amino acid analysis of purified GCSF

Example 39

N-terminal amino acid sequencing was performed by blotting GCSF separated by C4 reversed phase HPLC onto PVDF membrane. It was consistent with the N-terminal amino acid sequence of pure GCSF as confirmed in the following experimental conditions (NH ₂ -Thr-Pro-Leu-Gly-Pro-COOH).

(This analysis was commissioned by the Seoul Branch of the Institute for Basic Science).

The model name of the protein sequencing apparatus used for analysis was Milligen 6600B, and PTH-amino acid derivatives were prepared by Edman degradation method and analyzed by HPLC.

Mobile phase A: 35 mM ammonium acetate buffer (pH 4.8)

Mobile phase B: 100% acetonitrile

Temperature: 50 ℃

The amino acid sequence was also determined using the polymer coupling method. Grasp the membrane (membrane disk, PVDF) with tweezers and spot the A) solution on each side by 5ml and dry with a dryer for 15 ~ 20 seconds. Place on a 55 ° C. hotplate, spot 30 ml of solution B) and dry for 7 minutes (do not exceed 10 minutes).

Hold the membrane (Membrane disk) with tweezers, spot the C) solution on each side by 5ml, and dry it with a dryer for 15 ~ 20 seconds. Place on a 55 ° C. hotplate, spot 30 ml of D solution, dry for 5 minutes, spot 20 ml of solution, and dry for 5 minutes. Wash with ethanol, water and methanol.

A) PITC solution (10 nmol / μl in ethyl acetate)

B) buffer (2% v / v triethylamine in 50% v / v methanol)

C) DITC solution (0.1% w / v in ethyl acetate)

D) Polymer solution [(0.1% w / v polyarylamine hydrochloride (low molecular weight) in solution B)]

As a result of analyzing the amino acid sequence by the above method, threonine was detected in the first cycle, proline in the first cycle, leucine in the third cycle, lysine in the fourth cycle, and proline in the fifth cycle. Therefore, the GCSF prepared in the present invention has an amino acid sequence corresponding to NH-Thr-pro-Leu-Gly-Pro, which is consistent with pure human GCSF having physiological activity.

Claims (11)

It has a yeast-derived secretion signal consisting of a yeast-derived promoter, a complex promoter of a GAL1-10UAS regulatory region and an MFα-factor promoter, and a yeast-derived secretion signal composed of an amino terminus of IL-1β. Expression vector YEpαkt. The expression vector YEpαkt according to claim 1, wherein the toxic toxin secretion signal is a toxic toxin secretion signal optimized by codons of proteins expressed in yeast in large quantities. In addition to the yeast-derived promoter and yeast-derived secretion signal of claim 1, the expression vector YEp2-k having a restriction map of FIG. 12 further comprising a GDPH transcriptional terminator and a GAL4 gene. Expression vector YEp2kIL20GC having restriction map of FIG. Expression vector pIL20GC having restriction map of Article 14. Transformant Saccharomyces cerevisiae K2GC obtained by transforming Saccharomyces cerevisiae with the expression vector YEp2kIL20GC (Accession No. KCTC 0190BP). Transformant Saccharomyces cerevisiae GC1 obtained by transforming Saccharomyces cerevisiae with the expression vector pIL20GC (Accession No. KCTC 0193BP). An expression vector having a restriction map of FIG. 15 in which the MFα1 promoter and killer toxin sequence of the expression vector YEp2-k are replaced with a promoter and a heat sequence of the heat shock protein 150. Expression vector YEpHSPGC with restriction map of FIG. Transformant Saccharomyces cerevisiae HGCA obtained by transforming Saccharomyces cerevisiae with the expression vector YEpHSPGC (Accession No. KCTC 0194BP). A method for preparing GCSF by culturing the transformant of claim 6, 7 or 10.