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WO2000071580A1 - Bibliotheque de proteine secretoire et methode de production de ladite proteine - Google Patents

Bibliotheque de proteine secretoire et methode de production de ladite proteine Download PDF

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Publication number
WO2000071580A1
WO2000071580A1 PCT/JP2000/003214 JP0003214W WO0071580A1 WO 2000071580 A1 WO2000071580 A1 WO 2000071580A1 JP 0003214 W JP0003214 W JP 0003214W WO 0071580 A1 WO0071580 A1 WO 0071580A1
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Prior art keywords
serum
protein
antibody
proteins
secretory
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PCT/JP2000/003214
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English (en)
Japanese (ja)
Inventor
Shin-Ichi Kojima
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Sumitomo Pharmaceuticals Company, Limited
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Publication of WO2000071580A1 publication Critical patent/WO2000071580A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention relates to a secretory protein library useful as a material for searching for a therapeutic agent for various diseases and a method for producing the same.
  • the Genetics Institute has developed DiscoverEase (a library of secreted protein cDNA and recombinant protein cloned by signal, sequence, and trap using yeast as a host) and has begun to use it for drug discovery. I have.
  • Human Genome Sciences has closed the gene for the therapeutic protein and I'm making an library.
  • the secretory protein library can significantly shorten the process from the measurement of biological activity to the identification of a substance.
  • Once constructed it can be evaluated in a wide variety of measurement systems, making it easier to search for new useful proteins. In other words, high throughput can be achieved in the search for new useful proteins.
  • construction of an appropriate secretory protein library is also important for improving the efficiency of biopharmaceutical development.
  • the power of isolating and purifying bioactive proteins is essential for the construction of secretory protein libraries.
  • the method that has been repeatedly performed in the past has been to use a culture medium of several tens to several thousands of liters containing a secretory protein in a volume of 1-1-10. g) It is an extremely inefficient method of separating one type of bioactive protein of interest, and all secretory proteins other than the target protein are discarded, making it unsuitable for library construction. is there.
  • the present inventor predicts that there is a possibility that an unknown bioactive protein may be contained in a large part other than the purpose discarded, and removes all of the culture supernatant containing countless bioactive substances. Isolation and purification were performed as raw materials, and construction of a novel secretory protein library was attempted. In other words, the culture solution of one cell line is expected to contain more than 1,000 types of secreted proteins, so that by culturing several 10 types of cell lines, a total of 10,000 types can be obtained. A close secretory protein library can be constructed.
  • the present inventors cultured about 80 types of cell lines, obtained a culture supernatant, and used it as a material for a secretory protein library.
  • the present inventor tried to concentrate the bioactive protein contained in the culture solution by several hundred to several thousand times to prepare a secretory protein library free from the influence of components such as a medium. If this becomes possible, it will be possible to detect proteins secreted into the culture solution even if they are trace protein components. Eventually, it was thought that if a library of isolated and purified secreted proteins could be constructed, active proteins could be more easily obtained, evaluated, and analyzed.
  • the present inventors have conducted intensive studies on a method for constructing a library of nearly 10,000 secreted proteins to solve the above-mentioned problems.
  • the present invention has been completed by constructing a more practical secretory protein library.
  • the progress up to the completion of the present invention, the construction of the secretory protein library and the steps thereof will be described in detail below.
  • the present invention is based on the establishment of a method for removing serum-derived proteins used for culture and a method for removing media-derived proteins. For example, when a cell line is cultured in a medium of 1 L to 100 L with serum and then cultured as it is or without a secretion inducer and serum-free, the amount of the secreted protein may be reduced.
  • the present inventors have conducted various studies, and by establishing the following antibody column chromatography treatment, serum- and medium-derived proteins were detected to the extent that they were hardly detected by SDS-polyacrylamide gel electrophoresis, that is, It has been found that the secretory protein library intended by the present invention can be removed to the extent that it can function effectively.
  • serum-containing medium is used for cell culture, serum-derived proteins are first removed.
  • the serum-supplemented medium is a medium to which serum is added, and examples thereof include 0.5% to 10% of serum, poma serum, and egret serum. Even when a serum-free medium is used in the final stage of cell culture, a small amount of serum is contaminated, so serum-derived proteins are removed.
  • an antibody column for the serum-derived proteins. Using an antiserum antibody gel (10 L of gel 1 LZ culture solution), pass the culture supernatant through a column filled with the gel to remove serum proteins. It is desirable to use an antibody gel that can trap the amount of protein at the time of the addition of the antibody force ram.
  • the anti-puff serum BS antibody gel (BS amount of 100 mg or less at the time of addition / lL of gel), the anti-puff serum albumin BSA antibody gel (the BSA at the time of addition) l OOO mg or less Z gel 1 L), anti-serum serum immunoglobulin antibody genole (additional serum immunoglobulin 1 OOO mg at the time of addition 1 lmg), anti-fetuin antibody gel (fetuin amount at the time of addition 1 OOO) mg or less Z Genore 1 L), anti-anti Proteins derived from serum are removed using an antibody gel such as trypsin antibody gel (antitrypsin amount at the time of addition of 100 mg or less / gel per liter) or a gel to which protein A or protein G is bound.
  • an antibody gel such as trypsin antibody gel (antitrypsin amount at the time of addition of 100 mg or less / gel per liter) or a gel to which protein A or protein G is bound.
  • an anti-transferrin antibody gel (transferrin amount of 100 mg or less at the time of addition of Z-gel 1 L), an anti-insulin antibody gel (at the time of addition) Insulin amount 1
  • the secreted protein is fractionated by anion exchange column chromatography and cation exchange column chromatography, or by isoelectric focusing.
  • fractionation is performed by a molecular weight fractionation method using gel filtration, and the secreted proteins are grouped into about one thousand fractions, and the secreted protein concentration is increased about one thousand times. If necessary, fractionate and purify each fraction by hydrophobic chromatography or reverse phase chromatography.
  • the secretory protein library of the present invention can be constructed.
  • the present invention has been completed based on the above findings.
  • a secreted protein library obtained by fractionating and purifying from a cell culture solution in the following steps: (a) performing antibody column chromatography on the culture supernatant,
  • the anion exchange column chromatography column condition is pH7-10, and the cation exchange column chromatography column condition is PH2-7, and the adsorbed protein is eluted by the chromatographic method in which the salt concentration is increased from 0 M to 2M.
  • the column conditions for anion exchange column chromatography were pH 7-10 and the column conditions for cation exchange column chromatography were pH 2-7, and adsorption was performed by the chromatographic method in which the salt concentration was increased from 0M to 2M.
  • a method for constructing a secretory protein library comprising fractionating by a molecular weight fractionation method, and further comprising, if necessary, further fractionating by hydrophobic chromatography or reverse phase chromatography. How to construct a protein library,
  • Antibody used in the antibody column chromatography method is anti-serum serum antibody, anti-serum serum albumin antibody, anti-serum serum immunoglobulin antibody, anti-transferrin antibody, anti-insulin antibody, anti-fusin antibody, anti-antitrypsin
  • a secretory protein library obtained by purifying and fractionating from a cell culture solution, and having the following characteristics (a) to (d);
  • the fraction can be directly used for determining the structure of the protein
  • a secretory protein library obtained by purifying and fractionating from a cell culture solution and having the following characteristics (a) to (d);
  • a protein derived from a medium and a protein derived from serum can be used to determine the structure of a desired secreted protein. Has been removed to an extent that does not affect it,
  • the fraction can be directly used for determining the structure of the protein
  • a first aspect of the present invention is a practicable secretory protein library constructed from a bioactive protein obtained by fractionating and purifying a protein secreted from a cell line by the following steps.
  • the cell line is cultured in a serum-supplemented medium, and then cultured in a serum-free medium or a serum-free medium containing a secretion inducer.
  • a second aspect of the present invention is a method for producing a secretory protein library obtained by fractionating and purifying a protein secreted from a cell line by the following steps.
  • the cell line is cultured in a serum-supplemented medium, and then cultured in a serum-free medium or a serum-free medium containing a secretion inducer.
  • the animal cell line referred to in the present invention is, for example, a cell derived from an animal tissue, and preferably a cell line that can be subcultured.
  • ordinary cell culture conditions can be used (1, Method ed. Enzymology I, Volume LVIII, Cell Culture, edited by William Vijayakovi et al., 1979, 2, Cell Culture Handbook) Ed., Edited by Suzuki et al., September 30, 1993, 3, cultivated animal cells, edited by Earl Ian Fresney et al., 1994).
  • Preferred animal cell lines include human cell lines, mouse cell lines, and rat cell lines, of which more preferred are human cell lines.
  • Examples of the serum-supplemented medium include 0.5% to 10% of poma serum or poma serum.
  • the serum-free medium is a medium that does not contain serum, and examples thereof include Hybridoma-SFM medium, CD-CH0 medium, and ASF-104 medium. Furthermore, in a serum-free medium, for example, cell trophic factors such as transferrin and insulin may be added in order to maintain cell survival more effectively.
  • the concentration added at this time is a concentration sufficient to maintain the cells, but is usually 1 to 10 Omg // 1, preferably 5 to 20 mgZl.
  • Secretion inducers are agents that act on cultured cells to induce protein secretion.
  • Examples of the secretion inducer of the present invention include differentiation inducers such as phorbol ester and indole alkaloid, for example, dalcocorticoids such as dexamethasone and prezodone, and polar solvents such as DMSO and Noscal. Comes out.
  • Antibody power to remove proteins contained in serum and medium components Ram chromatography is an antibody gel using antibodies corresponding to the proteins contained in the serum and medium used, and a column filled with this is used. It is intended to adsorb and remove the corresponding protein by making it and passing it through the culture supernatant. Therefore, it is necessary to select the antibody gel to be used in accordance with the serum used and the protein contained in the medium. It is desirable to use an antibody gel that can trap the amount of protein during the addition of the antibody column.
  • the relationship between the protein concentration and the amount ratio of the antibody gel at the beginning of use is, for example, in the case of an anti-pseudo-serum protein antibody gel, the amount of the p-serum protein is 1000 mg or less at the time of addition, and 1 L of Z-gel is preferable. Is 1 L of gel containing 10 Omg or less of serum protein at the time of addition.
  • the amount of transferrin at the time of addition is at least lOOOmg or less, and the amount of transferrin at the time of addition is 10 Omg or less.
  • the amount of insulin is at least 1000 mg or less at the time of addition, and the amount of insulin at the time of addition is 10 Omg or less.
  • the amount of fetuin at the time of addition is 100 Omg or less at no gel 1 L, and the amount of fetuin at the time of addition is 10 Omg or less.
  • the amount of anti-trypsin at the time of addition is 1000 mg or less and the Z-gel is 1 L, and the amount of anti-trypsin at the time of addition is 1 OO mg or less / 1 L of gel.
  • the culture supernatant is prepared and passed as follows.
  • the column conditions are such that the culture supernatant is cooled to 4 ° C at a flow rate of 1 to 30 times the column volume per hour and passed through the culture supernatant.
  • the solution passed through the antibody column is concentrated as it is or by ultrafiltration, and purification is performed by isoelectric focusing.
  • Isoelectric point fractionation is a method of fractionation using the charge of proteins.More specifically, it is a method of chromatographic fractionation using columns packed with cation-exchanged genole and anion-exchanged genole, respectively.
  • the anion exchange column is, for example, DEAE Sepharose, DEAE Sephadex, CM Sepharose, QAE Sephadex, Q Sepharose, Resource Q, preferably Poros Q, Mono Q.
  • Examples of the cation exchange column include CM Sepharose, CM Sephadex, and SP Sephadex, and preferably, Poros S and Mono S.
  • a buffer of pH 7-10 for anion exchange columns
  • a buffer solution of pH 2-7 is used, and the adsorbed protein is eluted by a chromatographic method in which the salt concentration is increased from OM to 2 M.
  • it is a method of isoelectric focusing without using a support such as a gel to fractionate proteins. For example, fractionation using Mini Four (manufactured by Protein Technology Co., Ltd.).
  • the molecular weight fractionation method is, for example, a method of fractionation by Genofiltration method or SDS-PAGE method.
  • the genole filtration method and the SDS-PAGE method of the present invention can be performed, for example, according to the method described in Biochemistry Experiment Course 1 “Protein Chemistry I”.
  • Gels used for gel filtration include, for example, TSK-G3000SW column manufactured by Tosoichi Co., HiLoad Superdex S-200 column manufactured by Pharmacia, HiLoad Superdex S-75 column, Superdex S-200 column, Superdex S-200 column. 75 columns and the like.
  • the salt concentration is used phosphate buffer 300mM from 50 mM, at a flow rate of 0. 05ml / ml from 5 ml / m l, eluting with low temperature or room temperature.
  • Examples of the gel used for SDS—PAGE include polyacrylamide gel.
  • the secreted proteins from the human cells are separated into groups of about 100 to 1,000 fractions, and the secreted protein concentration is about 100 to 1000 times concentrated.
  • the hydrophobic fractionation method refers to hydrophobic chromatography or reversed-phase chromatography. Using this method, each fraction obtained above is further fractionated and purified as necessary.
  • the hydrophobic chromatography of the present invention is a chromatography method in which a filler having a hydrophobic residue as a ligand is used to separate proteins utilizing the difference in hydrophobicity of proteins.
  • Reversed-phase chromatography is a chromatographic method that separates proteins using the difference in the hydrophobicity of proteins, similar to hydrophobic chromatography. It uses a reversed-phase carrier and uses an organic solvent as an eluent.
  • the secretory protein library of the present invention prepared as described above comprises the following (a)
  • the fraction can be directly used for determining the structure of the protein
  • the structure of the protein can be determined only by subjecting the fraction to hydrophobic chromatography or reverse phase chromatography.
  • the phrase "removed to such an extent that it is almost not detected by SDS-polyacrylamide gel electrophoresis” means that most of the protein derived from the culture medium and the protein derived from serum are removed. And a state in which the secretory protein library of the present invention functions effectively. More specifically, it means that the protein derived from the culture medium and the protein derived from serum have been removed to such an extent that the determination of the structure of each secretory protein subjected to the molecular weight fractionation in the above (d) is not hindered.
  • the basis of “at least 10 types” in the above (b) is based on Examples 3 and 4, but originally about 500 to 1,500 secreted proteins are contained in a culture solution of one type of cell line. In fact, it is thought that it is actually an aggregate of more secreted proteins.
  • the secretory protein library of the present invention can be used as a secretory protein library derived from a specific cell depending on the purpose, or can be used as a library of secretory protein derived from several types of cells having different origins. It can also be used on a regular basis. In the following examples, a library of secreted protein derived from 10 kinds of cells is provided.
  • fractionation when there are several types of proteins present in one fraction, it is common to fractionate them into a single substance by reversed phase chromatography, A similar fractionation is also possible. Furthermore, fractionation can also be performed by other fractionation methods, that is, ion exchange column chromatography or gel filtration.
  • secretory protein library of the present invention is that it is an aggregate of secretory proteins having an activity as shown in Examples. Therefore, the present invention
  • the secretory protein library can be directly used for various activity measurements. Furthermore, since the fraction that has been positive in the activity measurement can be directly used for structure determination, there is an advantage that searching for a novel useful protein can be performed very quickly and easily.
  • secretory protein library of the present invention secretory proteins from human cells are grouped and separated into about 100 to 1,000 fractions, and the secretory protein concentration is about 100- to 1000-fold concentrated. The test can be performed by the usual function evaluation method, and the presence or absence of the corresponding protein can be checked.
  • the library of the present invention is used as in Example 1, and the secretory protein of the present invention is used. It was possible to identify the presence of activin in the library.
  • the physiologically active protein or the pharmaceutically acceptable salt thereof found from the secretory protein library of the present invention can be used for therapy, and in that case, as a pharmaceutical composition, It can be administered orally or parenterally (eg, intravenous, subcutaneous, or intramuscular injection, topical, rectal, transdermal, or nasal).
  • parenterally eg, intravenous, subcutaneous, or intramuscular injection, topical, rectal, transdermal, or nasal.
  • compositions for oral administration include tablets, capsules, pills, granules, powders, solutions, suspensions, and the like.
  • compositions for parenteral administration include injections Aqueous or oily agents, ointments, creams, lotions, aerosols, suppositories, patches, and the like.
  • preparations can be prepared using conventionally known techniques, and can contain nontoxic and inert carriers or excipients commonly used in the field of preparations.
  • dosage forms are prepared by combining the pharmaceutically acceptable usual carriers, excipients, binders, stabilizers, buffers, solubilizing agents, isotonic agents and the active ingredients of the present invention. be able to.
  • the dosage and frequency of administration vary depending on the patient's symptoms, history, age, body weight, dosage form, etc.For example, when orally administered to an adult (body weight 60 kg), usually lg to 1000 mg per day, preferably 5 ⁇ g
  • the dose may be suitably adjusted in the range of 500 to 500 mg, particularly preferably 10; zg to 200 mg, and may be administered once or in several divided doses.
  • FIG. 1 is a photograph showing the results of analyzing proteins secreted by Chang Liver cells by two-dimensional electrophoresis.
  • FIG. 2 is a photograph showing a result of analyzing a protein secreted by U87MG cells by two-dimensional electrophoresis.
  • FIG. 3 is a photograph showing a result of analyzing a protein secreted by CCd-112C0N cells by two-dimensional electrophoresis.
  • HS602 cells cervical lymph node
  • 581-13 ⁇ 4 cells (676; 61 ⁇ 01) 1331: 01 1 13
  • DU4475 cells ma crypt gland
  • CCRF-SB cells lymphoblast
  • FCS is fetal serum.
  • HybridomaSFM is capable of transferring human transferrin and periinsulin each 10 ⁇ g / ml.
  • CD-CHO is a complete protein-free medium, to which 300 mg / ml glutamine has been added.
  • HybridoraaSFM CD-CHO
  • the survival rate was greatly reduced.
  • the survival maintenance experiment in the HybridomaSFM, after growth in RPMI1640ZIO% FCS, the medium was exchanged, and the cell number and viability were almost maintained in both conditions with and without stimulation. The attained density was about 60% that of those cultured in RPMI1640 Z10% FCS.
  • CD-CH0 cell density and viability decreased after the medium was changed.
  • growth medium using EMEM + 1 0% FCS + 1/50 P- S, at 3 7 ° C, 5% C 0 2, and cultured at passage frequency of 2-3 times / week.
  • the culture solution was removed by suction, PBS (-) was added, and the cell adhesion surface was gently washed. Then, PBS (-) was removed by suction. This operation was repeated twice to remove serum.
  • the trypsin-EDTA solution was added, the trypsin-EDTA solution was immediately removed by suction, and the cells were detached by being left at room temperature or at 37 ° C.
  • the cells were suspended in growth medium, into an appropriate cell concentration, and cultured at 37 ° C, 5% C 0 2 below.
  • the confluent cultured cells were diluted in a growth medium, inoculated at a concentration of 60,000 cells / ml, and placed in 170 flasks of 9 Om 1 225 cm 2 flask and cultured at 37 ° C for 3 days.
  • An anti-BSA / BS antibody column treatment and an anti-transferrin antibody column treatment were performed on 15 L of Chang Liver culture supernatant to remove proteins derived from the medium.
  • a gel in which 10 ml of an anti-human transferrin antibody manufactured by Cappel was bound to 10 g of CNBr-Sepharose4FF gel was used.
  • 0.5 L of the solution passed through the anti-BS A & B S antibody column was passed through the anti-human transferrin TR antibody column at a flow rate of 40 ml / min.
  • 50 ml of water was flushed and coalesced as samples.
  • the column was washed with 100 mL of 0.1 M citrate buffer and regenerated. 100 mL of 50 mM Tris-HCl pH 8.0 was flowed through the flask, equilibrated, and used again.
  • the following fractionation operation was performed on the solution that had been subjected to anti-B SA & B S antibody column chromatography and anti-human transferrin TR antibody column chromatography.
  • the filtrate was chromatographed twice. 6 L of the filtrate (46 mg of total protein) was adjusted to pH 9 with 5N NaOH, diluted 2-fold with 6 L of water, and passed through an HQ / M column (20 ⁇ , 10 ⁇ 100 hidden, 8 ml) at 12 ml / min.
  • the adsorbed components were eluted with increasing NaCl concentration. Flow rate 5ml / min, 0-0.5M
  • the solution was passed through 12ral / tnin and chromatographed in the same manner as above.
  • the adsorbed components were eluted with increasing NaCl concentration.
  • the fractionation was performed at a flow rate of 5 ml / min, 0-0.5 NaCl / 50 min, 0.5 M-0.75 M NaCl / 12.5 min, 0.75 M-1 M NaCl / 6.25 min, 1 M NaCl / 10 min, pH 7.5, 5 ml / min / Fr.
  • the anion exchange Q column eluate Fr. 9-64 was combined with 4 Fr each to obtain a total of 14 fractions.
  • a total of 20 ml each of 5 ml was concentrated 5 times to 4 ml with a centrifugal UF membrane (MW3000cut).
  • the purified secretory protein fraction (BSA-free sample) was purified by reverse phase chromatography and subjected to RP-HPLC fractionation.
  • fraction 4F in which neurite outgrowth activity was observed use a VydacC4 (4.6X250 ⁇ ) column and eluate A solution: 0.1% TFA,
  • B solution 0.07% TFA 10%, MeCN 90%, flow rate 0.5ml / min, 20% -50% B solution / 5min, 50% -750% B solution / 30min, 75% -100% B solution /
  • the gradient was eluted with 100% B solution / 5min for 5min, and fractionated at 0.5ml / min / Fr.
  • One kind of protein was eluted in fr.43-44.
  • FCS Fetal serum
  • the GLEXDGKVN I XXKKQFFVSFK sequence was obtained as a result of analyzing the ⁇ -terminal amino acid sequence using Applied Biosystems' protein sequencer type 477. This protein was identified as activin based on the agreement with the ⁇ -terminal amino acid sequence of activin.
  • Cell culture U-87 MG cells (ATCC Number: HTB-14, human brain-derived glioblastoma cells) were used as cells.
  • the growth medium contains DMEM containing 10% FCS, and the production medium contains PMA 20ng / ml (Porbo 12-Myristate 13-Acetate: Wako Pure Chemicals, 20 ⁇ g / ml solution (dissolved in DMS0)) Serum-free medium Hybri doma-SFM medium (Gibco) was used.
  • a 0.25% trypsin-0.02% EDTA solution (Gibco) was used.
  • Preparation of anti-transferrin antibody rams was performed as follows. An anti-human transferrin-Goat antibody manufactured by Cappel was used as an antibody, and activated CNBr-Sepharose4FF manufactured by Pharmacia was used as a carrier. After pretreatment of 70 g of the dried gel, 70 ml of the antibody was bound. Approximately 250 ml of the swollen gel is applied to a Pharmacia XK50 column. I filled it. As a result of measuring the transferrin adsorption ability, about 50 mg of transferrin was bound to 250 ml of genole (5 L for Hybridoma-SFM medium).
  • Ultrafiltration membrane manufactured by Millipore Ultrafiltration and concentration were performed using a Millipore Prep / Scal-TFF (.W. 3000 cut) at a circulation flow rate of about 100 ml / min. About 300 ml of the ultrafiltration concentrate was passed through an anti-serum antibody column (filled with about 750 ml of anti-BSA / BS gel) at a flow rate of 20 to 30 ml / min. Water was passed until the absorbance decreased, and the water was combined. 292 mg of protein was obtained.
  • the primary purified product (135 mg / 450 ml total protein) was filtered through a Millipore ⁇ $ 3 ⁇ 4 filter (0.22 jum), adjusted to pH 8.5 with IN NaOH, diluted twice with 450 ml of mi 11 iQ water, and passed through.
  • the solution was passed through a PorosHQZM column (20, 7.5 ⁇ 200 awake) at 10 ml / min.
  • the adsorption component was eluted with increasing NaCl concentration [concentration gradient: 0-0.4M NaCl / 50min, 0.4M-0.8NaCl / 20min, 0.8M-1M NaCl / lOmin, 1M NaCl / 10min].
  • the pH was adjusted to 8.0, the flow rate was 5 ml / min, and fractionation was performed at 5 ml / min / Fr.
  • the protein eluted in Fr. 6-68.
  • the solution passed through the PorosHQZM column was adjusted to pH 5.5 with 5N HC1, and passed through a PorosH S / M column (10 ⁇ 100 ⁇ , 8 ml) at 15 ml / min to elute the NaCl concentration.
  • Human CCD112C0N cells were reconstituted from frozen cells and subcultured.
  • a DMEM medium containing 10% serum FCS was used as a growth medium, and a Hybridoma SFM serum-free medium (GIBC0) containing 20 ng / ml TPA solution (dissolved in DMS0) was used as a secretion medium.
  • GIBC0 Hybridoma SFM serum-free medium
  • the confluent cultured cells were diluted in a growth medium, inoculated at a concentration of 60,000 cells / ml, in a volume of 90 ral / 225 cm 2 flask, and placed in 170 plates, and cultured at 37 ° C for 3 days.
  • an ion exchange column After removing the serum-derived protein added to the culture medium by passing the serum-free culture supernatant of the CCD112C0N cells through the anti-transferrin antibody force column and the anti-serum antibody force column (primary purification), an ion exchange column is used. And about 800 fractions by gel filtration Fractionated (secondary purification). A portion of this fraction was added with 0.1% BSA to prevent adsorption and stabilize, and then filtered with a 0.45 ⁇ filter to obtain a sample for Atssey. The rest was stored frozen.
  • the protein quantification was performed by the Bio-Rad protein assay method. From 744 mg of protein in culture supernatant, 291 mg of primary purified protein was obtained. SDS-PAGE is a non-reducing electrophoresis on Daiichi Kagaku Multigel “10/20”.
  • the pH of the solution passed through the anion exchange PorosQ column was adjusted to 5.5.
  • the solution was passed through an HSM column (10 ⁇ 100 8 ml) at 15 tnl / tnin to elute the NaCl concentration. pH 6 ⁇ , flow rate 5ml / min, 0-0.25M NaCl / 30min, 0.25M-0.5M NaCl / 15min, 0.5M-1 NaCl / 15min, pH 6, 5ml / min / Fr Painted.
  • VGF8A VGF Protein Precusor
  • the three gel filtration samples were passed through a Vydac C4 column (2.1 x 250 mm) and chromatographed at a flow rate of 0.2 ml / min.
  • a 0.1% trifluoroacetic acid (TFA) solution was used as the solution A, and a 0.08% TFA / 90% acetonitrile solution was used as the solution B.
  • the protein concentration was eluted by increasing the concentration of acetonitrile. A portion of this fraction was filtered with a 0.2 / xm filter after adding 0.1% BSA to stabilize and prevent adsorption, and the library was purified by reversed-phase column chromatography containing a single protein.
  • Approximately 20 L of each of RF-1 cells, CCRF-SB cells, WERI cells, FHs783BL cells, DU4475 cells, U118MG cells, and HS683 cells were cultured according to the method for preparing a CCD-112C0N cell-derived protein library of Example 3.
  • the cell culture supernatant was purified to prepare a secretory protein library.
  • an ion exchange column and gel It was fractionated into about 800 fractions by filtration. If necessary, further fractionation was performed by reversed-phase liquid chromatography.
  • a part of this fraction was adsorbed and added with 0.1% BSA for stabilization, and then filtered through a 0.45 ⁇ filter to obtain a sample for Atsushi.
  • the sample for Atsey was dispensed into a 96-well plate made of polypropylene and supplied to Atsey. The residual solution was stored frozen and used for analysis.
  • the human monocyte cell line THP-1 cells are cultured in RPMI 1640 containing 10% FCS, 100 units of penicillin, and 100 / ig / ml of streptomycin to a concentration of 4 to 6 ⁇ 10 5 cells / ml and 500 g After submerging the cells by centrifugation for 5 minutes, wash once with PBS, and in a fresh medium (medium supplemented with 0.25% BSA instead of FCS) to a cell density of 1 ⁇ 10 5 cels / nil. And suspended.
  • diluted secreted protein samples to an appropriate concentration 100 after i 1 were added to each Ueru of a 96-well microtiter plates (Costar), cell suspensions of equal amounts after mixing C0 2 incubator The cells were cultured at 37 ° C for 3 days. After adding 10 liters of WST-1 reagent (manufactured by Dojinka ⁇ F R & D Laboratories) and culturing for 5 hours, the absorbance at 450 nm was measured with a microtiter spectrophotometer, and used as an index of the proliferation activity. Proliferation activity was shown as a relative value, with the absorbance at the time of culturing without addition of a sample taken as 100.
  • the THP-1 cell proliferation activity protein is IL-6 from the Shi-87-MG cell library (Q6-9 / G7) and urokinase-plasminogen activator receptor from the CCD112C0N cell library (S6-7 / G16 / R17).
  • (Monocyte activation antigen 03j, IGF-II was identified from the CCD112C0N cell library (S25-27 / G19 / R22).
  • Osteoblast MG63 cells were cultured until 10% FCS, the concentration of 4 ⁇ 6 X 10 5 eel ls / ml in RPMI1640 medium containing penicillin 100 units, the scan streptomycin 100 // g / ml, 500g, 5 minutes After submerging the cells by centrifugation, wash once with PBS, and adjust the cell density to 1 x 10 5 cells / ml with fresh medium (medium supplemented with 0.25% BSA instead of FCS). Suspended.
  • IL-6 As a proliferative protein for osteoblast MG63 cells, IL-6 was identified from U87-MG cell library (Q6-9 / G7).
  • Smooth muscle cells are cultured in a medium containing 10% FCS, 100 units of penicillin and streptomycin lOO / zg / ml to a concentration of 4 to 6 x 10 5 cells / ml.
  • the cells were suspended to a cell density of 10 5 cells / ml. Then secreted protein samples 5 mu 1 to 96-well microplate: were added to each Ueru of (Coaster ne t3 ⁇ 4), 100 // 37 ° C , 3 days 1 cell suspension after mixing C0 2 incubator Cultured. After 100 hours of Alamar-I blue reagent was added and the cells were cultured for 5 hours, the absorbance at 450 nm was measured with a microtiter spectrophotometer, and used as an indicator of the proliferation activity. Proliferation activity was shown as a relative value, with the absorbance when the cells were cultured without adding a sample taken as 100.
  • Amphiregulin was identified from (Q56-59 / G19).
  • a plasmid in which the upstream region of the PAI-1 gene is linked to the luciferase gene is introduced into the NRK-49F cell line. Then, a stable transformed cell NRK-D15 was prepared. Suspend the transformed cells in DMEM containing 10% FBS to a concentration of 5 ⁇ 10 4 cels / ml, and add 100 ⁇ l / we 11 to a 96-well white plate (Corning Coaster, Cat. No. 3917). I did it. This C0 2 incubator base in one coater 37 ° C, after incubation for 20-24 hours, the medium is aspirated off, 0.
  • TNF-] 3 from CCRF cell library (Q6-10 / G6 / R80) and TNF-N terminal 59 from CCRF cell library (S12-13 / G8 / R46) A missing group was identified.
  • 293 cells were inserted into the tandem tandem with three NF-kB binding sequences in the upstream region of the mouse immunoglobulin ⁇ chain gene.
  • the resulting strain was transformed into a stable transformant cell 293-kB-N5.
  • 293- KB_N5 cell lines were cultured in 10% FBS, 60 / ig / ral DMEM supplemented with kanamycin, 5 X 10 4 cel ls / well become as 96 well white plate (co one Jung Costar, Cat No. 3917) was added at 100 ⁇ l / well.
  • TNF- ⁇ inhibitor was identified as a TNF_ ⁇ inhibitory protein from the Chang Liver cell library (S7-8 / G18 / R6).
  • a plasmid in which the upstream region of the 2,5AS gene was ligated to the luciferase gene was introduced into the HepG2 cell line to produce a stable transformed cell.
  • Sustained transformed cells were suspended in DMEM at 2 x 10 5 cells / ml in a 96-well white plate (Corning Coaster, Cat. No.
  • Pigment Epithelium-Derived Factor was identified as a 2,5AS gene activating protein from the CCD112C0N cell library (Q20-22 / G17-18 / R84).
  • a secretory protein library useful as a material for searching for a therapeutic agent for various diseases can be provided.
  • the relevant active protein can be searched.
  • One feature of such a protein library is that the protein is a native protein, and further, the following functional analysis and partial structure analysis are easy.
  • the function of a secreted protein can be analyzed by accessing the secreted protein library by various evaluation methods. Furthermore, it is possible to screen bioactive proteins at high speed.
  • the physiological activities include, for example, cell proliferation activity, cell differentiation activity, cytotoxic activity, enzyme activity, enzyme inhibitory activity, ligand binding activity, ligand Binding inhibitory activity, receptor binding activity or receptor binding inhibitory activity.
  • the structure of the secretory protein such as the amino acid sequence
  • the secretory protein library by various preparative methods.
  • the N-terminal amino acid sequence can be structurally analyzed by the Edman method.
  • the partial structure of the amino acid sequence can be analyzed by the mass spectrum method for the constituent peptides produced by enzymatic digestion of the protein.
  • the partial structure is analyzed after further purification by a suitable chromatography method, for example, reverse phase column chromatography.
  • a protein mixture can be directly digested with an enzyme, and the resulting peptide fragments can be analyzed by mass spectrometry to analyze the partial structure of the amino acid sequence.
  • a probe can be prepared based on the partial structure of the amino acid sequence obtained as described above, cDNA can be cloned, and the entire structure of the amino acid sequence of the corresponding secreted protein can be analyzed.

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  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une méthode de construction d'une bibliothèque de protéines sécrétoires, qui consiste à mettre en culture une ligne cellulaire humaine dans un milieu contenant du sérum, puis dans un milieu exempt de sérum contenant éventuellement un agent inducteur de sécrétion. Le procédé consiste ensuite à éliminer les éléments moyens du surnageant de la culture contenant des protéines sécrétoires par la technique de chromatographie sur colonne par anticorps, à l'aide d'une résine à laquelle les anticorps anti-protéines contenus dans le sérum et les éléments moyens sont liés. Le procédé consiste enfin à fractionner et purifier les protéines sécrétoires provenant de la cellule par fractionnement isoélectrique, par fractionnement de masse moléculaire ou par fractionnement hydrophobe.
PCT/JP2000/003214 1999-05-21 2000-05-19 Bibliotheque de proteine secretoire et methode de production de ladite proteine WO2000071580A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999005293A1 (fr) * 1997-07-25 1999-02-04 The Mount Sinai School Of Medicine Of The City University Of New York Procede de preparation de banques d'expression genique enrichies

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999005293A1 (fr) * 1997-07-25 1999-02-04 The Mount Sinai School Of Medicine Of The City University Of New York Procede de preparation de banques d'expression genique enrichies

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
YUKI ISHIDA ET AL.: "Bunshi seibutsugaku kenkyu no tameno tanpaku jikkenhou", JIKKEN IGAKU BESSATSU BIO MANUAL UP SERIES (JAPAN), KABUSHIKI KAISHA YOUDOSHA, 30 October 1998 (1998-10-30), pages 228 - 236, 245-253, XP002946517 *

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