WO1998035049A1 - Disulfure-isomerase de proteine de levure recombinee et son procede de preparation - Google Patents
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/8215—Microorganisms
- Y10S435/911—Microorganisms using fungi
- Y10S435/94—Saccharomyces
- Y10S435/942—Saccharomyces cerevisiae
Definitions
- the present invention relates to a yeast-derived enzyme, a recombinant protein of protein disulfide isomerase (protein didisulfide, diisomersomese) (hereinafter referred to as “PDI”), and a method for producing the same.
- PDI protein didisulfide, diisomersomese
- the present invention provides a recombinant yeast PD I by modifying the coding region of the C-terminal endoplasmic reticulum localization signal in the yeast PDI gene and expressing the gene in an expression vector.
- the enzyme is secreted outside the cells while maintaining sufficient enzyme activity, thereby enabling mass production.
- the production method of the present invention further comprises obtaining a yeast PD I recombinant protein under a culture condition in which the pH is near neutral by using a host cell that can be cultured even under a condition in which the pH is near neutral. It is. Background art
- PDI is an enzyme that catalyzes disulfide exchange in proteins (EC 5.3.3.4).
- the PDI protein is localized in the lumen of the eukaryotic endoplasmic reticulum and has an activity of catalyzing the disulfide bond binding of a secreted protein translated by the membrane-bound ribosome and sent to the endoplasmic reticulum.
- PDI a useful enzyme that can be applied to improve the properties of protein-containing foods such as dough, ham and sausage, seafood paste products, and tofu.
- the PDI protein from L. cerevisiae has a molecular weight of about 70,000 and an isoelectric point of about 4.0—4.1, with an optimal pH of about 8.5-8.75. (J. Biochem., 108, 846 (1990), JP-A-4-197176).
- yeast PDI is an enzyme protein present in the cells.To obtain the enzyme, after culturing the cells, the cells are disrupted, extracted from the cell disruption solution, crudely extracted, and collected. It had to go through a complicated process of obtaining. This cumbersome extraction process simply adds to the cost problem.
- yeast PDI is an enzyme having an unstable property that is easily deactivated by heat or heavy metal ions, etc., and thus, accompanying the complexity of the extraction process, it is necessary to maintain the activity throughout the entire process. Careful and complicated operations are required.
- a means for secreting and producing the enzyme which is a substance in the cell, out of the cell is effective. That is, if cells can be continuously produced in a culture solution outside the cells from the cells once cultivated, the number of times of culturing the cells is reduced, and the step of crushing the cells becomes unnecessary.
- purification from a culture solution containing few impurities simplifies the purification process, reduces the effect on activity, and enables efficient mass production.
- yeast PDI retains an extracellular secretory signal at the time of its precursor, and is produced in the endoplasmic reticulum, the origin of the secretory pathway. However, even if an attempt is made to express yeast PDI so that it is secreted extracellularly, it is not secreted into cells in the wild-type (M. Lamantiia et al, Cell, 74, 899, 1993). This is presumed to be due to the retention of the C-terminal ER localization signal (eg, in the case of the yeast Saccharomyces cerevisiae, the amino acid sequence is HDEL). In addition, yeast—there is only one PDI gene per cell—the amount of PDI produced per cell is extremely small and cannot be obtained at a sufficient concentration even if secreted into the culture solution.
- yeast PDI activity is unstable in the acidic region, the optimal pH of normal yeast is in the weakly acidic region, and PDI is inactivated in this weakly acidic region (pH 6.0 or less).
- yeast cells cannot normally maintain activity at pH in the region where PDI is not inactivated.
- general yeast culture medium components also contain substances that reduce or inactivate PDI activity.If a medium containing such components is used, extracellularly produced PDI will be lost. Due to such various reasons, a method for mass-producing yeast PDI in an active state has not been developed before the present invention. Summary of the Invention
- the present invention provides a method suitable for producing large quantities of biologically active recombinant yeast protein disulfide isomerase. Specifically, the production method of the present invention is characterized in that yeast PDI protein is produced and secreted extracellularly by a genetic engineering technique using the yeast PDI gene.
- the present invention also provides a recombinant yeast protein disulfide isomerase produced by the production method of the present invention.
- FIG. 1 is a diagram showing the progress of culture of Saccharomyces cerevisiae P1 and Y3 near neutral pH.
- FIGS. 2 to 4 are a series of diagrams showing the base sequence of the PDI gene derived from Saccharomyces cerevisiae.
- Figure 5 shows a restriction map of the yeast expression vector YEp1GII: Figures 6 to 10 show the complete nucleotide sequence of the yeast expression vector Yp1GII. is there.
- FIG. 11 (A) is an image image of electrophoresis of a culture solution of various transformed yeasts using SDS-PAGE gel; (B) is an illustration of (A) It is. Detailed description of the invention
- the present inventors have made intensive studies to solve the above problems, and as a result, by preparing yeast PDI by genetic engineering, extracellular cells with sufficient enzyme activity are maintained. At the same time, it has enabled mass production.
- chromosome D from the yeast Saccharomyces cerevisiae
- the yeast PDI gene obtained from NA H. Tachikiwawaeta, J.
- Biochemical em., 110, 306-313, 199 1) (donated by Tachikawa) was transformed into a C-terminal 4-amino acid sequence H ( Histidine) D (aspartic acid) E (glutamic acid) L (leucine) Modified so that it does not encode, connect to a strong promoter, and use a multi-copy vector such as YE p-type.
- the cells were introduced into host cells capable of growing in a medium and transformed.
- the present invention was completed by expressing yeast PDI having enzymatic activity in a large amount in a culture solution outside the cells.
- the present invention provides a method for producing a biologically active recombinant yeast protein disulphide isomerase
- the secreted enzyme is further concentrated or purified.
- the endoplasmic reticulum localization signal is a C-terminal 4-amino acid sequence (HDEL).
- the expression vector is the yeast expression vector YEp1GII shown in FIG.
- the host cells are cultured while maintaining the pH at 6.5-8.
- the present invention also provides a recombinant yeast protein disulphide isomerase produced by the production method of the present invention.
- the present invention also includes an expression vector and a transformed host cell used in the above production method.
- the yeast PDI gene is not limited, and for example, a PDI gene derived from yeast Saccharomyces' Celepiche described in SEQ ID NO: 1 (Fig. 2-4) can be used.
- the PDI gene derived from the yeast Saccharomyces cerevisiae is also disclosed in H. Tachikawaeta 1., J. Biochem., 110, 306—313, 1991, and JP-A-4-19771. And so on.
- PDI is considered to be present in many yeasts besides Saccharomyces cerevisiae.Enzymes present in such other yeasts also have an endoplasmic reticulum localization signal in the natural state. Can be used for the method.
- a PDI gene derived from yeast such as Picia or Kluyveromyces can also be used.
- the PDI gene can be obtained by a technique commonly used based on the above-mentioned prior art documents and the like, for example, a hybridization method, a PCR method and the like.
- secretion of proteins is usually accomplished by the following pathways.
- the secreted protein is translated from mRNA in the ribosome, translocates into the endoplasmic reticulum after or during translation, is further transported to the Golgi apparatus, and is distributed to vacuoles, cell membranes, cell walls, or extracellular cells. Proteins that should remain in the endoplasmic reticulum follow the same transport pathway as secretory proteins, but are transported to the Golgi and then back to the endoplasmic reticulum.
- these proteins located in the endoplasmic reticulum have a specific amino acid sequence consisting of a few residues in their structure to remain in the endoplasmic reticulum. Consensus sequence) or a consensus sequence called "motif" in which amino acid residues having similar properties may be conservatively substituted. In the present specification, to remain in the endoplasmic reticulum,
- a part or all of the endoplasmic reticulum localization signal is encoded by deleting, substituting or adding one or more bases in the region encoding the endoplasmic reticulum localization signal of the PDI protein. Are modified so that they do not.
- the endoplasmic reticulum localization signal is the C-terminal four amino acid residues (HDEL).
- Other yeasts may have endoplasmic reticulum localization signals of different sequences.
- the modified recombinant yeast yeast DI of the present invention has its gene modified so that it does not encode part or all of the ER localization signal, and the expressed recombinant PDI protein is converted into the ER. It is not localized but is secreted outside the cell.
- Modification of the region encoding the ER localization signal can be achieved by any of a number of known techniques. Mutations can be introduced at specific positions by synthesizing mutant sequence-containing oligonucleotides flanking restriction sites that can be ligated to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes a variant having the desired amino acid insertion, substitution or deletion in the ER localization signal region.
- oligonucleotide-directed site-directed mutagenesis producers can be used to provide altered genes with specific codons altered by the necessary deletion, substitution or addition.
- Techniques for making such changes include Walder et al. (Gene 42: 133, 196 8); Bauer et al. (Gene 37: 73, 198 5); C raik ( Biotechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1989); and U.S. Pat. Included are those described in US Pat. Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference.
- the modified recombinant yeast PDI of the present invention has a gene modified so as not to encode part or all of the ER localization signal, but is still biologically active. That is, it retains the enzymatic activity of catalyzing disulfide exchange in proteins. Furthermore, the region other than the ER localization signal region differs from the natural amino acid sequence. Even so, analogs having the biological activity of PDI are included in the recombinant yeast PDI of the present invention.
- An analog can include, for example, a conservatively substituted sequence, wherein one or more amino acid residues of a native PDI protein is replaced with a different residue, but the conservatively substituted The PDI protein is shown to retain essentially the same desired biological activity as the native protein.
- conservative substitutions include amino acid substitutions that do not alter the secondary and / or tertiary structure of PDI.
- Those skilled in the art can easily delete, substitute, or delete one or more nucleotide sequences in a portion other than the coding region of the endoplasmic reticulum localization signal of the yeast PDI gene using known genetic engineering techniques as described above. Addition can be performed to obtain an analog of yeast PDI.
- naturally occurring analogs of yeast PDI such as allyls, are also included in yeast PDI of the present invention.
- it can be modified to form yeast PDI derivatives by forming covalent or aggregate bonds with other chemical residues such as glycosyl groups, lipids, phosphates, acetyl groups, etc. it can.
- the present invention provides a recombinant expression vector for expressing recombinant yeast PDI, and a host cell transformed with the expression vector.
- Any suitable expression system can be used.
- Yeast expression systems are preferred.
- the expression vector contains a gene encoding a modified yeast PDI that is operably linked to an appropriate transcriptional or translational regulatory nucleotide sequence, such as one derived from a microorganism such as yeast, a mammal, a virus or an insect gene. Including. Examples of regulatory sequences include transcriptional promoters, operators or enhancers, mRNA ribosome binding sites, and appropriate sequences that control the initiation and termination of transcription and translation.
- a strong transcription promoter sequence in the expression vector in order to enable large-scale expression of a modified yeast PDI.
- an origin of replication that confers the ability to replicate in the desired host cell and a selection gene that identifies transformed cells are included in the expression vector.
- the yeast PDI gene, promoter The DNA having the role of as described in J.o.1.Biol., 96, 171-184, 1974, for example, EcoR —
- It can be prepared by digesting with a restriction enzyme such as I and BamHI and then ligating with a ligase such as T4DNA ligase.
- Suitable host cells for expression of the yeast PDI variant include yeast, prokaryotic or higher eukaryotic cells.
- Suitable cloning and expression vectors for use with yeast, bacterial, fungal and mammalian cell hosts are described, for example, in Pouwe 1 s et al., Cloning Vectors: AL aboratory Manual, El Sepia, NY (1980).
- Yeast PDI can preferably be expressed in a yeast host, particularly preferably in the genus Saccharomyces (eg, Saccharomyces cerevisiae). Other genera of yeast, such as the genus Pichia or the genus Kluyveres (Kluyveromyces), may be used. Yeast vectors often have a 2 // origin of replication sequence from yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a sequence for polyadenylation, a sequence for transcription termination, and one gene for a selectable marker.
- ARS autonomously replicating sequence
- Promoter sequences suitable for yeast vectors include, in particular, meta-mouth thionein, 3-phosphodaricerate kinase (PGK) (Hitzeman et al., J. Biol. Chem. 255: 207). 3,1980), or enolase, glyceraldehyde-1-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-16-phosphate isomerase, 3-phosphoglycerate Other glycolytic enzymes such as mutase, pyruvate kinase, triosephosphate isomerase, phosphodulcose isomerase and dalcokinase (Hess et al., J.
- PGK 3-phosphodaricerate kinase
- enolase glyceraldehyde-1-phosphate dehydrogenase
- hexokinase hexo
- shuttle vectors capable of replicating in both yeast and E. coli were used to select DNA sequences from pBR322 (ATCC 370 17) (Amp resistance gene and replication) for selection and replication in E. coli. (Origin) can be inserted into the above yeast vector.
- yeast-derived YEpGII Japanese Patent Application Laid-Open No. 7-289267 shown in FIG. 5-10 is preferable.
- a prokaryotic organism such as a Gram-negative or Gram-positive microorganism, for example, Escherichia coli or Bacillus (Bac11i) can be used as a host cell.
- Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis (B aci 1 1 ussubti 1 is ), Salmonella typhimurium (S a 1 mone 1 latyphi mu ri um) 3 ⁇ 4 sequence (this shoe - Pseudomonas genus ( Pseudomonas), Streptomyces and various other species within the genus Staphylococcus.
- Expression vectors for use in prokaryotic host cells generally include one or more phenotypic selectable marker genes.
- a phenotype selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or auxotrophy.
- useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids, such as the cloning vector pBR322 (ATCC 37017). Other commercially available vectors include, for example, pKK23-3-3 (Pharmacia Fine Chemi. Ca 1 s, Uppsala, Sweden) and pGEM1 (Promega Biotec). , Madison, WI, USA).
- Promoter sequences commonly used in recombinant prokaryotic host cell expression vectors include:] 3-lactamase (penicillinase), a lactose promoter system (Chang et al., Nature 275: 615, 19). 78; and Goedde 1 et al., Nature 281: 544, 1979), a tryptophan (trp) motor series (Goeddel et al., Nucl. Acids Res. 8: 405) -
- C 1 oning: AL aboratory Ma nual , C old S pring H arbor L aboratory, 4 1 2 page, 1 9 8 2) is:
- the phage lambda P L promoter and c I 8 5 7 ts heat labile repressor Arrays can also be used.
- mammalian or insect host cell culture systems can also be used to express recombinant yeast PDI variants.
- Expression vectors for use in mammalian host cells can be constructed, for example, as disclosed by Ocama and Berg (Mo 1. Cell Biol. 3: 280, 1983).
- host cells selected from a wide range can be used in the present invention, but yeast PDI is inactivated under weakly acidic (pH 6.0) conditions.
- Preferred host cells can also be obtained by known genetic engineering techniques, crossing techniques, and the like.
- preferred host cells can be selected from naturally-occurring mutants.
- yeast usually has an optimal pH for growth in a weakly acidic region, but the method described in Example 1 can screen yeast capable of growing in a medium having a pH near neutrality.
- Saccharomyces cerevisiae P1 manufactured by Oriental Yeast
- yeast cells prokaryotic cells, mammalian or insect cell culture systems or the like, which can be grown and cultured under conditions in which pH is near neutral, can be selected and used.
- the intended host cell selected can be transformed using a known technique.
- a yeast transformation protocol is described in Hinnen et al., Proc. Natl. Acad. Sci. US A75: 1929, 1997. 8
- the transformed host cells are cultured under conditions suitable for the expression of the recombinant yeast PDI variant, depending on each host cell.
- the cultivation is performed under conditions where the pH is near neutral, preferably under the condition of pH 6.5-8.0.
- the recombinant PDI variant protein expressed by the method of the present invention is secreted out of the host cell.
- the pH is maintained near neutrality, for example, by using a buffer such as HEPES in the culture medium of the transformant.
- perilla-free minimal medium (6.7 g / L yeast nitrogenbasewith outaminoacid (D ifco), 20 g / L glucose, 20 mg ZL adenine, l O mg / LL-histidine, 60 m g / LL-leucine, 20 mg gZL L-tryptophan, 100 mM HE PES, 10 mM EDTA-2 Na, pH 7.5) can be used as the culture medium.
- the modified yeast PDI protein produced by the method of the present invention can be purified by a known method.
- the medium is first concentrated using a commercially available protein concentration filter, for example, an Am icon or Millipore Pel 1 icon ultrafiltration device. can do.
- the concentrate can be applied to a purification matrix such as a gel filtration base.
- a purification matrix such as a gel filtration base.
- an anion exchange resin such as a matrix or support having pendant getylaminoethyl (DEAE) groups can be used.
- the matrix can be acrylamide, agarose, dextran, cellulose or other types commonly used in protein purification.
- a cation exchange step can be used.
- Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups.
- RP-HPLC reversed-phase high-performance liquid chromatography
- Secreted recombinant proteins from yeast host cell fermentations can be obtained, for example, by methods similar to those disclosed by Urda 1 et al. (J. Chromatog. 296: 171, 1984). Can be purified.
- the modified yeast PDI protein is purified, for example, by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) so that protein bands corresponding to other proteins cannot be detected. Protein bands can be viewed by silver staining, Coomassie blue staining (if the protein is radiolabeled) by autoradiography.
- SDS-PAGE SDS-polyacrylamide gel electrophoresis
- the recombinant yeast PDI variant protein of the present invention has a modified endoplasmic reticulum localization signal portion, but retains the PDI biological activity.
- the enzymatic activity of the PDI protein can be performed by a known technique.
- the activity of in vitro can be measured by, for example, restoring the activity using a denatured and reduced enzyme as a substrate. For example, but not limited to, using the method described in Example 4, which is a modification of the method of Mizunaga et al. (J. Biochem., 108, 846, 1990). Can be.
- a host cell for producing PDI having an activity even at a pH near neutral was selected by the following method.
- L-tryptophan 2.0% Next, main culture was performed near pH neutral.
- l YPAD medium (10 gZL yeast extract, 20 g / L peptone, 140 mg / L adenine, 2 QgZL glucose) adjusted to pH 7.5 by adding O OmM HEP ES l
- O OmL to Sakaguchi flask
- 1 mL of the above precultured moromi was added thereto, and shaking culture was performed at 30 ° C. and 105 rpm.
- Saccharomyces cerevisiae p1 (manufactured by Oriental Yeast Co., Ltd .; MAT a1 eu2-3, 112. 289 ura 3-1 and 2 his 3- ⁇ 32 his 4-5 16 ade 2) were obtained.
- Example 2 Modification of C-terminal ER localization signal of yeast PDI W yeast (Saccharomyces cerevisiae) Replaces the nucleotide sequence “CACGATGAATTG” encoding the C-terminal ER localization signal “HDEL” of PDI with “CACG ATTAATTG” and inserts a stop codon in the middle of the ER localization signal. was modified to be
- the Sal I-Eco RV cleavage site containing the termination gene and the structural gene region of PD I was cut with the S a1 I-Sma I of the commercially available E. coli vector pUC19. Inserted between the sites to construct pUCPDIC1.
- a stop codon (TAA) was introduced on the sequence encoding the endoplasmic reticulum localization signal by PCR site-directed mutagenesis.
- the primers for PCR are shown in Table 2: Table 2 Primers used for PCR Primer Nucleotide sequence
- the two amplified primary amplification products were mixed, annealed to form ⁇ , and then subjected to secondary PCR using a combination of RV and M4 as primers.
- the resulting secondary amplification products containing the two DNA fragments were treated with SphI and EcoRI.
- the SphI-EcoRI fragment was inserted between the Sphl-EcoRI cleavage sites of pUC19 to construct pUCPDIC2.
- YEp1GII As a multicopy type vector for expression in yeast, YEp1GII (JP-A-7-289926) having the nucleotide sequence shown in FIG. 5-10 was used. YEp1GII contains a GAP (daricelaldehyde triphosphate dehydrogenase) promoter with high promoter activity.
- GAP daricelaldehyde triphosphate dehydrogenase
- yeast PDI expression vector obtained in Example 3 host cells for production of PDI, Saccharomyces cerevisiae P1, were transformed by a conventional cell method using lithium phosphate according to a conventional method.
- E0 the strain into which YEpGIPI has been introduced
- E1 the yeast strain into which YEpGIPIDI has been introduced
- E2 the yeast strain into which YEpGIPIDIC has been introduced
- the obtained transformant, E2 was sent to FERM P-1 595 by the Institute of Biotechnology and Industrial Technology, Institute of Advanced Industrial Science and Technology (1-3-1, Higashi 305, Tsukuba, Ibaraki, Japan) on January 15, 1996. Deposited as one.
- FERM P-159951 was transferred from the original deposit to a deposit based on the Budapest Treaty on January 28, 1998, and became a deposit number F ERM BP-6240.
- the PDI activity measurement method was performed by modifying the method of Mizunaga et al. (J. Biochem., 108, 846, 1990). In other words, the activity of RNase A whose activity was replicated by PDI was measured using the method of Uchida (Biochemistry Laboratory Course 2, pp 68, Tokyo Kagaku Dojin, 1977).
- PD I activity measurement sample (or ultrapure water) 40 / L, 2. 5-fold concentration PE buffer (1 2 5 mM N a H 2 P_ ⁇ 4, 6. 2 5 mM EDTA- 2 Na, p H 7 5) 40 ⁇ L, 0. Mix 10 ⁇ L of ImM dithiothreitol (within 2 hours after preparation) in a 1.5 mL eppendorf tube, and pre-incubate at 30 ° C for 5 minutes. I did it.
- PDI 1 unit (1U) is pH 7.5, 30. C, defined as the enzyme activity that restores RNase A 1 U in 1 minute.
- the “RNaseA 1 unit (1 U)” is defined as the above method (by stopping and diluting the RNase A reaction solution (by diluting 200/150 ⁇ 1040/40 times))
- the absorbance at 260 was measured and defined as an enzyme activity that increases by 1 per minute.Example 6 Selection of culture medium that does not inhibit PDI activity
- Yeast culture medium for producing yeast PDI was selected by mixing the medium with a crude enzyme solution extracted from yeast cells and measuring PDI activity.
- a minimal medium without ⁇ rasil (6.7 g / L yeast nitrogenbase with out am inoacid (D ifco), 20 g / L glucose, 20 mg ZL adenine, l OmgZL L-histidine, 60 mg ZL L—mouth isine, 20 mg / LL —Tributophan, 100 mM HEPE S, 10 mM EDTA—2 Na, pH 7.5) Inoculate one platinum loop of yeast (Saccharomyces' Celepiche) into a 16 mm diameter test tube containing 5 mL.
- a crude enzyme solution was prepared from the cells by shaking culture at 30 ° C for 3 days.
- the above-mentioned minimal medium containing no peracil, YAD medium (10 g / L yeast extract, 20 gZL glucose, 20 mg L adenine, lOOmM HEPES, 10 mM EDTA—2Na, pH7
- Table 4 shows the results of comparing 5).
- Table 4 Selection of culture medium that does not inhibit PDI activity
- Minimal medium 100 not containing From Table 4 a minimal medium containing no peracil was selected as a medium not inhibiting PDI activity.
- Example 7 Culture of transformed host cells
- the transformed yeast host cells obtained in Example 4 were inoculated with one platinum loop into a 16 mto diameter test tube containing 5 mL of YPAD medium, and cultured at 30 ° C for 3 days with shaking.
- the culture was centrifuged at 3000 rpm for 5 minutes, and washed twice with ice-cold 4-fold concentration PE buffer (200 mM NaH2P04, 10 mM EDTA-2 Na, pH 7.5). .
- PE buffer 200 mM NaH2P04, 10 mM EDTA-2 Na, pH 7.5
- 2 mL of a PDI production medium having the above composition prepared by adding a buffer and a chelating agent to a minimal medium containing no peracil, was added, and cultured with shaking at 30 ° C for 15 hours.
- Example 8 PD I activity of recombinant yeast PD I protein Yeast serum albumin (BSA) (manufactured by Wako) was added to the culture solution obtained from the yeast PDI production culture to a final concentration of 0.1 lg / L. ipore, Ultrafree C 3 LGC, molecular weight cut off 10,000), and concentrated at 600 rpm at 5 ° C until the total volume became about 100 ⁇ L to obtain a yeast-containing PDI solution.
- BSA yeast Yeast serum albumin
- the PDI protein was secreted and expressed in the medium, and the PDI activity was measured and compared by the method of Example 4, and the results are shown in Table 5.
- Table 5 PDI activity of ⁇ 0, — ⁇ 1, _ ⁇ 2 strains Sample PDI activity ml L) —
- yeast PDI in the culture was also performed by SDS-PAGE: the culture was concentrated approximately 60-fold in the same way as described above, without adding BSA.
- the equipment used was a double minislab electrophoresis apparatus (AE 6450) manufactured by ATTO.
- the method was the Laemmii method according to the attached manual. Separation gel concentration was 7.5%.
- Staining was performed using BLU PRINT Fast-PAGE Stain (GI BCOB RL).
- Figure 11 shows an image of the stained SDS-PAGE gel.
- a band specific to the PDI-introduced strains (El, E2) is observed around 70,000 daltons.
- the yeast PD I molecule 1 is about 70,000 daltons, which is considered yeast PDI.
- the band concentration was E 2> E 1, and the effectiveness of the present invention was also confirmed by SDS-PAGE. The invention's effect
- a large amount of yeast PDI protein is secreted into the culture solution in an active state, and can be recovered by a simple purification method.
- ATC AAG CAA AGC CAA CCG GCT GTC GCC GTT GTT GCT GAT CTA CCA 450 lie Lys Gin Ser Gin Pro Ala Val Ala Val Val Ala Asp Leu Pro
- GGT CTA ATG AAC TTT GTT AGC ATC GAT GCC AGA AAA TTC GGC AGA 900 Gly Leu Met Asn Phe Val Ser lie Asp Ala Arg Lys Phe Gly Arg
- ATC CAC GAC ATG ACT GAA GAC TTG AAG TAC GGT TTG CCT CAA CTC 990 lie His Asp Met Thr Glu Asp Leu Lys Tyr Gly Leu Pro Gin Leu
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EP98901535A EP0974665A4 (en) | 1997-02-07 | 1998-02-06 | RECOMBINANT PDI FROM YEAST AND METHOD FOR THE PRODUCTION THEREOF |
US09/368,588 US6387683B1 (en) | 1997-02-07 | 1999-08-05 | Recombinant yeast PDI and process for production thereof |
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Cited By (6)
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KR100414182B1 (ko) * | 2001-07-02 | 2004-01-07 | 대한민국 | 누에 배양세포로부터 분리한 프로테인 디설피드이소머레이즈를 코딩하는 cDNA 유전자의 염기서열 및그의 연역 아미노산 |
JP2010051196A (ja) * | 2008-08-26 | 2010-03-11 | Tokyo Univ Of Agriculture | 小麦加工製品の改質剤及び小麦加工製品の製造方法 |
WO2010058527A1 (ja) * | 2008-11-18 | 2010-05-27 | アサヒビール株式会社 | グルタミン酸高含有酵母の製造方法 |
WO2010058616A1 (ja) * | 2008-11-18 | 2010-05-27 | アサヒビール株式会社 | グルタミン酸高含有酵母の製造方法 |
JP2010517537A (ja) * | 2007-02-02 | 2010-05-27 | グローブイミューン, インコーポレイテッド | 酵母ベースのワクチンを生成するための方法 |
JP2011177059A (ja) * | 2010-02-26 | 2011-09-15 | Tokyo Univ Of Agriculture | 小麦加工製品の改質剤及び小麦加工製品の製造方法 |
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EP1863921B1 (en) * | 2005-03-24 | 2011-10-05 | BioGeneriX AG | Expression of soluble, active eukaryotic glycosyltransferases in prokaryotic organisms |
US20240392279A1 (en) * | 2021-09-17 | 2024-11-28 | The University Of North Carolina At Chapel Hill | Modified protein disulfide isomerase and uses thereof |
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JPH0530967A (ja) * | 1991-11-21 | 1993-02-09 | Agency Of Ind Science & Technol | ヒト・リゾチームの製造法 |
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JPH07289267A (ja) | 1994-04-25 | 1995-11-07 | Oriental Yeast Co Ltd | 発現増強方法 |
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1998
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- 1998-02-06 EP EP98901535A patent/EP0974665A4/en not_active Withdrawn
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1999
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JPH04197176A (ja) * | 1990-11-28 | 1992-07-16 | Tonen Corp | 酵母プロテインジスルフィドイソメラーゼ |
JPH0530967A (ja) * | 1991-11-21 | 1993-02-09 | Agency Of Ind Science & Technol | ヒト・リゾチームの製造法 |
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KR100414182B1 (ko) * | 2001-07-02 | 2004-01-07 | 대한민국 | 누에 배양세포로부터 분리한 프로테인 디설피드이소머레이즈를 코딩하는 cDNA 유전자의 염기서열 및그의 연역 아미노산 |
JP2010517537A (ja) * | 2007-02-02 | 2010-05-27 | グローブイミューン, インコーポレイテッド | 酵母ベースのワクチンを生成するための方法 |
US9066893B2 (en) | 2007-02-02 | 2015-06-30 | GlobelImmune, Inc. | Yeast-based vaccines |
US9549970B2 (en) | 2007-02-02 | 2017-01-24 | Globeimmune, Inc. | Methods for producing yeast-based vaccines |
JP2010051196A (ja) * | 2008-08-26 | 2010-03-11 | Tokyo Univ Of Agriculture | 小麦加工製品の改質剤及び小麦加工製品の製造方法 |
WO2010058527A1 (ja) * | 2008-11-18 | 2010-05-27 | アサヒビール株式会社 | グルタミン酸高含有酵母の製造方法 |
WO2010058616A1 (ja) * | 2008-11-18 | 2010-05-27 | アサヒビール株式会社 | グルタミン酸高含有酵母の製造方法 |
JP2010148517A (ja) * | 2008-11-18 | 2010-07-08 | Asahi Breweries Ltd | グルタミン酸高含有酵母 |
JP4503700B1 (ja) * | 2008-11-18 | 2010-07-14 | アサヒビール株式会社 | グルタミン酸高含有酵母の製造方法 |
JP4757944B2 (ja) * | 2008-11-18 | 2011-08-24 | アサヒビール株式会社 | 酵母エキス |
US9005683B2 (en) | 2008-11-18 | 2015-04-14 | Asahi Group Holdings, Ltd. | Method for producing yeast with high glutamic acid content |
JP2011177059A (ja) * | 2010-02-26 | 2011-09-15 | Tokyo Univ Of Agriculture | 小麦加工製品の改質剤及び小麦加工製品の製造方法 |
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US6387683B1 (en) | 2002-05-14 |
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