CN1149291C - Process for preparing recombinant proteins using highly efficient expression vector from accharomyces cerevisiae - Google Patents

Process for preparing recombinant proteins using highly efficient expression vector from accharomyces cerevisiae Download PDF

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CN1149291C
CN1149291C CNB97182214XA CN97182214A CN1149291C CN 1149291 C CN1149291 C CN 1149291C CN B97182214X A CNB97182214X A CN B97182214XA CN 97182214 A CN97182214 A CN 97182214A CN 1149291 C CN1149291 C CN 1149291C
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dna
yeast
expression vector
gene
hgcsf
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CN1256711A (en
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张基龙
文载雄
裵千淳
梁斗硕
李志远
成百麟
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South Korea (strain) - Synthetic Fiber
Hanil Synthetic Fiber Co Ltd
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Abstract

The present invention relates to a method using yeast, particularly to yeast expression vectors to prepare human granulocyte colony stimulating factors (hGCSF) and human growth hormone (hGH) through recombinant DNA technology. A hybrid promoter contained in each expression vector comprises two heterogeneic promoters from yeast, namely a yeast killing toxin leader peptide and an amino terminal of IL-1 beta. In addition, the present invention also relates to a method using expression vectors containing HSP150 promoters and secretion signals to prepare hGCSF.

Description

Be used to home-brewed wine zymic expression vector and prepare the method for recombinant protein
Background of invention
The present invention relates to utilize recombinant DNA technology to prepare the method for recombinant protein by yeast.More particularly, the present invention relates to utilize Yeast expression carrier to prepare the method for recombinant protein, this carrier contains the hybrid promoter be made up of two primary yeast inducible promoters and kills and wounds the secretion signal that the aminoterminal of toxin and ripe interleukin-11 β (IL-1 β) is formed by zymic.
In addition, the present invention relates to utilize the Yeast expression carrier of the promotor that comprises yeast heat shock protein 150 and secretion signal to prepare the method for hGCSF and hGH.
In addition, the present invention relates to utilize the Yeast expression carrier that has the XbaI cleavage site to prepare the method for recombinant protein, inserting the XbaI site is to insert the recombinant protein gene for convenience.
The described expression vector of the application of the invention can prepare Filgrastim (hGCSF) and human growth hormone (hGH), and make it efficient secretion.Marrow differentiation and multiply test has disclosed neutrophilic granulocyte or mononuclear macrophage can form colony, people knew and in vivo had G CFS (J.Cell.Comp.Physiol.66:319 (1965) afterwards; Aust.J.Exp.Biol.Med.Sci.44:287 (1966)).
The factor that is called as G CFS (representing with CSF below in the literary composition) can be divided into following a few class according to its biological activity characteristics:
(i) GM-CSF (granulocyte-macrophage CSF) makes the stem cells hyperplasia and the differentiation of granulocyte and mononuclear macrophage, forms colony at last; (ii) M-CSF (scavenger cell CSF) makes mononuclear macrophage form colony; (iii) multi-CSF (polyphyly CSF) stimulates undifferentiated multipotential stem cell, finally forms the pluripotent cell colony; (iv) G-CSF (granulocyte CSF) makes granulocyte form colony (J.B.C.252:1998-2033 (1977), J.B.C.252:4045-4052 (1977), journal of biological chemistry 185:341-343 (1980), J.B.C.258:9017-9021 (1983)).
GCSF is the glycoprotein of a kind of about 20kDa, and it derives from monocyte, monocyte scavenger cell, epithelial cell, inoblast etc.And people GCSF (representing with hGCSF below in the literary composition) gene is present on the 17th karyomit(e).Knownly GCSF is used in combination the generation that can stimulate neutrophilia colony and protoblast, macrophage colony with IL-3, and obtains some sophisticated HL-60s external.GCSF can increase neutrophil(e) cell and monocytic quantity external.
HGCSF has following clinical application:
At first, when the treatment neutrophil(e) cell reduced concurrent advanced solid tumor of disease and patients with blood cancer, hGCSF can increase neutrophil's quantity in dose-dependently ground.
Secondly, the neutrophil(e) cell that hGCSF can cure some patient rapidly reduces disease, and these patients' neutrophil(e) cell reduces disease and malignant lymphoma, lung cancer, carcinoma of testis, urothelioma and acute leukemia etc. are carried out chemotherapy causes.
The 3rd, when doing bone marrow transplantation for acute nonlymphocytic leukemia and chronic myeloid leukemia patient, increase neutrophil's quantity with hGCSF.
The 4th, the neutrophil(e) cell that hGCSF can cure some patient rapidly reduces disease, and it is that the osteomyelodysplasia syndromes causes that these patients' neutrophil(e) cell reduces disease.
The 5th, the neutrophil(e) cell that hGCSF can cure some patient rapidly reduces disease, and it is that aplastic anemia causes that these patients' neutrophil(e) cell reduces disease.
The 6th, hGCSF can be used for treating heredity and the primary neutrophil(e) cell reduces disease.
The 7th, hGCSF can suppress or reduce the anti-tumor chemotherapeutic mucositis that causes and the neutrophil(e) cell that generates heat and reduce the incidence (medicine yearbook (Drug Evaluations Annual) 1993, the p2232-2333 of American Pharmaceutical Association (American Medical Associations)) of disease.
Human growth hormone (representing with hGH below in the literary composition) is a kind of non-glucosides protein of being made up of 191 amino acid, and it is by the prepituitary gland excretory.HGH contains two intramolecular disulfide bonds, and its molecular weight is 22000 dalton.It at first is synthesized and is its precursor, and secretion is come out from cell after processing.
The people produces a large amount of hGH before growing up, and generation is all arranged in life.
HGH is that normal growth and growth are necessary, but hGH generation level is crossed the low nanism that can cause several types, can association acromegaly or gigantosoma and excessively produce hGH.
HGH shows multiple biological activity, and multiple tissue is directly or indirectly exerted an influence.It can influence the length growth rate and the lactation of bone, and demonstrates diabetogenic para-insulin activity.In addition, it can promote protein synthesis, and influences the metabolism of lipid and carbohydrate.
Below be the clinical application of hGH:
We know, lack the nanism that causes for hGH, if take hGH in childhood, can make misgrowth restore (Raben, M.S., J.Clin.Endocr.18:901-904 (1958)).Know that also hGH can be used for treatment of obesity, and can treat (Proc.of NIAMDD Symp.Publ.No.74-612 (Raiti, S. compiles) (Baltimore, Maryland, 1973)) such as multiple slight illness such as fracture, skin burn, hemorrhagic ulcers effectively.
Known the base sequence of hGH DNA by gene being carried out cDNA clone, and existing report hGH DNA at expression in escherichia coli (Martial etc., science 205:602-605 (1979)).
There are many gene engineering method to attempt to make recombinant protein to cross expression.
At first, set up a kind of clone goal gene after in expression in escherichia coli method of protein (science 232:61-64 (1986)).But use in the method for intestinal bacteria as the host at this, have some shortcomings as described below.
In human body, protein is synthesized earlier to its precursor, is processed into mature form by proteolysis then.
But when expression in escherichia coli protein, removed effectively by aminopeptidase the N-of institute's synthetic protein end methionine(Met) can not resemble in human body, therefore in colibacillary tenuigenin, have and do not have the protein of methionine(Met) to exist simultaneously.So just being difficult to not to have the protein of methionine(Met) and the protein that methionine(Met) is arranged to separate.
In many cases, protein is expressed as inactivation or insoluble form, should be translated into bioactive protein by renaturation (folding again) process then, and this process reduces the recovery of protein amount sometimes significantly.
The pollution problem that also exists bacterial endotoxin to cause in the purge process.
In addition, in intestinal bacteria, can not carry out proteinic posttranslational modification (for example glucosidesization of hGCSF).
Secondly, at zooblast, such as the goal gene of having expressed the clone in CHU-2 (people produces the tumor cell line of GCSF) or the Chinese hamster ovary cancer cells.
But as host's method some shortcomings are arranged also with zooblast, use the culture condition of expensive blood serum medium very complicated such as need, and, reclaim very low usually (the EMBO magazine 5:871-876 (1980) of output owing to generally from the substratum of significant volume, can only be purified to a small amount of recombinant protein; (KR91-5624)).
What set up is a kind of reasonable solution of above problem with yeast as host's expression system.According to Loison and other people, can from recombination yeast, obtain a large amount of desired polypeptides or protein (biology/technology 4:433-437 (1986) in this way; Rose and Harrison compile, yeast, the 3rd volume, 349-420 page or leaf, Burrow, " baker's yeast ", AcademicPress, London (1970)).Adopt zooblast or intestinal bacteria to compare as host's expression system with other, there is tangible advantage in the expression of recombinant yeast system.
The inventor has studied and has used yeast to prepare the method for hGCSF.US FDA declares that yeast is not the pathogenic bacterium of human body, and the most gene expression regulation principles in the yeast are disclosed (Strathern etc., yeast belong zymic molecular biology, metabolism and genetic expression, cold spring harbor laboratory, New York (1982)).
As host cell some advantages are like this arranged with yeast, it has been generally acknowledged that promptly yeast is the biology to human body safety, might from the high-cell-density cultivation thing, produce a large amount of hGCSF, also have purge process to simplify, this is because soluble protein can be secreted from cell under the guiding of signal peptide.
The method (Valensuela etc., the natural 298:347-350 (1982) that express some heterologous protein such as hepatitis B virus, Interferon, rabbit, ox rennin, Urogastron have been reported in yeast recently; Hitzeman etc., NAR11:2745-2763 (1983); McAleer etc., natural 307:178-180 (1 984); Tuite etc., EMBO magazine 1:603-608 (1982); Mellor etc., gene 24:1-14 (1983); Urdea etc., PNAS80:7461-7465 (1983)).
But in general the expression level of heterologous protein in recombination yeast compared very low with the zymic homologous protein, so people did, and extensive work is set up efficient expression vector so that improve the expression level (Chen etc., NAR12:8951-8970 (1984)) of heterologous protein in yeast.
For example, EP84303833 discloses and has a kind ofly had the cloning vector of external goal gene and yeast GAL1 promotor by use, prepares the method for galactokinase-Niu renninogen fused protein from yeast.Can also be under yeast GAL4 gene be inserted into situation in the expression vector that contains foreign gene and GAL1 promotor, by semi-lactosi transcriptional level control approach GAL4 protein expression amount is improved, thereby can improve synthetic (Laughon etc., the PNAS79:6827-831 (1982)) of heterologous protein.
EP84302723 discloses a kind of by the signal sequence of use mating factor α and the method for promotor expressing human Interferon, rabbit, human serum albumin, Bov IFN α-1, α-2, tissue plasminogen activator, rennin and human insulin-like growth factor in yeast.
Summary of the invention
The purpose of this invention is to provide a kind of by using recombinant DNA technology from yeast, to prepare the method for recombinant protein.More precisely, the invention provides a kind of method of utilizing Yeast expression carrier to prepare recombinant protein, this carrier contains the hybrid promoter be made up of two primary yeast inducible promoters and kills and wounds the secretion signal that the aminoterminal of toxin and ripe interleukin-11 β (IL-1 β) is formed by yeast.
The purpose of this invention is to provide the method that a kind of expression vector that constitutes by the promotor and the secretion signal that use by yeast heat shock protein 150 prepares hGCSF.
The purpose of this invention is to provide the method that a kind of Yeast expression carrier that has an XbaI cleavage site by use prepares recombinant protein, inserting the XbaI site in described carrier is to insert the recombinant protein gene for convenience.
The drawing summary
In the accompanying drawings:
Fig. 1 shows the preparation process of YEp2-K.
Fig. 2 shows the preparation process of YEp2KIL20GC.
Fig. 3 shows the preparation process of pIL20GC.
Fig. 4 shows the preparation process of YEpHSPGC.
Fig. 5 shows an aminoacid sequence, and this aminoacid sequence is made up of N-end 24 residues, hGCSF and the signal peptidases that kill and wound toxin leader sequence, IL-1 β and the cleavage site of KEX2 peptase.
Fig. 6 is presented at the SDS-PAGE analytical results of the hGCSF that expresses in the yeast.
Fig. 7 shows the Western trace result utilize the hGCSF that Yeast expression carrier YEpHSPGC expresses in yeast
Fig. 8 is showed cell, ethanol and hGCSF concentration and plasmid stability time history plot.
Fig. 9 is presented at the SDS-PAGE analytical results of the hGCSF that expresses in the yeast.
Figure 10 is showed cell, ethanol and hGH concentration and plasmid stability time history plot.
Figure 11 is presented at the SDS-PAGE analytical results of the hGH that expresses in the yeast.
Figure 12 shows an aminoacid sequence, and this aminoacid sequence is made up of N-end 24 residues, hGH and the signal peptidases that kill and wound toxin leader sequence, IL-1 β and the cleavage site of KEX2 peptase.
Figure 13 is the collection of illustrative plates of pIL20XGH.
Figure 14 shows the purge process of the hGCSF in the yeast culture liquid.
Figure 15 shows the process through Sephacryl S-200 column chromatography purification hGCSF.
Figure 16 shows the final step of hGCSF purge process.
Detailed Description Of The Invention
What the inventor paid close attention to is such fact, namely for high-level Restruction hGCSF from yeast, should improve its secernment efficiency and expression.
In order to secrete finished hGCSF, once the aminoterminal with multiple secretion signal and hGCSF merged, but secretion and unsuccessful.
Simultaneously once the someone reports and utilizes secretion signal to secrete efficiently interleukin-11 β (EMBO magazine 6:229-234 (1987)) from yeast. What the inventor paid close attention to is the possibility of utilizing the N-terminal amino acid of IL-1 β to come to secrete finished hGCSF from yeast cells. The inventor is final when finding to place in the front of hGCSF gene a fusogenic peptide that is comprised of 24 amino acid that kill and wound toxin secretion signal and IL-1 β, can successfully express and secrete hGCSF from cell. At this moment, between 24 residues of IL-1 β N-end and hGCSF, insert dibasic KEX2 cleavage site, thereby the effect by KEX2 enzyme (endopeptidase) discharges ripe hGCSF in secretory pathway.
Above-described expression vector has 24 amino-acid residues-KEX2 cleavage site-this arrangement mode of ripe hGCSF that kills and wounds toxin secretion signal-IL-1 β.Utilize above-mentioned expression vector expressed protein to secrete: to be transported in the process of golgi body at synthetic good protein by following steps, signal peptide is cut by signal peptidase, the ripe hGCSF that to have correct amino terminal sequence is finally secreted in KEX2 peptase cutting IL-1 β district.
Below describe the used hGCSF expression vector of the present invention in detail.
The hGCSF expression vector comprises: replace the CYC-1 promotor among the Yeast expression carrier YEpsecl-hI1 (C.Baldari etc., EMBO magazine 6:229-234 (1987)) mating factor-α 1 promotor, hold 24 heterozygosis secretion signal, hGCSF gene and GAL4 (it is activation of GAL gene) that residue is formed by killing and wounding toxin leader sequence (this sequence is optimized by zymic codon use habit) and IL-1 β N-.With this expression vector transformed saccharomyces cerevisiae, in the minimum medium that lacks uridylic, pick out transformant then as the bacterial strain that produces hGCSF.Recombination is expressed in the high-cell-density cultivation thing of this transformant of choosing, and the result produces the outer hGCSF of a large amount of born of the same parents, and cell growth and the culture condition that produces hGCSF have obtained systemic optimization in the fermentor tank.
The inventor has also made up an expression vector, and it contains the promotor and the signal leading peptide of heat shock protein; By means of this carrier, the expression of reorganization hGCSF is subjected to the regulation and control of temperature inversion.Different with other derivable Yeast promoters (such as the ADHII promotor that is subjected to semi-lactosi inductive GAL promotor, phosphate starvation inductive Pho5 promotor, glucose induction) is that the promotor of heat shock protein (HSP) is only come regulatory transcription and protein synthesis subsequently by temperature regulation (37-42 ℃).And HSP by leader sequence from cell, secrete (PNAS, 89:3671-3675).The inventor has set up a kind of utilization is prepared hGCSF by the expression vector of HSP150 promotor and HSP leader sequence structure method.
In addition, for convenience other heterologous genes are inserted in the above-mentioned expression vector that has a mating factor α promotor, the contriver has inserted the XbaI site between the IL-1 of this carrier beta amino acids sequence and KEX2 cleavage site.
Above-mentioned expression vector can be used to produce other recombinant proteins.Particularly in the present invention, utilize the XbaI-BamHI fragment structure gene of hGH to be inserted above-mentioned expression vector, successfully give expression to hGH from selected transformant as cloning site.Equally, in the high-cell-density cultivation thing of the above-mentioned transformant of selecting, successfully synthesized hGH, and be secreted in a large number in the outer nutrient solution of born of the same parents in the fermentor tank.
Answer emphasis to mention herein, although only provide the method for producing hGCSF and hGH in the present invention, above-mentioned expression vector can be used to produce other recombinant proteins.
The output of hGH surpasses 1g/L in above-mentioned high cell density fermentation, and other fermentation yields that derive from the zymic recombinant protein of report were compared with the past, and this is a relatively very high level.Therefore, will contain the sequence of killing and wounding toxin leader sequence-24 residue-hGH of IL-1 β aminoterminal expression vector, and can from yeast, express and also secrete other recombinant proteins efficiently as the heterozygosis signal peptide by using.
Describe the present invention in detail by embodiment.
Embodiment only is used for illustrating the present invention, does not limit the scope of claim of the present invention.
I. prepare YEp2-K
Expression vector YEpsecl-hI1 is by GAL1, the upstream activating sequence of 10 genes, CYC-1 promotor, Kluyveromyces lactis kill and wound toxin leader sequence [M.J.R.Stark etc., NAR12:6011-6031 (1984)] and il-1 β genomic constitution, and il-1 β is by inductor (semi-lactosi) abduction delivering.
For the efficient that makes carrier YEpsecl-hI1 is higher, will kills and wounds the toxin leader sequence and optimize, and replace the CYC-1 promotor with more effective MF α 1 promotor.
Transcribe in order to stop mRNA, added the transcription terminator of GAPDH at 3 of hGCSF ' end, and cloned Gal4 gene (it is activation of Gal gene), and this gene has been added in the expression vector.
1) optimizes the codon that kills and wounds the toxin leader sequence
Preparation YEpsec-ok
The embodiment 1 synthetic oligonucleotide that kills and wounds the toxin leader sequence
In order to replace the codon that kills and wounds the toxin leader sequence among the Yeast expression carrier YEpsecl-hI1 with the codon that is coded in mistake expressed protein in the yeast saccharomyces cerevisiae, with synthesizer (ABI, 392 DNA/RNA synthesizers) synthesized oligonucleotide (J.Bennetzen, B.Hall journal of biological chemistry 257:3026-3031) with sequence shown in the SEQ ID NO:1.
Follow following reaction so that the synthetic oligonucleotide is inserted a site, make to cut out and kill and wound the toxin leader sequence among the YEpsecl-hI1 in this site.Use T 4Polynucleotide kinase (NEB) contains reaction soln (70mM Tris-HCl (pH7.6), the 10mMMgCl of ATP at 30 μ l 2, 5mM DTT (dithiothreitol (DTT))) in oligonucleotide each 5 ' hold in 37 ℃ of phosphorylations 1 hour.Two parts of reaction solns are mixed, placed 20 minutes.In slowly being cooled to 30 ℃ process, oligonucleotide is annealed.
Embodiment 2 digestion YEpsecl-hI1
With restriction enzyme (SacI, KpnI; NEB) in 40 μ l reaction solns (20mM Tris-acetic acid, 10mM magnesium acetate, 50mM Potassium ethanoate) with 1 μ gYEpsecl-hI1 in 37 ℃ digestion 1 hour, then in 1% agarose gel plate through electrophoretic separation.After the separation, the band that downcuts 8.4kb utilizes Jetsorb, and (GENOMED cat#110300), elutes DNA, and carries out purifying from the DNA band that scales off.
The connection of embodiment 3DNA and conversion
With 100 T of unit 4(this solution is by 50mMTris-HCl (pH7.6), 10mM MgCl in 30 μ l reaction solns for dna ligase 2, 10mM DTT and 1mM ATP form) annealed among the embodiment 1 is killed and wounded the YEpsecl-hI1 that gets well with restriction enzyme (SacI and KpnI) digestion among toxin leader sequence and the embodiment 2 be connected in 16 ℃ and spend the night.According to molecular cloning: (Sambrook, Fritch Mantiatis the 2nd edition CSH), passes through CaCl to experiment guide 2Method ligation liquid transformed into escherichia coli XL-1 Blue (supE44 hsdR17 recA1 endA1gyrA46 thi relA1 Iac-F ' (proAB +LacI qLacZ Δ M15 Tn (tetr))).After the conversion, the intestinal bacteria that will be transformed are layered on the LB-Amp agar lithographic plate substratum (10g/l Tryptones, 5g/l yeast extract, 10g/l NaCl, 100 μ g/ml penbritins), and in 37 ℃ of incubations 20 hours.With amicillin resistance transformant (Amp R) bacterium colony in 1.5ml liquid LB-Amp substratum, cultivate after, by the alkaline lysis method of extracting plasmid, and be purified with RPM rotary filter (BIO101).Pick out the plasmid (because by codon optimized step, the restriction enzyme SmaI site of killing and wounding in the toxin leader sequence has disappeared) that is not limited enzyme SmaI digestion, and with gained plasmid called after YEpsec-ok.
Embodiment 4 single stranded DNAs
In order to identify the base sequence that replaces for the optimizing codon purpose, measure the sequence of YEpsec-ok.Be prepared as follows the required single stranded DNA of order-checking.
With gained YEpsec-ok among the restriction enzyme BamH1 digestion embodiment 3, with SacI digestion, electrophoresis on 1.5% sepharose downcuts the gel that has the 0.66kb dna fragmentation again.With GENE CLEAN test kit II DNA is eluted from sepharose subsequently.Get 1 μ g carrier M13mp19 with restriction enzyme BamH1 and SacI digestion, be purified with GENECLEAN test kit II then.In linked system, use T 4Dna ligase is connected with M13mp19 the dna fragmentation of 0.66kb and spends the night in 16 ℃.Behind reaction soln transformed competence colibacillus intestinal bacteria XL-1Blue, according to molecular cloning: experiment guide (the same) separates single stranded DNA.More particularly, XL-1Blue solution, 40 μ l X-Gal (20mg/ml is in diformamide) and the 4 μ l IPTG (200mg/ml) of 200 μ l incubated overnight are mixed with agar, mixed solution is layered on the LB Agar Plating.With the intestinal bacteria that transform behind 37 ℃ of incubations, white colony of picking on agar plate.Infect 200 μ l XL-1Blue, 250rpm cultivation 5 hours with bacterium colony of choosing and 20ml LB in 37 ℃.After this,, in supernatant liquor, add the PEG (being dissolved in the 20%PEG8000 of 2.5M NaCl) of 1/5 volume, placed 15 minutes on ice medium centrifugal.After centrifugal, abandon supernatant.The M13 virus throw out that precipitates is resuspended in 200 μ l TE damping fluids (10mM Tris-HCl (pH7.6), 1mM EDTA), uses phenol/chloroform/primary isoamyl alcohol (25: 24: 1) to extract protein then.After centrifugal, in supernatant liquor, add 2 volume of ethanol and come deposit D NA, and precipitate with 70% washing with alcohol DNA.After will precipitating vacuum-drying, be dissolved in again in the 20 μ l distilled water.
Embodiment 5 sequential analyses
Analyze the base sequence of the single stranded plasmid of preparation among the embodiment 4 by dideoxy chain-termination method.With the used primer of the synthetic order-checking of ABI synthesizer.
The few DNA that is used to analyze base sequence has the sequence shown in the SEQ ID NO:2.
The result of base sequence analysis shows that the base of codon has been replaced to the optimizing codon listed as SEQ IDNO:3.
2) transcription terminator (YEpsec-term) of preparation GAPDH (Glycerose 3-phosphate dehydrogenase).
Embodiment 6 synthetic oligonucleotides
In order to stop by GAL1,10UAS (upstream activating sequence)-MF α 1 promotor guiding transcribe synthetic GAPDH transcription terminator (journal of biological chemistry 245:839-845 (1979)) shown in SEQ ID NO:4.
After synthetic, through OPC (oligonucleotide purification column) purification of oligonucleotides.And according to embodiment 1 with its phosphorylation and annealing.
Embodiment 7 digestion YEpsecl-hI1
For the transcription terminator with GAPDH is inserted into by the downstream of expressing gene, in 37 ℃ YEpsecl-hI1 was digested 1 hour with restriction enzyme BamH1 and SacI.With plasmid electrophoresis in 1% agarose gel plate of digestion, downcut the band of 9kb, and DNA is eluted from the band that downcuts with Jetsorb.
The connection of embodiment 8DNA and conversion
Use T in 16 ℃ 4Dna ligase will link together with the transcription terminator oligonucleotide of phosphorylation in embodiment 6 and annealed GAPDH with the YEpsecl-hI1 of restriction enzyme BamH1 and SacI digestion.
(this bacterium colony is according to CaCl with the bacterium colony of amicillin resistance 2Method reaction solution transformed into escherichia coli XL-1 Blue acquisition) cultivation is in 1.5ml LB-amp substratum.After the cultivation, through RPM filter plasmid purification.
With restriction enzyme BamH1, SacI digested plasmid, and in 8%PAGE (polyacrylamide gel), carry out electrophoresis.The plasmid called after YEpsec-term that will have the 70bp dna fragmentation.
3) preparation YEpsec-kt
Embodiment 9 digestion YEpsec-ok
With restriction enzyme KpnI, SalI with 1 μ g YEpsec-ok in 37 ℃ of digestion 1 hour, and in 1% agarose gel plate, separate.Downcut the band of about 8.3kb and use the Jetsorb purify DNA.
Embodiment 10 digestion YEpsec-term
With restriction enzyme KpnI, SalI 1 μ g YEpsec-term was digested 1 hour in 37 ℃.Afterwards, separation quality grain in 1% agarose gel plate, band and the eluted dna of cutting-out 0.6kb.
The connection of embodiment 11DNA and conversion
The 8.3kb carrier that wash-out goes out among the 0.6kb fragment that wash-out among the embodiment 10 is gone out and the embodiment 9 is dissolved in 30 μ l and is connected in the solution, and uses T 4Dna ligase links together them.With reaction soln through CaCl 2Method transformed into escherichia coli XL-1 Blue.The bacterium colony of amicillin resistance is cultivated in 1.5ml LB-amp substratum, isolated plasmid with the RPM rotary filter.Use the restriction enzyme digested plasmid, will have the plasmid called after YEpsec-kt of the transcription terminator of YEpsec-ok.
4) replace CYC-1 promotor (preparation Yep-α kt) with MF α 1 promotor
Primary YEpsecl-hI1 is by the promotor complex body, the GAL1 that comprise CYC-1 promotor and control region, 10 UAS (Gal4 protein (activation of semi-lactosi metabolism related gene) is combined in above it) form, but in the present invention, the CYC-1 promotor is replaced by MF α 1 promotor, the latter has more effective transcription initiation effect (Kurjan etc., cell 30:933-943 (1982)).
The PCR (polymerase chain reaction) of embodiment 12MF α 1 promotor
For the ease of cloning, obtain MF α 1 promotor through PCR with the primer that has suitable restriction site.
At restricted enzyme SalI of 5 of each primer ' end and SacI cleavage site.
The primer of MF α 1 transcriptional initiation sequence of being used to increase has the sequence shown in SEQ ID NO:5 and SEQ ID NO:6 respectively.
Template used among the PCR is the p70 α T carrier that contains MF α 1 promotor.The reaction soln (10mM KCl, the 10mM (NH that contain 200 μ M dNTP to every kind of primer of 50pmol and 100 μ l 4) 2SO 4, 20mM Tris-HCl (pH8.8), 2mM MgSO 4, 0.1%triton X-100) in add the Vent archaeal dna polymerase of 2 units, utilize PCR ROBOT (Fine Co.) to make reaction according to following temperature program(me) circulation 35 times then:
Pre-treatment: 94 ℃, 300 seconds;
Annealing: 53 ℃, 40 seconds;
Extend: 72 ℃, 40 seconds;
Sex change: 94 ℃, 40 seconds;
Afterreaction: 53 ℃, 300 seconds
With MF α 1 promotor electrophoresis in 1.5% agarose gel plate of amplification, identify the 150bpDNA band, and it is eluted from gel.Digest the DNA that elutes with restriction enzyme SalI and SacI.
Embodiment 13 digestion YEpsec-kt
With restriction enzyme XhoI and SacI 1 μ g YEpsec-kt was digested 1 hour in 37 ℃.The plasmid that digestion is good separates on 1% agarose gel plate, about 8.8kb DNA band is downcut, and use the Jetsorb eluted dna.
Embodiment 14 connects and transforms
Use T 4The 8.3kb carrier that dna ligase digests with XhoI and SacI in MF α 1 promotor of SalI and SacI digestion preparation and embodiment 13 in 30 μ l connect in the solution embodiment 12 links together.With reaction soln transformed into escherichia coli XL-1 Blue, the conversion bacterium colony of amicillin resistance is cultivated in 1.5ml LB-amp substratum, and plasmid purification.When the XhoI site correctly was connected with the SacI site, plasmid can not be by XhoI and SacI cutting, and therefore picking out can not be by the plasmid of XhoI and SacI cutting, and with it called after YEp α kt.
5) Gal4 gene (preparation YEp α ktGAL4)
When semi-lactosi during as carbon source, GAL4 albumen activates semi-lactosi metabolism related gene (Gal7,10,1, Gal2, the Mel1) expression in yeast (Johnstone etc., Proc. Natl. Acad. Sci.USA 79:6971-6975 (1982)).This induces process from each gene transcription level, and with GAL4 albumen as transcription activator.UAS (it is the binding site of GAL4, Cirton etc., bacterium magazine 158:269-278 (1984)) is contained in the Gal1 of YEpsecl-hI1 (kinases)-Gal10 (epimerase) site.YEpsecl-hI1 is yeast 2 a μ annular high copy number plasmid.Because GAL4 albumen is by the chromosomal DNA coding, when inducing with semi-lactosi, the GAL4 protein concentration that can be combined on the GAL1-10 UAS is very low.Therefore be difficult to carry out that GAL4 is proteic efficiently expresses.In order to keep the GAL4 albumen of q.s, in YEpsecl-hI1, insert the GAL4 gene.
The PCR of embodiment 15GAL4 gene
Synthesized have sequence shown in SEQ ID NO:7 and the SEQ ID NO:8 respectively primer so that carry out the PCR of GAL4 gene.
Template is the genomic dna of yeast saccharomyces cerevisiae 2805.
This PCR has used each primer of following component: 50pmol, reaction soln (10mM KCl, the 10mM (NH that 100 μ l contain 200 μ MdNTP 4) 2SO 4, 20mM Tris-HCl (pH8.8), 6mM MgSO 4, 0.1%triton X-100), used polysaccharase is the Vent archaeal dna polymerase of 2 units, utilizes PCR ROBOT (Fine Co.) to make reaction according to following temperature program(me) circulation 35 times then:
Pre-treatment: 94 ℃, 300 seconds;
Annealing: 53 ℃, 50 seconds;
Extend: 72 ℃, 260 seconds;
Sex change: 94 ℃, 50 seconds;
Afterreaction: 53 ℃, 300 seconds
With GAL4 gene electrophoresis in 1% agarose gel plate of amplification, identify the DNA band of about 3.5bp, DNA is eluted and digests with limiting enzyme EcoRI from gel.
Embodiment 16 preparation pUCGAL4
PUC18 after 1 hour, is used CIP (calf intestinal phosphatase enzyme, the plasmid dephosphorylation that NEB) will digest in 37 ℃ of digestion with limiting enzyme EcoRI.Utilize the Jetsorb will be with the plasmid purification of EcoRI digestion.Use T 4Dna ligase connects in the solution GAL4 gene spent the night in 16 ℃ with the plasmid that digested with EcoRI at 30 μ l and is connected.According to CaCl 2Method transformed into escherichia coli XL-1 Blue cultivates bacterium colony, and through RPM rotary filter plasmid purification.Pick out the plasmid that when digesting, has 3.5kb DNA with EcoRI, and with it called after pUCGAL4.
Embodiment 17 YEp α ktGAL4
For the GAL4 gene being inserted YEp α kt, carried out following operation.
Digest pUCGAL4 with limiting enzyme EcoRI, and in 1% agarose gel plate electrophoresis, the DNA of about 3.5kb band is downcut, elute and purifying from glue.With limiting enzyme EcoRI with 1 μ g YEp α kt in 37 ℃ digestion 1 hour, and with CIP (the calf intestinal phosphatase enzyme, NEB) with the plasmid dephosphorylation, the carrier purifying that utilizes Jetsorb to digest.Use T 4Dna ligase is connected with YEp α kt with EcoRI digestion the GAL4 gene in connecting solution and spends the night in 16 ℃.According to CaCl 2Method transformed into escherichia coli XL-1 Blue cultivates bacterium colony, plasmid purification then.Behind EcoRI cutting plasmid, pick out 3.5kb DNA band, and called after YEp α ktGAL4.
6) preparation Yep2-k
Embodiment 18 preparation Yep2-k
Two cleavage sites of restricted enzyme KpnI on YEp α ktGAL4.One at the end that kills and wounds the toxin leader sequence, and another is on selective marker leu2-d gene.In the present invention, owing to use URA3, be positioned at that KpnI cleavage site on the leu2-d gene is destroyed have been fallen as the zymic selective marker.After YEp α ktGAL4 partly digested with KpnI, use T 4Dna ligase is at 50 μ l reaction soln (10mM Tris-HCl (pH8.0), 5mM MgCl 2, 5mMDTT, 100 μ M dDNP, 50 μ g/ml BSA) in will digest the site and fill and lead up 1 hour in room temperature, the KpnI site becomes flat terminal like this.Utilize T 4Dna ligase and ATP will put down terminal KpnI site and be connected in 16 ℃ and spend the night.According to CaCl 2Method transformed into escherichia coli XL-1 Blue cultivates bacterium colony in 1.5ml LB-Amp substratum, then plasmid purification.Use the restriction enzyme digested plasmid, will not have the plasmid called after YEp2-k in the KpnI site on the leu2-d gene.
II. clone GCSF (preparation YEp3KGC)
Embodiment 19 preparation hGCSF
Synthesized have sequence shown in SEQ ID NO:9 and the SEQ ID NO:10 respectively oligonucleotide so that carry out the PCR of GCSF.
Template used among the PCR of hGCSF is scavenger cell cDNA library (Clontech).The reaction soln (10mM KCl, the 10mM (NH that contain 200 μ M dNTP to every kind of primer of 50pmol and 100 μ l 4) 2SO 4, 20mM Tris-HCl (pH8.8), 2mM MgSO 4, 0.1%TritonX-100) in add 2 Vent of unit archaeal dna polymerases, utilize PCR ROBOT (FineCo.) to make reaction according to following temperature program(me) circulation 35 times then:
Pre-treatment: 94 ℃, 300 seconds;
Annealing: 53 ℃, 30 seconds;
Sex change: 94 ℃, 30 seconds;
Afterreaction: 53 ℃, 300 seconds
With hGCSF gene electrophoresis in 1% agarose gel plate of amplification, identify the 0.5kbDNA band, carry out purifying and use restriction enzyme KpnI and BamHI digestion.
Embodiment 20 digestion YEp2-k
After KpnI and BamHI YEp2-k digestion with 1 μ g, the DNA that digested is carried out electrophoresis on 1% agarose gel plate, discard IL-1 β fragment, other fragments of carrier are eluted.
The connection of embodiment 21DNA and conversion
Use T 4Dna ligase links together the GCSF gene in 30 μ l connection solution with the YEp2-k that digests with KpnI and BamHI.With reaction soln transformed into escherichia coli XL-1Blue, cultivate bacterium colony, plasmid purification then.During with restriction enzyme KpnI and BamHI digested plasmid, inserted the segmental plasmid of GSCF and be named as YEp2KGC.
III. prepare YEpGalMF
In order in general cloning vector YEp352 (J.E.Hill etc., yeast 2:163-167 (1986)), to express GCSF, carried out following operation.Obtain to express the required fragment of GCSF from YpGX265Gal4, construction of expression vector YEpGalMF also is used to express GCSF.
Embodiment 22GAL4-GAL1, the PCR of 10 UAS-MF α 1
With obtaining GAL4-GAL1 by YpGX265GAL4 (United States Patent (USP) 5013652) through PCR with GAL4 and MF α 1 promotor complementary primer, the UAS-MF α 1 in 10.Has the sequence shown in SEQ ID NO:11 and the SEQ ID NO:12 respectively with MF α 1 promotor complementary primer with GAL4 complementary primer.
The Vent archaeal dna polymerase that adds 2 units in every kind of primer of 500pmol and 100 μ l contain the reaction soln of 200 μ M dNTP, utilize PCR ROBOT (Fine Co.) to make reaction according to following temperature program(me) circulation 35 times then:
Pre-treatment: 94 ℃, 300 seconds;
Annealing: 53 ℃, 30 seconds;
Extend: 72 ℃, 30 seconds;
Sex change: 94 ℃, 30 seconds;
Afterreaction: 53 ℃, 300 seconds
With the GAL4-GAL1 of amplification, 10 UAS-MF α, 1 gene electrophoresis in 1% agarose gel plate identifies the DNA band of about 4kb, it is eluted from gel and digests with restriction enzyme SacI and EcoRI.
Embodiment 23 connects YEp352
With SacI and EcoRI with the YEp352 of 1 μ g in 37 ℃ of digestion 1 hour, in 1% agarose gel plate,, the DNA band of about kb is downcut, and utilizes Jetsorb its wash-out with the plasmid electrophoresis.
Pre-treatment: 94 ℃, 300 seconds;
Annealing: 50 ℃, 30 seconds;
Extend: 72 ℃, 30 seconds;
Sex change: 94 ℃, 30 seconds;
Afterreaction: 53 ℃, 300 seconds
Connection and the conversion of embodiment 24 DNA
Use T 4Dna ligase connects at 30 μ l will be from the GAL4-GAL1 of YpGX265GAL4 in the solution, and 10 UAS-MF α 1 and the YEp352 that digests with SacI and EcoRI link together.With reaction soln transformed into escherichia coli XL-1 Blue, cultivate bacterium colony plasmid purification then.During with restriction enzyme SacI, EcoRI digested plasmid, pick out DNA band and the called after YEpGalMF of 3.5kb.
IV. prepare YEp2kIL20GC
Embodiment 25 preparation IL20GCSF
1) synthetic have the primer of sequence shown in SEQ ID NO:13 and the SEQ ID NO:14 respectively for use in the PCR that kills and wounds toxin leader sequence, IL-1 β aminoterminal 24AA and endopeptidase KEX2 cleavage site.
As template, in every kind of primer of 50pmol and 100 μ l contain the PCR reaction soln of 200 μ MdNTP, add 2 Vent of unit archaeal dna polymerases with YEp2-k, make reaction by following program loop 35 times then:
Pre-treatment: 94 ℃, 300 seconds;
Annealing: 50 ℃, 30 seconds;
Extend: 72 ℃, 30 seconds;
Sex change: 94 ℃, 30 seconds;
Afterreaction: 53 ℃, 300 seconds
With amplification kill and wound toxin leader sequence, IL-1 β aminoterminal 24AA electrophoresis in 1.5% agarose gel plate, the DNA band of about 80bp is scaled off wash-out, and carry out purifying from gel.The dna sequence dna that PCR increased is shown in SEQ ID NO:15.
2) utilize above 1) in the PCR product described and with GCSF carboxyl terminal complementary oligonucleotide through the PCR GCSF that increases.
Synthetic following and the carboxyl terminal complementation of GCSF and oligonucleotide with sequence shown in SEQ ID NO:16.
The YEp2KGC that will be obtained by embodiment 21 is as template.Kill and wound to every kind of primer of 50pmol, coding and to add 2 Vent of unit archaeal dna polymerases in the dna fragmentation of toxin leader sequence and IL-1 β 24A and the reaction soln that 100 μ l contain 20 μ M dNTP, make reaction according to following temperature program(me) circulation 35 times then:
Pre-treatment: 94 ℃, 300 seconds;
Annealing: 50 ℃, 30 seconds;
Extend: 72 ℃, 30 seconds;
Sex change: 94 ℃, 30 seconds;
Afterreaction: 53 ℃, 300 seconds
With the IL20GCSF electrophoresis in 1% agarose gel plate that increased, the DNA that downcuts about 0.66kb from gel is with, wash-out, and carry out purifying.With SacI, BamHI digestion IL20GCSF, and purifying.
Embodiment 26 digestion YEp2-k
With SacI and BamHI 1 μ g YEp2-k was digested 1 hour in 37 ℃.With the plasmid electrophoresis in 1% sepharose that digested, the DNA of about 12kb band is scaled off, and use the Jetsorb eluted dna.
The connection of embodiment 27DNA and conversion
Use T 4The YEp2-k that dna ligase digested with SacI and BamHI in 30 μ l connect in the solution IL20GCSF and embodiment 26 links together.
With reaction soln transformed into escherichia coli XL-1 Blue, cultivate bacterium colony and with plasmid purification.Use the restriction enzyme digested plasmid, only select the plasmid that contains IL20GCSF and it called after YEp2kIL20GC.
V. prepare pIL20GC
Embodiment 28 digestion YEp2kIL20GC
In order from YEp2kIL20GC, to obtain the IL20GCSF-GAPDH transcription terminator, this plasmid was digested 1 hour in 37 ℃ with KpnI and SalI.Isolate the good plasmid of digestion by method same as described above, and with the DNA purifying of about 0.66kb size.
Embodiment 29 digestion YEpGalMF
With KpnI and SalI 1 μ g YEpGalMF was digested 1 hour in 37 ℃.By separating the plasmid that had been digested with purifying with top identical method.
The connection of embodiment 30DNA and conversion
Use T 4Dna ligase connects in the solution and will link together with IL20GCSF-GAPDH transcription terminator and the YEpGalMF that KpnI and SalI digested at 30 μ l.Use the reaction soln transformed into escherichia coli.Cultivate bacterium colony and plasmid purification.Use the restriction enzyme digested plasmid, pick out the plasmid that inserted the IL20GCSF-GAPDH transcription terminator and it called after pIL20GC.
VI. prepare YEpHSPGC
HSP150 (heat shock protein) is a kind of by yeast excretory glycoprotein under 37 ℃ to 42 ℃ culture condition.Utilize this characteristic of HSP150, can regulate and control the expression of hGCSF at an easy rate.At first clone HSP150 promotor and secretion signal, prepare YEpHSPGC by inserting the hGCSF gene then.
The PCR of embodiment 31HSP150 promotor and leader sequence
The synthetic primer that has sequence shown in SEQ ID NO:17 and SEQ ID NO:18 respectively is used for the PCR of HSP150 promotor and leader sequence.
Adopt this primer by genes of brewing yeast group DNA through PCR increase HSP150 promotor and the leader sequence of about 0.4kb.Behind the purifying, digest this plasmid with KpnI and SalI.
Embodiment 32 preparation YEpHSPGC
Replace MF (mating factor) α 1 promotor among the YEp2kGC and kill and wound the toxin leader sequence with HSP150 promotor and leader sequence, and with gained plasmid called after YEpHSPGC.
VII. utilize pIL20GC and YEp2kIL20GC to express hGCSF
Embodiment 33 zymic transform
In order in yeast, to express hGCSF, with pIL20GC and YEp2kIL20GC transformed yeast.
With yeast saccharomyces cerevisiae 2805 (a, pep4 ∷ HIS3, pro1-, can1, GAL1, his3 ura3-52) is inoculated in the YEPD substratum (1% yeast extract, 2% peptone, 2% glucose) of 3ml, in 30 ℃, 250rpm incubated overnight.Cultured cells inoculates in the 15ml YEPD substratum.Work as OD 600Be about at 1.0 o'clock, culture is centrifugal, and the explanation according to alkaline kation-yeast conversion test kit (Bio101) prepares the competence yeast then.With TE damping fluid washing yeast sedimentation, precipitation is suspended in the Lithium Acetate solution, and in 30 ℃, 120rpm cultivation.Behind the centrifugal suspension, precipitation is suspended in the TE damping fluid, and with transform plasmid, carrier DNA and histamine mixing in little plastic centrifuge tube.Mixing solutions after room temperature was placed 15 minutes, to wherein adding PEG, and is placed gained solution 10 minutes in 30 ℃.Reaction solution is handled in 42 ℃ of heat shocks of carrying out 5 minutes, centrifugal, be suspended in then in the 200 μ l SOS substratum.Aaerosol solution is layered on the SD Agar Plating (the complete fill-in substratum-URA of 0.8g/l (Bio101), 6.7g/l yeast do not have amino acid nitrogen basis (DIFCO), 2% glucose, 1.5% agar),, picks out URA in 30 ℃ of cultivations 3 days +Bacterium colony.The yeast called after yeast saccharomyces cerevisiae GCl that will have been transformed by pIL20GC, the yeast called after yeast saccharomyces cerevisiae k2GC that has been transformed by YEp2kIL20GC, two bacterial strains all are deposited in Korea S typical case culture collection center (KRIBB, Taejon, Korea) (preserving number is respectively KCTC0193BP and KCTC0195BP) September 27 nineteen ninety-five.
The expression of embodiment 34GCSF
With colony inoculation in the SD substratum, and in 30 ℃, 250rpm incubated overnight.Behind medium centrifugal, precipitation is suspended in the 1ml YEPGal substratum (1% yeast extract, 2% peptone, 2% semi-lactosi), cultivates 15 hours so that express hGCSF in 30 ℃, 250rpm.Culture is centrifugal, get 0.5ml supernatant liquor and 10 μ g/ml BSA and 10%TCA mixing, put 20 minutes on ice.Then it was precipitated hGCSF in centrifugal 10 minutes in 4 ℃, 13000rpm.
With resolution of precipitate in the solution that contains 20 μ l distilled water and 20 μ l, 2 * SDS dyestuff (125mMTris-HCl (pH6.8), 4%SDS, 20% glycerine, 10%2-mercaptoethanol), this solution 16%SDS (sodium laurylsulfonate)-PAGE gel (Laemmli, nature 227:680-684) electrophoretic analysis is dyeed glue with Coomassie blue.The result shows the hGCSF band of 18.7kDa.
Embodiment 35 fermentation culture are produced hGCSF
A) bacterial strain and substratum
In the zymic fed batch cultivation thing that has been transformed by recombinant plasmid pIL20GC, express and produce hGCSF.Seed culture medium contains 20g glucose, 6.7g YNB (yeast does not have the amino acid nitrogen basis) and 0.8g CSM-Ura (no uridylic replenishes mixture fully) for every liter.Be used for as follows with the nutrient media components of fed batch cultivation in batches:
(1) batch culture (every liter)
a) KH 2PO 4 10g
(NH 4) 2SO 4 2g
CaCl 22H 2O 0.5g
NaCl 0.5g
Trace metal solution 10ml
Vitamin solution 1ml
Casamino acids 5g
Tween 80 0.6g
b) MgSO 4·7H 2O 0.5g
C) glucose 10g
Component a), b) and c) respectively 121 ℃ the heating 15 minutes autoclavings.
(2) fed batch cultivation
1) vegetative period (every liter)
a) (NH 4) 2SO 4 3g
KH 2PO 4 5g
Vitamin solution 3.5ml
Trace metal solution 5ml
The casamino acids variable
Tween 80 0.6g
b) MgSO 4·7H 2O 4g
C) glucose variable
The concentration ratio of sequence and casamino acids is 0.5 to 4.5 in the vegetative period substratum.
2) induce or produce yeast phase (every liter)
a) (NH 4) 2SO 4 3g
KH 2PO 4 5g
Vitamin solution 3.5ml
Trace metal solution 5ml
The yeast extract variable
Tween 80 0.6g
b) MgSO 4·7H 2O 4g
C) semi-lactosi variable
The concentration ratio of glucose and yeast extract is 0.5 to 3 in the inductive phase substratum.
Component a), b) and c) respectively 121 ℃ the heating 15 minutes autoclavings.
Every liter of trace metal solution comprises: 2.78g FeSO 4, 1.36g ZnCl 22H 2O, 0.8gCuSO 45H 2O, 2.42g Na 2MoO 42H 2O, 2.38g CoCl 26H 2O and 1.69gMnSO 4
Every 100ml vitamin solution comprises: 0.6g inositol, 0.12g calcium pantothenate, 0.12g pyridoxine hydrochloride, 0.12g VitB1 and 0.01g vitamin H.
B) cultivate and produce hGCSF
Recombination yeast is cultivated on the Agar Plating identical with the seed culture medium component, afterwards, be suspended in bacterium colony in 15% glycerine solution and be stored in-70 ℃.When cultivating, the recombination yeast that is stored in-70 ℃ is layered on the above-mentioned Agar Plating, and cultivated 48 hours in 30 ℃.Then with colony inoculation to shaking in bottle (250ml) seed culture medium, cultivate in 30 ℃, 250rpm.After 24 hours, inoculum is inoculated in the batch culture base with the highest 5% inoculum size, (30 ℃, pH5.5) cultivates in the 5L fermentor tank.When the glucose in the substratum exhausts, add the substratum in vegetative period that is used for fed batch cultivation by the pump that links to each other with the fermentor tank control cabinet.Control medium feed speed so that the concentration of glucose is remained on below the 100mg/l in order to following design formula.
μ = μ 0 ( S ) ( 1 - X X m )
F i = μ X i V i S 0 Y x / s
V i+1＝V i+F i(Δt)
X i+1V i+1＝X iV iexp(μΔt)
S 0: the glucose concn in the supplemented medium (g/L)
X: the yeast concn in the nutrient solution (g/L)
X m: cellular biomass suppresses constant (g/L)
μ: specific growth rate (hr -1)
V: volume of culture (L)
F: volume feeding rate (L/hr)
Δ t:30 second
In vegetative period, when zymic concentration reaches 25 to 35g/L, supplemented medium should be changed into substratum inductive phase.The control volume feeding rate is so that make galactose concentration remain on 10 to 35g/L.Jolt speed and airflow rate remains on more than 40% gas saturation oxyty in the fermented liquid by control.From fermentor tank, gather culture sample and be used for analysis of cells density, alcohol concn, hGCSF concentration and plasmid stability.As a result, the hGCSF concentration that produces in fermented liquid reaches 230mg/L, and alcohol accumulation is suppressed, and plasmid stability maintains more than 90%.
VIII. express hGCSF with YEpHSPGC
Embodiment 36 transformed yeasts and expression GCSF
Come transformed saccharomyces cerevisiae 2805 with YEpHSPGC, pick out Ura by means of alkalescence-positively charged ion yeast conversion test kit (Bio101) +Transformant.The yeast called after yeast saccharomyces cerevisiae HGCA that has been transformed by YEpHSPGC, and be deposited in Korea S's typical case culture collection center (KRIBB, Korea S, land for growing field crops) (preserving number: KCTC0194BP) September 27 nineteen ninety-five.The yeast colony that will grow on the SD of no uridylic substratum is inoculated in the 3ml liquid nutrient medium, and in 36 ℃, 250rpm incubation.The throw out that centrifugal back deposit was suspended in the 1mlYEP Gal substratum (1% yeast extract, 2% peptone, 2% semi-lactosi), in 37 ℃, 250rpm cultivation 18 hours.Behind medium centrifugal, in precipitation and supernatant liquor, respectively add the SDS dyestuff, and through the 16%SDS-PAGE analysing protein.In supernatant liquor, do not show derivative recombinant protein band, but in the precipitation part, the new main band of a 20.1kDa size occurred.With hGCSF Ab (R ﹠amp; D System) and anti-mouse IgG-alkaline phosphatase do the Western trace, prove that the protein of this 20kDa size has immunoreactivity.
The expression of IX.hGCSF
Embodiment 37 preparation pIL20XGC
For the ease of the clone, in pIL20GC, insert the XbaI site.
1)PCR
At first, with the synthetic oligonucleotide that has shown in SEQID NO:19 sequence and comprise the XbaI site of synthesizer (ABI, 392DNA/RNA synthesizer).
The primer that has sequence shown in SEQ ID NO:20 with above-mentioned primer and following and mating factor α complementary is PCR.
With pIL20GC as template.Contain to 100 μ l and to add 2 Vent of unit archaeal dna polymerases in the PCR reaction soln of 200 μ M dNTP and every kind of primer of 50pmol, make reaction according to following condition circulation 35 times then:
Pre-treatment: 90 ℃, 60 seconds;
Annealing: 45 ℃, 5 seconds;
Extend: 72 ℃, 15 seconds;
Sex change: 94 ℃, 5 seconds;
Afterreaction: 53 ℃, 30 seconds
Kill and wound toxin leader sequence-IL-1 β-24-AA dna fragmentation with 1.5% sepharose through what electrophoretic separation went out amplification, the DNA band of 80bp is eluted and carry out purifying.The DNA that is obtained by PCR has the base sequence shown in SEQ ID NO:21.
When inserting the XbaI site by PCR, aminoacid sequence does not change, and has only the replacement of base sequence level.
2) with as above synthetic PCR product and with hGCSF gene C-end complementary oligonucleotide by PCR acquisition hGCSF gene.
Has the sequence shown in SEQ ID NO:22 with hGCSF gene C-end complementary oligonucleotide.
With pIL20GC as template.In 100 μ l contain the reaction soln of the dna fragmentation that kills and wounds toxin leader sequence-IL-1 β-24AA-XbaI-KEX2 of 50pmol primer, amplification and 20 μ M dNTP, add 2 Vent of unit archaeal dna polymerases, make reaction according to following condition circulation 35 times then:
Pre-treatment: 95 ℃, 60 seconds;
Annealing: 55 ℃, 5 seconds;
Extend: 72 ℃, 15 seconds;
Sex change: 94 ℃, 7 seconds;
Afterreaction: 53 ℃, 30 seconds
Going out amplified production with 1% sepharose through electrophoretic separation (kills and wounds toxin leader sequence-IL-1 β N and holds (24AA)-XbaI-KEX2-hGCSF), the DNA of 0.66kb size band under the wash-out from the gel, with restriction enzyme SacI and BamHI digestion, carry out purifying at last.1 μ g pIL20GC was digested 1 hour in 37 ℃ with SacI and BamHI.Plasmid electrophoresis in 1% sepharose with digesting elutes DNA with Jetsorb.Add T 4Dna ligase reacts the PCR product and the plasmid pIL20GC that digested through SacI and BamHI in 30 μ l ligation solution.With reaction mixture transformed into escherichia coli XL-1 Blue.Cultivate bacterium colony, plasmid purification then.Pick out the plasmid and the called after pIL20XGC that can be limited enzyme XbaI digestion.
Embodiment 38 zymic transform
In order in yeast, to express hGCSF, use the pIL20XGC transformed yeast.With yeast saccharomyces cerevisiae 2805 (a, pep4 ∷ HI53, pro1-δ, can1, GAL1, his3 δ ura3-52) is inoculated in the 3mlYEPD substratum, in 30 ℃, 250rpm incubated overnight.It is inoculated in the 15ml YEPD substratum again.Work as OD 600Be about at 1 o'clock, culture is centrifugal.Explanation according to alkaline kation-yeast conversion test kit (Bio101) prepares the competence yeast then.Wash yeast sedimentation with the TE damping fluid, the gained yeast sedimentation is suspended in the Lithium Acetate solution, and in 30 ℃, 120rpm vibration.Behind the centrifugal suspension, precipitation is suspended in the TE damping fluid, and adding contains in the little plastic centrifuge tube that transforms plasmid, carrier DNA and histamine.After room temperature is placed 15 minutes, in this solution, add PEG solution, and the solution that obtains was placed 10 minutes in 30 ℃.With mixed solution in 42 ℃ of heating 5 minutes and centrifugal after, precipitation is suspended in the 200 μ l SOS substratum, be layered on subsequently on the SD Agar Plating, cultivated 3 days in 30 ℃.Pick out URA at last +Bacterium colony.
This yeast strain has been deposited in Korea S typical case culture collection center (KRIBB, Taejon, Korea) (preserving number is KCTC0330BP) on May 9th, 1999.
Embodiment 39 expresses hGCSF
With colony inoculation in 3ml SD substratum, and in 30 ℃, 250rpm incubated overnight.Behind the centrifugation medium, precipitation is suspended in the 1ml YEPGal substratum, cultivated 15 hours, last abduction delivering hGCSF in 30 ℃, 250rpm.2 * SDS the dyestuff (125mM Tris-HCl (pH6.8), 4%SDS, 20% glycerine, 10%2-mercaptoethanol) that in nutrient solution, adds equivalent, and with solution heating 5 minutes.In 15%SDS-PAGE, isolate protein (Laemmli, natural 227:680-684) and use Coomassie blue stain.The result shows that the hGCSF expression level is identical with the situation of using pIL20GC.
X. the expressed hGCSF protein of purifying
Embodiment 40 ammonium sulphate precipitations
Yeast cell culture after 10,000 * g is centrifugal 10 minutes, is used (NH 4) 2SO 4Make the supernatant liquor saturation ratio reach 85% and placed 24 hours in 4 ℃.After 10,000 * g is centrifugal 30 minutes, the precipitation that obtains is dissolved in 50mMTris (pH7.8) damping fluid that contains 0.1mM EDTA and 1mM DTT, removed insolubles in centrifugal 10 minutes in 10,000 * g.All above-mentioned experiments are all carried out at 4 ℃.Further come the hGCSF of purifying by above operation acquisition by gel permeation chromatography.
Embodiment 41 gel permeation chromatographies
With 50mM Tris (pH7.8) the damping fluid detergent gel permeation chromatography medium Sephacryl-S-200 (Pharmacia) that contains 1mM DTT and 0.1mM EDTA, and with described medium dress post (1.6 * 100cm).By above-mentioned (NH 4) 2SO 4Precipitation process concentrates the protein in the 20ml nutrient solution, and with sample on the concentrated solution in pillar, with containing 50mM Tris (pH7.8) buffer solution elution of 1mM DTT and 0.1mMEDTA.Concentrate protein in each peak (at the peak that 280nm photoabsorption place produces) by freeze-drying.According to the SDS-PAGE analytical results of each peak sample, contain hGCSF and some other protein in first peak sample, contain nutrient media components peptide/protein (Figure 15) in second peak sample.Most substratum peptide/protein are removed by the Sephacryl-S-200 gel permeation chromatography.Will be through gel permeation chromatography and partially purified hGCSF sample carries out following C4 reversed-phase HPLC again and handles.
Embodiment 42 C4 reversed-phase HPLCs
To do final purification through the partially purified hGCSF sample of gel permeation chromatography with reversed-phase HPLC C4 post.The C4 pillar is the product of Vydac company, and column volume is 1.0 * 25cm.Flow velocity is 2ml/min.After injecting about 100 μ g media, use 0.1%TFA/ water elution pillar 5 minutes, use the 0.1%TFA/ acetonitrile of 0 to 100% linear gradient.HGCSF under 90%0.1%TFA/ acetonitrile gradient wash-out.Confirm hGCSF purifying (Figure 16) fully through SDS-PAGE.In purification process described in the embodiment 44-46, the hGCSF rate of recovery is 8%, obtains the pure hGCSF of about 18mg from the 1L nutrient solution.
XI. analyze the n terminal amino acid of purifying hGCSF
The n terminal amino acid analysis of embodiment 43 pure hGCSF
To be transferred on the pvdf membrane through the isolating hGCSF of C4 reversed-phase HPLC, and measure its n terminal amino acid sequence.
N terminal amino acid sequence (the NH of this n terminal amino acid sequence and ripe hGCSF 2-Thr-Pro-leucine-glycine-proline(Pro)-COOH) conform to.This analysis is to carry out under the technical assistance at Korea S basic science center.Analyzing used protein sequencer is Milligen6600B, analyzes the PTH-amino acid derivative of Edman edman degradation Edman preparation with HPLC.
Mobile phase A: 35mM ammonium acetate buffer (pH4.8)
Mobile phase B: 100% acetonitrile
Temperature: 50 ℃
Time (minute) Flow velocity (ml/min) ％A ％B Curve
Initial 0.7 95 5 *
0.7 0.7 75 25 6
1.4 0.7 73 27 6
2.8 0.7 73 27 6
5.7 0.7 55 45 6
7.4 0.7 55 45 6
8.1 0.7 52 48 6
12.0 0.7 30 70 6
20.0 0.7 95 5 6
Aminoacid sequence is also determined with the polymkeric substance coupling method.With 5ml solution A point on each limit of lamina membranacea (PVDF), with lamina membranacea drying 15 to 20 seconds.Lamina membranacea is placed on 55 ℃ the hot-plate, gets 30ml solution B point thereon, dry 7 minutes (drying is not above 10 minutes).With 5ml solution C point on each limit of lamina membranacea, dry 15 to 20 seconds.Lamina membranacea is placed on 55 ℃ of hot-plates, gets 30ml solution D point in the above, dry 5 minutes.Go up the 20ml solution B again, dry 5 minutes, with ethanol, water and washed with methanol lamina membranacea.
A) PITC solution (10nmol/ μ l is dissolved in ethyl acetate)
B) (2% triethylamine is dissolved in 50% methyl alcohol to damping fluid, v/v)
C) DITC solution (0.1%w/v is dissolved in ethyl acetate)
D) polymers soln (0.1%w/v, the many arylamine of hydrochloric acid (lower molecular weight) are dissolved in B solution)
Amino acid analysis result according to being obtained by above method detects Threonine in the first round, and second takes turns and detect proline(Pro), and third round detects leucine, and four-wheel detects glycine, and the 5th takes turns and detect proline(Pro).Therefore, the n terminal amino acid sequence of the hGCSF of the present invention's preparation is NH 2-Thr-Pro-leucine-glycine-proline(Pro), the n terminal amino acid sequence of it and real people hGCSF is coincide.
XII. express hGH with pIL20XGH
The PCR of embodiment 44 human growth hormones (hGH)
In order to be the PCR of hGH, synthetic have respectively as SEQ ID NO:23 and shown in SEQ IDNO:24 sequence and hold the complementary oligonucleotide with the N-end and the C-of ripe human growth hormone.
The reaction soln from 200 μ M dNTP to 100 μ l (10mMKCl, the 10mM (NH that contain each primer of 50pmol and 4) 2SO 4, 20mM Tris-HCl (pH8.8), 2mM MgSO 4, 0.1%TritonX-100) in add 2 Vent of unit archaeal dna polymerases, personnel selection hypophysis cDNA library is as template, makes PCR according to following condition circulation 35 times:
Pre-treatment: 94 ℃, 60 seconds;
Annealing: 60 ℃, 5 seconds;
Extend: 72 ℃, 10 seconds;
Sex change: 94 ℃, 7 seconds;
Afterreaction: 53 ℃, 30 seconds
With the DNA zone purification of about 0.6kb of manifesting in 1% sepharose, and with restriction enzyme XbaI and BamHI digestion.With restriction enzyme XbaI and BamHI with 1 μ g pIL20XGC in 37 ℃ of digestion 1 hour, in 1% sepharose, separate.Remove the hGCSF fragment then, select remaining carrier part and elute.Use T 4Dna ligase links together the hGH gene in 30 μ l ligation liquid with the pIL20XGC that digested with XbaI and BamHI.Behind reaction mixture transformed into escherichia coli XL-1 Blue, cultivate bacterium colony, plasmid purification then.With restriction enzyme XbaI and BamHI digested plasmid, choose the plasmid and the called after pIL20XGH that contain the hGH gene.
Embodiment 45 transformed yeasts
In order in yeast, to express hGH, use the pIL20XGH transformed yeast.With yeast saccharomyces cerevisiae 2805 (a, pep4 ∷ HIS3, pro1-δ, can1, GAL1, his3 δ ura3-52) is inoculated in the 3ml YEPD substratum, in 30 ℃, 250rpm overnight incubation.Nutrient solution is inoculated in the 15ml YEPD substratum, work as OD 600Be about at 1 o'clock, culture is centrifugal, and the operation instruction according to alkaline kation-yeast conversion test kit (Bio101) prepares the competence yeast then.Wash yeast sedimentation with the TE damping fluid, the gained precipitation is suspended in the Lithium Acetate solution, and in 30 ℃, 120rpm vibration.Centrifugal suspension is suspended in precipitation in the TE damping fluid, and joins and contain in the little plastic centrifuge tube that transforms plasmid, carrier DNA and histamine.Mixing solutions after room temperature is placed 15 minutes, is added PEG in mixed solution, and solution was placed 10 minutes in 30 ℃.Above-mentioned solution is handled in 42 ℃ of heat shocks of carrying out 5 minutes, be suspended in then in the 200 μ l SOS substratum, be layered on the SD Agar Plating, and, pick out URA at last in 30 ℃ of cultivations 3 days +Bacterium colony.
This saccharomycete has been deposited in Korea S typical case culture collection center (KRIBB, Taejon, Korea) (preserving number is KCTC0331BP) on May 9th, 1997.
Embodiment 46 expresses hGH
With colony inoculation in the SD substratum, and in 30 ℃, 250rpm incubated overnight.Centrifugation medium is suspended to precipitation in the 1ml YEPGal substratum, cultivates 15 hours so that induce the expression of hGH in 30 ℃, 250rpm.In nutrient solution, add 2 * SDS dyestuff (125mMTris-HCl (pH6.8), 4%SDS, 20% glycerine, 10%2-mercaptoethanol), and be heated 5 minutes, through 16%SDS-PAGE gel (Laemmli, natural 227:680-684) electrophoresis.With Coomassie blue glue is dyeed, detect the hGH band that is equivalent to about 22kDa size.
Embodiment 47 fermentation culture are produced hGH
(a) bacterial strain and substratum
Produce hGH with the yeast that has been transformed by recombinant plasmid pIL20XGH through fed batch cultivation.Used identical in the composition of seed culture medium and the hGCSF fermentation.Be used for as follows with the medium component of fed batch cultivation in batches:
(1) batch culture (every liter)
Identical with embodiment 35
(2) fed batch cultivation
1) vegetative period (every liter)
a) (NH 4) 2SO 4 3g
KH 2PO 4 5g
Vitamin solution 3.5ml
Trace metal solution 5ml
Casamino acids 136g
Tween 80 0.6g
b) MgSO 4·7H 2O 4g
C) glucose 409g
2) inductive phase or fermentative production phase (every liter)
a) (NH 4) 2SO 4 3g
KH 2PO 4 5g
Vitamin solution 3.5ml
Trace metal solution 5ml
Yeast extract 167g
Tween 80 0.6g
b) MgSO 4·7H 2O 4g
C) semi-lactosi 333g
Component a), b) and c) respectively 121 ℃ the heating 15 minutes autoclavings.
Identical among the composition of trace metal solution and vitamin solution and the embodiment 35.
B) cultivate and produce hGH
In vegetative period, when zymic concentration reaches 25 when the 35g/L, supplemented medium should be changed into above-mentioned inductive phase of substratum.The feeding rate of control substratum is so that make the galactose concentration in the nutrient solution remain on about 18g/L.Unless otherwise indicated, the preservation of culture is identical with embodiment 35 with cultural method.As a result, the ethanol accumulation of denier is only arranged, plasmid stability is more than 80%, and the hGH concentration in the nutrient solution is increased to 1330mg/L.
Sequence table
(1) physical data:
(i) applicant: HANIL SYNTHETIC FIBER CO., LTD.
JANG.Ki-Ryong
MOON，Jae-Woong
BAE，Cheon-Soon
YANG，Doo-Suk
LEE，Jee-Won
SEONG，Baik-Lin
(ii) denomination of invention:
Be used to home-brewed wine zymic efficient expression vector and prepare the method for recombinant protein
(iii) sequence number: 24
(2) data of SEQ ID NO:1:
(i) sequence signature:
(A) length: 64 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:1:
CTATAA AACAATGAAC ATCTTCTACA TCTTCTTGTT CTTGTTGTCT TTCGTTCAAG 60
TCGAGATATT TTGTTACTTG TAGAAGATGT AGAAGAACAA GAACAACAGA AAGCAAGTTC
GTAC 64
(2) data of SEQ ID NO:2:
(i) sequence signature:
(A) length: 17 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:2:
GTTTTCCCAG TCACTAC 17
(2) data of SEQ ID NO:3:
(i) sequence signature:
(A) length: 81 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:3:
GAGCTCTATA AAACAATGAA CATCTTCTAC ATCTTCTTGT TCTTGTTGTC TTTCGTTCAA 60
GGTACCCGGG GATCACTGAA C 81
(2) data of SEQ ID NO:4:
(i) sequence signature:
(A) length: individual base pair
(B) type: nucleic acid
(C) chain: two strands
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:4:
GATCCCGGGT TTTTTATAGC TTTATGACTT AGTTTCAATT ATATACTATT TTAATGACAT 60
GGCCCA AAAAATATCG AAATACTGAA TCAAAGTTAA TATATGATAA AATTACTGTA
TTTCAGG 71
AAAGTCCAGC T
(2) data of SEQ ID NO:5:
(i) sequence signature:
(A) length: 35 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:5:
GTGCACTCGA GCCAAAAAGC AACAACAGGT TTTGG 35
(2) data of SEQ ID NO:6:
(i) sequence signature:
(A) length: 36 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:6:
TTAATGAGCT CTATTGTGTA TGAAATTGAT AGTTTG 36
(2) data of SEQ ID NO:7:
(i) sequence signature:
(A) length: 36 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:7:
AGTTTGAATT CCAACAGCAA GCAGGTGTGC AAGACA 36
(2) data of SEQ ID NO:8:
(i) sequence signature:
(A) length: 36 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:8:
TCGAAGAATT CTCACCTTCG TGAACTTCAG AGGCGA 36
(2) data of SEQ ID NO:9:
(i) sequence signature:
(A) length: 30 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:9:
AGGTAGGGTA CCACCCCCCT GGGCCCTGGCC 30
(2) data of SEQ ID NO:10:
(i) sequence signature:
(A) length: 33 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:10:
ATGGGAGGAT CCGGGCTTGG CTCAGGGCTG GGC 33
(2) data of SEQ ID NO:11:
(i) sequence signature:
(A) length: 36 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:11:
TTAATGAGCT CTATTGTGTA TGAAATTGAT AGTTTG 36
(2) data of SEQ ID NO:12:
(i) sequence signature:
(A) length: 36 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:12:
AGTTTGAATT CCAACAGCAA GCAGGTGTGC AAGACA 36
(2) data of SEQ ID NO:13:
(i) sequence signature:
(A) length: 21 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:13:
ACAATAGAGC TCTATAAAAC A 21
(2) data of SEQ ID NO:14:
(i) sequence signature:
(A) length: 57 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:14:
GGCAGGGCCC AGGGGGGTTC TCTTGTCCA AAGAAACAG GTTTCAGTT CATATGG 57
(2) data of SEQ ID NO:15:
(i) sequence signature:
(A) length: 285 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:15:
ACAATAGAGC TCTATAAAAC AATGACCATC TTCTACATCT TCTTGTTCTT GTTGTCTTTC 60
TGTTATCTCG AGATATTTTG TTACTTGTAG AAGATGTAGA AGAACAAGAA CAACAGAAAG
GTTCAAGGTT TGTCACTGAA CTGCACGCTC CGGGACTCAC AGCCAAAAAG CTTGGTGATG 120
CAAGTTCCAA ACAGTGACTT GACGTGCGAG GCCCTGAGTG TCGGTTTTTC GAACCACTAC
TCTGGTCCAT ATGGACTGAA AGCTGGTGTT TCTTTGGACA AGAGAACCCC CCTGGGCCCT 180
AGACCAGGTA TACCTGACTT TCGACCACAA AGAGATCTGT TCTCTTGGGG GGACCCGCGA
GCC 183
CGG
(2) data of SEQ ID NO:16:
(i) sequence signature:
(A) length: 33 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:16:
ATGGGAGGAT CCGGGCTTGG CTCAGGGCTG GGC 33
(2) data of SEQ ID NO:17:
(i) sequence signature:
(A) length: 33 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:17:
CTAGCAGTCG ACGATAAGTC GCCAACTCAG CCT 33
(2) data of SEQ ID NO:18:
(i) sequence signature:
(A) length: 33 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:18:
CTAGCAGGCA CCGGCCAAAG TAGTAGCGGC CAA 33
(2) data of SEQ ID NO:19:
(i) sequence signature:
(A) length: 22 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:19:
TCTCTTGTCT AGAGAAACAG CT 22
(2) data of SEQ ID NO:20:
(i) sequence signature:
(A) length: 21 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:20:
ACAATAGAGC TCTATAAAAC A 21
(2) data of SEQ ID NO:21:
(i) sequence signature:
(A) length: 165 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:21:
ACAATAGAGC TCTATAAAAC AATGAACATC TTCTACATCT TCTTGTTCTT GTTGTCTTTC 60
TGTTATCTCG AGATATTTTG TTACTTGTAG AAGATGTAGA AGAACAAGAA CAACAGAAAG
GTTCAAGGTT TGTCACTGAA CTGCACGCTC CGGGACTCAC AGCCAAAAAG CTTGGTGATG 120
CAAGTTCCAA ACAGTGACTT GACGTGCGAG GCCCTGAGTG TCGGTTTTTC GAACCACTAC
TCTGGTCCAT ATGGACTGAA AGCTGGTGTT TCTCTAGACA AGAGA 165
AGACCAGGTA TACCTGACTT TCGACCACAA AGAGATCTGT TCTCT
(2) data of SEQ ID NO:22:
(i) sequence signature:
(A) length: 33 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:22:
ATGGGAGGAT CCGGGCTTGG CTCAGGGCTG GGC 33
(2) data of SEQ ID NO:23:
(i) sequence signature:
(A) length: 43 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:23:
TGTTTCTCTA GACAAGAGAT TCCCAACCAT TCCCTTATCC AGG 43
(2) data of SEQ ID NO:24:
(i) sequence signature:
(A) length: 40 base pairs
(B) type: nucleic acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: oligonucleotide
(xi) sequence description of SEQ ID NO:24:
ATGCCAGGAT CCCAGCTAGA AGCCACAGCT GCCCTCCACA 40
(2) data of SEQ ID NO:25:
(i) sequence signature:
(A) length: 241 amino acid
(B) type: amino acid
(C) chain: strand
(D) topology: linear
(ii) molecule type: peptide
(xi) sequence description of SEQ ID NO:25:
Met Asn Ile Phe Tyr Ile Phe Leu Phe Leu Leu Ser Phe Val Gln Gly
5 10 15
Thr Arg Gly Ser Leu Asn Cys Thr Leu Arg Asp Ser Gln Gln Lys Ser
20 25 30
Leu Val Met Ser Gly Pro Tyr Glu Leu Lys Ala Gly Val Ser Leu Asp
35 40 45
Lys Arg Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met
50 55 60
Leu Arg Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu
65 70 75 80
Phe Glu Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln
85 90 95
Asn Pro Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser
100 105 110
Asn Arg Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile
115 120 125
Ser Leu Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg
130 135 140
Ser Val Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val
145 150 155 160
Tyr Asp Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly
165 170 175
Arg Leu Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr
180 185 190
Tyr Ser Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys
195 200 205
Asn Tyr Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu
210 215 220
Thr Phe Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly
225 230 235 240
Phe
241

Claims (14)

1. comprise the heterozygosis secretion signal and derive from the expression vector of zymic hybrid promoter, wherein said heterozygosis secretion signal comprise coding il-1 β N-terminal 24 amino acid whose DNA and derive from zymic and kill and wound the toxin secretion signal; The described zymic hybrid promoter that derives from comprises GAL1-10UAS and mating factor α-1 promotor.
2. the expression vector of claim 1, the wherein said toxin secretion signal that kills and wounds has obtained optimization according to yeast codon use habit.
3. the expression vector of claim 1, this carrier also comprises transcription terminator and GAL4 gene.
4. the expression vector of claim 3, wherein said transcription terminator is the transcription terminator of GAPDH.
5. the expression vector of claim 1, it is expression vector YEp2kIL20GC, this carrier comprises according to 5 ' → 3 ' direction comprise the dna sequence dna of following sequence successively:
A) UAS of GAL1 and GAL10 gene,
B) MF α-1 promotor,
C) kill and wound the toxin leader sequence,
D) dna sequence dna of 24 amino-acid residues of the N-terminal of coding IL-1 β,
E) KEX2 cleavage site,
F) GCSF gene,
G) GAPDH transcription terminator,
H) GAL4 gene.
6. the expression vector of claim 1, it is expression vector pIL20GC, and it prepares by insert the section of DNA sequence in cloning vector YEp352, and this dna sequence dna comprises successively by 5 ' → 3 ' direction:
A) UAS of GAL1 and GAL10 gene,
B) MF α-1 promotor,
C) kill and wound the toxin leader sequence,
D) dna sequence dna of 24 amino-acid residues of the N-terminal of coding IL-1 β,
E) KEX2 cleavage site,
F) GCSF gene,
G) GAPDH transcription terminator,
H) GAL4 gene.
7. the expression vector of claim 1, it is expression vector pIL20XGC, and it prepares by insert the section of DNA sequence in cloning vector YEp352, and this dna sequence dna comprises successively by 5 ' → 3 ' direction:
A) UAS of GAL1 and GAL10 gene,
B) MF α-1 promotor,
C) kill and wound the toxin leader sequence,
D) dna sequence dna of 24 amino-acid residues of the N-terminal of coding IL-1 β,
E) KEX2 cleavage site,
F) XbaI site
G) GCSF gene,
H) GAPDH transcription terminator,
I) GAL4 gene.
8. the expression vector of claim 1, it is expression vector pIL20XGH, and it prepares by insert the section of DNA sequence in cloning vector YEp352, and this dna sequence dna comprises successively by 5 ' → 3 ' direction:
A) UAS of GAL1 and GAL10 gene,
B) MF α-1 promotor,
C) kill and wound the toxin leader sequence,
D) dna sequence dna of 24 amino-acid residues of the N-terminal of coding IL-1 β,
E) KEX2 cleavage site,
F) XbaI site
G) human growth hormone gene,
H) GAPDH transcription terminator,
I) GAL4 gene.
9. with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the transformant K2GC of YEp2kIL20GC conversion, its preserving number is KCTC0195BP.
10. with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the transformant GC1 of pIL20GC conversion, its preserving number is KCTC0193BP.
11. with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the transformant XGC that pIL20XGC transforms, its preserving number is KCTC0330BP.
12. prepare the method for GCSF by cultivation claim 9,10 or 11 described transformants.
13. with yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) the transformant XGH that pIL20XGH transforms, its preserving number is KCTC0331BP.
14. prepare the method for human growth hormone by the described transformant of cultivation claim 13.
CNB97182214XA 1997-05-27 1997-05-27 Process for preparing recombinant proteins using highly efficient expression vector from accharomyces cerevisiae Expired - Fee Related CN1149291C (en)

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