WO2002033070A1 - Acide synthase n-acetylneuraminique - Google Patents

Acide synthase n-acetylneuraminique Download PDF

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Publication number
WO2002033070A1
WO2002033070A1 PCT/JP2001/008935 JP0108935W WO0233070A1 WO 2002033070 A1 WO2002033070 A1 WO 2002033070A1 JP 0108935 W JP0108935 W JP 0108935W WO 0233070 A1 WO0233070 A1 WO 0233070A1
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dna
protein
culture
cell
cells
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PCT/JP2001/008935
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English (en)
Japanese (ja)
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Satoshi Koizumi
Hiroshi Mizoguchi
Mari Ishikawa
Tetsuo Endo
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Kyowa Hakko Kogyo Co., Ltd.
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Priority to AU2001295917A priority Critical patent/AU2001295917A1/en
Priority to JP2002536439A priority patent/JPWO2002033070A1/ja
Publication of WO2002033070A1 publication Critical patent/WO2002033070A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01056—N-acetylneuraminate synthase (2.5.1.56)
    • 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
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Definitions

  • the present invention relates to a protein having N-acetyl neuraminic acid synthase activity, a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant having the recombinant DNA, The present invention relates to a method for producing a protein having N-acetyl neuraminic acid synthase activity using a transformant, and a method for producing N-acetyl neuraminic acid using the transformant. Background surgery
  • N-acetylneuraminic acid synthase an enzyme derived from a microorganism belonging to the genus Escherichia [Glycobiology, 1, 697 (1997) Biosci. Biotech. Biochem., Si, 2046 (1997)] has been obtained.
  • N-acetylneuraminic acid synthase gene a gene derived from a microorganism belonging to the genus Escherichia [J. Bacteriol., M, 312 (1995)], a gene derived from a microorganism belonging to the genus Streptococcus [J. Bacteriol., 181, 5176 (1999)], a gene derived from a microorganism belonging to the genus Campylopa [Mol. Microbiol., 35, 1120 (2000)], a gene derived from a microorganism belonging to the genus Legionella [Int. J. Med. Microbiol., 37 (2000)], but no gene derived from a microorganism belonging to the cyanobacteria or a gene belonging to the genus Nostock is known, and even reports suggesting the presence of the gene are known. Absent.
  • N-Acetylneuraminic acid has been shown to be involved in intercellular recognition at the non-reducing end of sugar chains in vivo [Essentials of Glycobiology, Gold Spring Harbor Laboratory Press ( 1999), Virology,, 19 (1997)], and application as pharmaceuticals are expected.
  • N-acetyl neuraminic acid a method of decomposing colominic acid, which is an N-acetyl neuraminic acid polymer [J. Biochem., 82, 1425 (1977)]
  • a method using an enzyme [ J. Am. Chem. Soc, iiil, 6481 (1988), J. Am. Chem. Soc ⁇ , 7159 (1988), U.S. Pat.No. 5,665,574, Carbohydrate Res., 306, 575 (1998), Kai 10- 4961 Glycobiology, 1, 697 (1997)]
  • An object of the present invention is to provide a protein having N-acetyl neuraminic acid synthase activity, a DNA encoding the protein, a recombinant DNA containing the DNA, and a transformant having the recombinant: DNA.
  • Another object of the present invention is to provide a method for producing a protein having N-acetyl neuraminic acid synthase activity using the transformant, and a method for producing N-acetyl neuraminic acid using the transformant.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and have used the sequence information of Nosiflfi punctiforme ATCC 29133, whose nucleotide sequence of genomic DNA is in progress, to obtain an N-acetylneuron derived from Escherichia coli.
  • searching for a sequence homologous to the amino acid sequence of laminate synthase a DNA encoding a novel N-acetylneuraminic acid synthase, which had not been identified, was found.
  • the present invention has been completed.
  • the present invention relates to the following (1) to (14).
  • a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 and having an N-acetyl neuraminic acid synthase activity .
  • a culture of the transformant of the above (10) or (11) or a processed product of the culture is used as an enzyme source, and the enzyme source, N-acetyl mannosamine and phosphoenolpyruvate are added to an aqueous solution.
  • N-acetylneuraminic acid wherein N-acetylneuraminic acid is produced and accumulated in the aqueous medium, and N-acetylneuraminic acid is collected from the aqueous medium.
  • the processed product of the culture is a concentrate of the culture, a dried product of the culture, a cell obtained by centrifuging the culture, a dried product of the cell, a lyophilized product of the cell, Cell treated with a surfactant, ultrasonically treated cell, mechanically milled cell, solvent-treated cell, enzyme-treated cell, cell-treated enzyme (13)
  • the method according to (13) above which is a protein fraction, an immobilized product of the cells, or an enzyme preparation obtained by extraction from the cells.
  • the protein of the present invention includes N-acetyl having the amino acid sequence represented by SEQ ID NO: 1.
  • a protein having a laminic acid synthase activity, or an amino acid sequence represented by SEQ ID NO: 1 comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, or added; and N-acetylneuraminic acid A protein having a synthase activity can be mentioned.
  • the deletion, substitution or addition of the above amino acids can be performed by the methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Gold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as current 'protocols' in molecular 'biology'), Nucleic Acids Res., IQ, 6487 (1982), Proc. Natl. Acad. Sci. USA, 11, 6409 (1982), Gene,, 315 (1985), Nucleic Acids Res., 11, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985). It can be carried out by introducing a site-specific mutation into a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 using a mutagenesis method.
  • the number of amino acids to be deleted, substituted or added is not particularly limited. , Preferably:! -20, more preferably 1-10, and even more preferably 1-5.
  • Mutations can be introduced into DNA encoding a protein consisting of the amino acid sequence represented by 1. That is, first, a sense primer corresponding to the 5 'end of the DNA and an antisense primer corresponding to the sequence immediately before (5' side) the mutation introduction site having a sequence complementary to the mutation sequence at the 5 'end. PCR is carried out using the DNA as type III, and a fragment A (3, having a mutation introduced at the end) from the 5th end to the mutation introduction site of the DNA is amplified.
  • the DNA is ligated with a sense primer corresponding to the sequence immediately after (3 'side) having the mutation sequence at the 5' end and an antisense primer corresponding to the 3 'end of the DNA.
  • the PCR is performed, and a fragment B from the mutation-introduced site of the DNA having a mutation introduced at the 5 ′ end to the 3 ′ end is amplified. If these amplified fragments are purified and mixed together and PCR is performed without adding Type I primers, Since the sense strand of fragment A and the antisense strand of amplified fragment B have a common mutation-introducing site, they are hybridized, and the PCR reaction proceeds as a primer / gun type, whereby the DNA into which the mutation has been introduced is amplified.
  • deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 means that any one or more amino acid residues in the same sequence
  • the deletion, substitution, insertion or addition may occur at the same time, and the amino acid to be substituted, inserted or added Residues may be natural or non-natural.
  • Natural amino acid remnants include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glucamic acid, glycine, L-histidine, L-isoleucine, L-leucine, and L-lysine.
  • L-arginine L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.
  • amino acid residues that can be substituted for each other.
  • Amino acid residues included in the same group can be substituted for each other.
  • Group A leucine, isoleucine, norleucine, norin, norparin, alanine, 2-aminobutanoic acid, methionine, ⁇ -methylserine, t-butylglycine, t-butylylalanine, cyclohexylalanine
  • Group B aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid
  • Group D lysine, arginine, orditin, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid
  • Group E Proline, 3-hydroxyproline, 4-hydroxyproline
  • Group F serine, threonine, homoserine
  • Group G fenirualanine, tyrosine
  • the protein of the present invention In order for the protein of the present invention to have N-acetyl neuraminic acid synthase activity, it must have at least 60%, usually at least 80%, and especially at least 95% identity with the amino acid sequence represented by SEQ ID NO: 1. It is preferable to have n
  • the amino acid sequence and nucleotide sequence identity can be determined by the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] by Karlin and Altschul. Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)].
  • the DNA of the present invention includes, under stringent conditions, DNA encoding the protein of the present invention, DNA having the nucleotide sequence represented by SEQ ID NO: 2, or DNA having the nucleotide sequence represented by SEQ ID NO: 2.
  • Examples of the DNA include a DNA which hybridizes and encodes a protein having N-acetyl neuraminic acid synthase activity.
  • the DNA hybridizing under the above-mentioned stringent conditions refers to a colony hybridization method using, for example, a DNA fragment of the present invention such as a DNA having the nucleotide sequence of SEQ ID NO: 2 or a partial DNA fragment thereof as a probe.
  • a DNA fragment of the present invention such as a DNA having the nucleotide sequence of SEQ ID NO: 2 or a partial DNA fragment thereof as a probe.
  • the DNA is obtained by using a filter in which DNA derived from colony or plaque is immobilized.
  • DNA that can be used have a homology of at least 60% or more with the nucleotide sequence represented by SEQ ID NO: 2 when calculated based on the above algorithm BLAST.
  • DNA DNAs having a homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more can be mentioned.
  • Nostoc uncti forme ATCC29133 is currently undergoing nucleotide sequencing for its genomic DNA
  • any amino acid sequence of a protein having N-acetyl neuraminic acid synthase activity can be used.
  • specific examples include the amino acid sequence of N-acetylneuraminic acid synthase derived from Escherichia coli [J. Bacteriol., 117, 312 (1995)].
  • search method a method of performing a homology search on the database using a program such as the above BLAST or FASTA can be exemplified.
  • N-acetyl neuraminic acid synthase is low among different kinds of organisms, and thus the sequence selected above is a protein that actually has N-acetyl neuraminic acid synthase activity. You need to make sure it is.
  • the DNA of the present invention can be prepared from a microorganism belonging to the cyanobacterium.
  • the microorganism belonging to the cyanobacterium include, for example, microorganisms belonging to Nostock.
  • the microorganisms belonging to the cyanobacterium include, for example, Nostoc uctifornte ATCC29133. Microbiology, Cliff, 3233 (1994)].
  • the chromosomal DNA of the microorganism is isolated and purified by a known method (for example, current 'protocols' in' molecular virology).
  • the fragment containing the DNA of the present invention can be obtained by preparing a primer based on the nucleotide sequence of the genome specified in the above (1), using the genomic DNA as a type II, PCR method [PCR Protocols, Academic Press ( 1990)].
  • the target DNA can be obtained by a hybridization method using synthetic DNA designed as a probe based on the base sequence of the genome.
  • the obtained DNA is used as it is, or after digestion with an appropriate restriction enzyme or the like, incorporated into a vector by a conventional method, and a commonly used nucleotide sequence analysis method, for example, the dideoxy method [Proc. Natl. Acad. Sci. USA, 74: , 5463 (1977)] or 373A.
  • DNA sequencer Perkin-Elma Inc. or the like can be used to determine the nucleotide sequence of the DNA.
  • Examples of the vector incorporating the DNA include pBluescript KS (+) (Stratagene), pDIRECT [Nucleic Acids Res., 18, 6069 (1990)], pCR-Script Amp SK (+) (Stratagene) PT7Blue (Novadin), pCR11 (Invitrogen) and pCR-TRAP
  • the desired DNA can be prepared by chemical synthesis using Perceptive's 8905-type DNA synthesizer manufactured by Biosystems.
  • Examples of the DNA having the novel nucleotide sequence obtained as described above include DNA having the nucleotide sequence represented by SEQ ID NO: 2, and the like.
  • Examples of Escherichia coli having a plasmid having a DNA having a nucleotide sequence represented by SEQ ID NO: 2 include Miaiiklik coli banded 522 / pNPl described below.
  • Examples of host E. coli harboring the recombinant DNA include, for example, Escherichia coli XLl-Blue, Escherichia coli XL2-Blue, Escherichia coli DHl, Rscherichia coli MC1000, Escherichia coli KY3276, Escherichia ooli W1485, Kscherichia.ooli JM109, EscherOKaHB Escherichia coli No. 49, Escherichia coli W311Q, Rscherichia coli NY49, Rscherichia coli MP347, Rscherichia coli NM522 and the like can be mentioned.
  • any method can be used as long as it is a method for introducing DNA into the above host cells.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], protoplast method (JP-A-63-248394), electroporation method [Nucleic Acids Res., I £, 6127 (1988)] and the like.
  • the protein of the present invention can be obtained by the method described in the above [1], for example, by the method described in Molecular 'Clothing Second Edition, Current' Protocols' in 'Molecular' Biology, etc. Can be produced by expressing the DNA of the present invention obtained by the method described above in a host cell.
  • a DNA fragment having an appropriate length containing a portion encoding the protein is prepared. Further, by substituting the base so that the nucleotide sequence of the portion encoding the protein has an optimal codon for expression in the host, the production rate of the protein can be improved.
  • the recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector.
  • a transformant producing the protein of the present invention By introducing the recombinant DNA into a host cell suitable for the expression vector, a transformant producing the protein of the present invention can be obtained.
  • any cells that can express the gene of interest such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
  • DNA it is capable of autonomous replication in the above-mentioned host cell or is assembled into a chromosome.
  • a promoter containing a promoter at a position where the DNA of the present invention can be transcribed is used.
  • the recombinant DNA containing the DNA encoding the protein of the present invention is capable of autonomous replication in the prokaryote, and at the same time, promotes ribosome binding. It is preferably a recombinant DNA comprising the sequence, the DNA of the present invention, a transcription termination sequence. A gene that controls the promoter may be included. PHelixl (manufactured by Roche Diagnostics), PKK233-2
  • Any promoter may be used as long as it functions in a host cell such as Escherichia coli.
  • tra promoter Pine
  • lac promoter Isseki one Plaa
  • P L promoter Isseki one P R promoter and P SE promoter
  • promoter derived from E. coli and file one di-, etc. SP 0 1 promoter
  • SP02 Promo pen P promoter and the like.
  • the two series are not the promoter Ptrp (Pke x 2), ia flop port motor, i AcT7 promoters artificially designed and modified promoters like l et I promoter, etc. can also be used.
  • a plasmid in which the distance between the Shine-Dalgarno sequence, which is the ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to ⁇ bases).
  • a transcription termination sequence is always required for expression of the DNA of the present invention.
  • a transcription termination sequence immediately below the structural gene Prokaryotes include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevipacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Kscherichi coli XLl-Blue, Esoheri nhi col i XL2-Blue, Escheri chi col i DH1, Escherichia coli MC1000, Escherichia noli KY3276, Escheri ctik mli W1485, Escherichia coli JM109, Escherichia coli HBi01, Escherichia col i No.49, Escherichia coli W3ll0s Esicaria rata , Serratia fonticola, Ser
  • any method for introducing the DNA into the above host cells can be used.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, M, 2110 (1972)], protoplast method (JP-A-63-248394), and electroporation method [Nucleic Acids Res., Ifi, 6127 (1988)].
  • YEpl3 ATGG37U5
  • YEp24 ATCC37051
  • YCp50 ATGC37419
  • pHS19, pHS15 and the like can be used as expression vectors.
  • Any promoter may be used as long as it functions in the yeast strain.
  • Examples of the host cell include yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybaceae, Trichosporon, Schizinomyces, Pichia, Candida, etc. Saccharomvces cerevi si ae.
  • any method can be used as long as it is a method for introducing the DNA into yeast.
  • the elect opening method [Methods Enzymol., M, 182 (1990)]
  • the lithium acetate method [J. Bacteriol., 153, 163 (1983)]
  • an expression vector for example, pcDNAI, pcDM8 (commercially available from Funakoshi), AGE107 (JP-3- 22979), P AS3-3 (JP 2-227075), pCDM8 [Nature , 840 (1987)], pcDNAI / Amp (manufactured by Invitrogen), PREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem, Dish, 1307 (1987)], pAGE2lO, pAMo, pAMoA and the like can be used. .
  • any promoter can be used as long as it functions in animal cells.
  • the promoter of the immediate early (IE) gene of cytomegalovirus (CMV) include a thionine promoter, a retrovirus promoter, a heat shock promoter, and an SR promoter.
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • Host cells include mouse myeloma cells, rat 'myeloma cells', mouse 'hybridoma cells', human Namalwa cells or Namalwa KJM-1 cells, human fetal kidney cells, human leukemia Cell, African green monkey kidney cell, CH0 cell which is a Chinese-hamster cell, HBT5637 (Japanese Patent Application Laid-Open No. 63-299), and the like.
  • Mouse / myeloma cells include SP2 / 0, NS0, etc.
  • rat's myeloma cells include YB2 / 0, etc.
  • human fetal kidney cells include HEK293 (ATCC: CRL-1573), etc.
  • human leukemia cells include BALL-1.
  • African green monkey kidney cells include COS-1 and COS-7.
  • any method can be used as long as it is a method for introducing DNA into animal cells.
  • an electroporation method [Cyto-7 echnology, 3, 133 (1990)]
  • the calcium phosphate method Japanese Patent Laid-Open No. 2-227075
  • the lipofusion method [Proc. Natl. Acad. Sci. USA, M, 7413 (1987)]
  • the method described in Virology, 52, 456 (1973). can be raised.
  • the recombinant gene transfer vector and baculovirus are co-transfected into insect cells to obtain recombinant virus in the insect cell culture supernatant, and then the recombinant virus is infected into insect cells to express the protein. Can be done.
  • Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacII (all manufactured by Invitrogen) and the like.
  • the baculovirus e.g., monkey in be used such as out publicly available, power Nuclear Doroshisu 'virus to single-poly is a virus that infects burglary Gaka insects (Autographa californica nuclear polyhedrosis virus) 0
  • insect cells single cells of eggs of Spofioptera. Fn] giperria, ovary cells of Ikhopliiaiaiii, cultured cells derived from silkworm ovary, and the like can be used.
  • BTI-TN-5B1-4 Cultured cells derived from silkworm ovaries, such as Invitrogen can be exemplified by mhyx moi.
  • Examples of the method for co-transferring the above-described baculovirus and the above-described recombinant gene transfer vector into insect cells for preparing a recombinant virus include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • an expression vector for example, Ti Bra Sumid, tobacco mosaic virus vector and the like can be mentioned.
  • Any promoter may be used as long as it functions in a plant cell, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the geneactin 1 promoter.
  • CaMV cauliflower mosaic virus
  • Examples of the host cell include plant cells of tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, wheat, and the like.
  • any method can be used as long as it is a method for introducing DNA into plant cells.
  • agropacterium toh kriM
  • electro-volatilization method JP-A-60-251887
  • method using a particle gun Gene gun
  • Patent No. 2606856, Patent No. 2517813 Patent No. 2606856, Patent No. 2517813
  • a sugar or sugar chain-added protein When a gene is expressed using yeast, animal cells, or insect cells as a host, a sugar or sugar chain-added protein can be obtained.
  • the protein of the present invention can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the protein of the present invention in the culture, and collecting from the culture.
  • the method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.
  • a culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can be efficiently cultured.
  • the carbon source may be any one that can be assimilated by the organism, such as glucose, fructose, sucrose, carbohydrates such as rice nectar, starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Acids, alcohols such as ethanol and propanol, and the like can be used.
  • Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate.
  • Ammonia, ammonium salts of inorganic or organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn starch, casein hydrolyzate, soybean meal and soybean meal Hydrolysates, various fermentation cells, and digests thereof can be used.
  • potassium (II) phosphate potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.
  • the cultivation is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culture temperature is
  • the temperature is preferably 15 to 40 ° C, and the culture time is usually 5 hours to 7 days.
  • the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.
  • an antibiotic such as ampicillin-tetracycline may be added to the medium during the culture.
  • an inducer may be added to the medium, if necessary.
  • an inducer may be added to the medium, if necessary.
  • indoleacrylic acid or the like may be added to the medium.
  • RPMI 1640 medium J. Am. Med. Assoc., Dist., 519 (1967)]
  • Eagle's MEM medium Science , 501 (1952)]
  • DMEM medium DMEM medium [Virology, S, 396 (1959)]
  • 199 medium Proc. Soc. Biol. Med., ⁇ , 1 (1950)]
  • fetal calf serum or the like was added to these mediums.
  • a medium or the like can be used.
  • Culture is usually pH 6-8, it performed 1-7 ⁇ under conditions such as 25 ⁇ 40 ° C, 5% C 0 2 presence.
  • an antibacterial substance such as kanamycin, penicillin or streptomycin may be added to the medium during the culturing.
  • culture media for transformants obtained using insect cells as a host include T ⁇ -FH media (Pharmingen) and Sf-900 II SFM media (Life's Technologies).
  • ExCell400, ExCell140 (all manufactured by JRH Biosciences), Grace's Insect Medium [Nature, 195, 788 (1962)] and the like can be used.
  • Cultivation is usually carried out for 1 to 5 days under conditions of pH 6 to 7 and 25 to 30 ° C.
  • an antibiotic such as genyumycin may be added to the medium during the culture.
  • a transformant obtained using a plant cell as a host can be cultured as a cell or after being differentiated into a plant cell or organ.
  • a medium for culturing the transformant commonly used Murashige and Skoog (MS) medium, white (White) medium, or a plant hormone such as auxin or cytokinin is added to these mediums.
  • MS Murashige and Skoog
  • White white
  • a plant hormone such as auxin or cytokinin
  • the cultivation is usually performed at pH 5 to 9 and 20 to 40 ° C for 3 to 60 days.
  • antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.
  • a transformant derived from a microorganism, animal cell, or plant cell having a recombinant DNA into which the DNA encoding the protein of the present invention has been incorporated is cultured according to a conventional culture method, and the protein is purified. By producing and accumulating the protein, and collecting the protein from the culture, the protein can be produced.
  • the method for producing the protein of the present invention includes a method of producing the protein in a host cell, a method of secreting the protein outside the host cell, and a method of producing the protein on the host cell outer membrane.
  • the protein of the present invention is expressed by adding a signal peptide in front of the protein containing the active site of the protein of the present invention using a gene recombination technique, whereby the protein of the present invention can be used as a host. It can be secreted extracellularly.
  • the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
  • an individual animal (transgenic non-human animal) or individual plant (transgenic plant) into which the gene has been introduced is created.
  • the protein of the present invention can be produced using an individual.
  • the transformant is an animal or plant individual
  • the protein is produced or cultivated according to a conventional method to produce and accumulate the protein, and the protein is collected from the animal or plant individual to obtain the protein. Can be manufactured.
  • the protein can be produced.
  • the production-accumulation site in the animal include milk (Japanese Patent Application No. 63-309192), eggs, and the like of the animal.
  • Any promoter that can be used in this case can be used as long as it functions in animals. Examples of such promoters include casein promoters, casein promoters, lactoglobulin promoters, and whey acid, which are breast cell-specific promoters.
  • sexual protein promoters and the like are preferably used.
  • Examples of the method for producing the protein of the present invention using plant individuals include, for example, transgenic plants into which DNA encoding the protein of the present invention has been introduced [Tissue culture, 0 (1994), Tissue culture, 21 (1995) , Pp. 45 (1997)], producing and accumulating the protein in the plant ⁇ , and collecting the protein from the plant to produce the protein. can give.
  • the protein produced by the transformant of the present invention is isolated and purified by a conventional method. Enzyme isolation and purification methods can be used.
  • the cells when the protein of the present invention is expressed in a dissolved state in a cell, the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, or the like. The cells are disrupted using a Manton Gaurin homogenizer, Dynomill, etc. to obtain a cell-free extract.
  • a normal enzyme isolation / purification method that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Luminoethyl (DEAE) —Anion exchange chromatography using resins such as Sepharose and DIAION HPA-75 (Mitsubishi Kasei), and cation exchange chromatography using resins such as S-Sepharose FF (Pharmacia) Electrophoresis such as chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenylsepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc.
  • a purified sample can be obtained using techniques such as electrophoresis alone or in combination.
  • the cells are similarly recovered, crushed, and separated from the precipitate fraction obtained by performing eccentric separation. After recovery, the insoluble form of the protein is solubilized with a protein denaturant.
  • a purified sample can be obtained by the same isolation and purification method as described above.
  • the protein of the present invention or its derivative such as a modified sugar is secreted extracellularly, the protein or its derivative such as a sugar chain adduct can be recovered in the culture supernatant.
  • a soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.
  • Examples of the protein obtained in this manner include a protein having the amino acid sequence shown in SEQ ID NO: 1.
  • polypeptide of the present invention is produced as a fusion protein with another protein
  • Purification can also be performed using affinity chromatography using a substance having an affinity for the combined protein.
  • the method of Lowe et al. Proc. Natl. Acad. Sci. USA, 2 £, 8227 (1989), Genes Develop., I, 1288 (1990)], JP-A-5-336963, W094 / 23021.
  • the polypeptide of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using imnoglobulin G.
  • polypeptide of the present invention can be produced as a fusion protein with an F1ag peptide and purified by affinity chromatography using an anti-F1ag antibody [Proc. Natl. Acad. Sci. USA , S £, 8227 (1989), Genes Develop., 4, 1288 (1990)]. Furthermore, the polypeptide can be purified by affinity chromatography using an antibody against the polypeptide itself.
  • the chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the Seven Boc method (71-butyloxycarbonyl method)
  • the protein of the invention can be produced. Advanced ChemTech, Parkin
  • Chemical synthesis can also be performed using peptide synthesizers such as Elma, Pharmacia, Protein Technology Instrumen, Synthecell-Vega, PerSeptive, and Shimadzu.
  • peptide synthesizers such as Elma, Pharmacia, Protein Technology Instrumen, Synthecell-Vega, PerSeptive, and Shimadzu.
  • a culture of the transformant obtained by the culture according to the above [2] and a processed product of the culture are used as an enzyme source, and the enzyme source, N-acetyl mannosamine and phosphoenolpyruvate are added to an aqueous medium.
  • N-acetylneuraminic acid can be produced in the aqueous medium.
  • Examples of the processed product of the culture include a concentrate of the culture, a dried product of the culture, cells obtained by centrifuging the culture, a dried product of the cells, a freeze-dried product of the cells, the cells Surfactant treated product, ultrasonically treated product of the cells, mechanically milled product of the cells, solvent-processed product of the cells, enzyme-treated product of the cells, protein fractionation of the cells Products, immobilized products of the cells or enzyme preparations obtained by extraction from the cells.
  • the enzyme source used in the production of N-acetylneuraminic acid is l ⁇ mol / min at 37 ° C per minute.
  • the activity capable of producing N-acetylneuraminic acid as 1 unit (U) is 1 mU / 1 to 1,000 U / 1, preferably a concentration of 10 mU / l to 100 U / 1. Used in
  • Aqueous media used in the production of N-acetylneuraminic acid include water, phosphates, carbonates, acetates, buffers such as borate, citrate and tris, and alcohols such as methanol and ethanol. And esters such as ethyl acetate, ketones such as acetone, and amides such as acetoamide. Further, a culture solution of the microorganism used as the enzyme source can be used as an aqueous medium.
  • a surfactant or an organic solvent may be added as necessary.
  • the surfactant include nonionic surfactants such as polyoxyethylene octadecylamine (for example, Nimeen S-215, manufactured by NOF Corporation), cetyltrimethylammonium bromide alkyldimethylbenzylbenzyl chloride, and the like.
  • cationic surfactants such as cation F2-40E, manufactured by NOF Corporation
  • anionic surfactants such as lauroyl'zalcosinate
  • alkyldimethylamine for example, tertiary amine FB, manufactured by NOF Corporation
  • the surfactant is usually used at a concentration of 0.1 to 50 g / 1.
  • the organic solvent include xylene, toluene, aliphatic alcohol, acetone, and ethyl acetate, which are usually used at a concentration of 0.1 to 50 ml / l.
  • the reaction for producing N-acetyl neuraminic acid is carried out in an aqueous medium at pH 5 to 10, preferably pH 6 to 8, and 20 to 50 ° G for 1 to 96 hours.
  • inorganic salts such as Mn C 1 2 if necessary.
  • the quantification of N-acetyl neuraminic acid formed in the aqueous medium can be performed using a sugar analyzer manufactured by Dionex or the like [Anal. Biochem., 189, 151 (1990)].
  • FIG. 1 is a diagram showing a process for constructing a plasmid pNP1 expressing N-acetyl neuraminic acid synthase.
  • a mp r Ampicillin resistance gene
  • n e u B N-Acetylneuraminic acid synthase gene
  • Example 1 Homology Search Using Genome D ⁇ ⁇ Sequence Database
  • an amino acid sequence represented by SEQ ID NO: 1 was obtained as an amino acid sequence having high homology to the amino acid sequence of N-acetylneuraminic acid synthase derived from Escherichia coli, and a DNA encoding the amino acid sequence was obtained.
  • DNA having the nucleotide sequence represented by SEQ ID NO: 2 was obtained.
  • Nostoc punctiforme ATCC29133 was cultured by the method described in Microbiology, 140, 3233 (1994).
  • the chromosome DNA of the microorganism was isolated and purified by the method described in Current “Protocols” in “Molecular Biology”.
  • Example 1 Using a DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4 synthesized using a Perceptive Biosystems Model 8905 DNA synthesizer, the DNA fragment containing the gene selected in Example 1 was used. The width was increased by the following method.
  • PCR is a reaction containing 0.1 mg of chromosomal DNA, 0.5 mmol / U of each primer, 2.5 units of Pfu DNA polymerase (manufactured by Stratagene), 4 ml of x10 buffer for Pfu DNA polymerase, and 200 imnol / 1 of deoxyNTP.
  • Pfu DNA polymerase manufactured by Stratagene
  • 4 ml of x10 buffer for Pfu DNA polymerase 4 ml of x10 buffer for Pfu DNA polymerase
  • 200 imnol / 1 of deoxyNTP Using 40 ml of the liquid, the process was repeated 30 times at 9.4 ° C for 1 minute, at 42 ° C for 2 minutes, and at 72 ° C for 3 minutes.
  • the DNA precipitate was dissolved in 20 ml of TE.
  • DNA fragments were separated by agarose gel electrophoresis, and a 1.0 kb: DNA fragment was purified using GeneClean II kit (Funakoshi). Collected.
  • Escherichia coli 522522 was transformed according to the above-mentioned known method, and the transformant was LB agar medium containing 50 mg / ml of ampicillin [Pactotripton (Difco) iOg / Yeast extract] (Manufactured by Difco), 10 g / l, sodium chloride 5 g / l, agarose 15 g], and cultured overnight at 28 ° C.
  • Plasmid was extracted from the thus grown transformant colonies according to the above-mentioned known method to obtain pNP1, an expression plasmid.
  • Fig. 1 shows the construction procedure and structure of the plasmid.
  • the Escherichia, coli ⁇ 522 / pNPl strain obtained in Example 2 was transformed with L containing 50 mg / ml of ampicillin.
  • a large test tube containing 8 ml of B medium was inoculated and cultured at 28 ° C for 17 hours.
  • the culture solution was placed at 1 ° / ⁇ in a large test tube containing 8 ml of LB medium containing 50 mg / ml of ampicillin. After inoculation and culturing at 28 ° C for 5 hours, 0.4 ml of the culture was centrifuged to obtain wet cells.
  • the wet cells could be stored at -20 ° C if necessary, and could be thawed before use.
  • reaction product was analyzed under the following analysis conditions using a sugar analyzer (DX-500, manufactured by Dionex), and 0.48 ol / l (150 mg / l) of N-acetylethyl It was confirmed that laminic acid was generated and accumulated.
  • N-acetyl neuraminic acid synthase can be produced in large quantities.
  • N-acetylneuraminic acid can be efficiently produced by using the enzyme.

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Abstract

L'invention concerne une nouvelle protéine présentant une activité d'acide synthase N-acétylneuraminique; un ADN codant cette protéine; un ADN recombiné contenant l'ADN précité; un transformant porteur dudit ADN recombiné; et enfin, une technique de fabrication de ladite protéine ou de l'acide N-acétylneuraminique au moyen de ce transformant.
PCT/JP2001/008935 2000-10-16 2001-10-11 Acide synthase n-acetylneuraminique WO2002033070A1 (fr)

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JP2008005794A (ja) * 2006-06-30 2008-01-17 Kyowa Hakko Kogyo Co Ltd シチジン‐５´‐一リン酸‐ｎ‐アセチルノイラミン酸およびｎ‐アセチルノイラミン酸含有糖質の製造法
EP1987137A1 (fr) * 2006-02-09 2008-11-05 Medicago Inc. Synthèse d'acide sialique dans des végétaux
US10378034B2 (en) 2014-05-27 2019-08-13 Universitetet I Tromsø—Norges Arktiske Universitet Use of a N-acetylneuraminate lyase derived from the bacterium Aliivibrio salmonicida in the production of neuraminic acid and derivatives thereof

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LINTON D. ET AL.: "Multiple N-acetylneuraminic acid synthase(neuB) genes in campylobacter jejuni: identification and characterization of the gene involved in sialylation of lipo-oligosaccharide", MOL. MICROBIOL., vol. 35, no. 5, March 2000 (2000-03-01), pages 1120 - 1134, XP002907706 *
MARU I. ET AL.: "Simple and large-scale production of N-acetylneuraminic acid from N-acetyl-D-glucosamine and pyruvate using N-acyl-D-glucosamine 2-epimerase and N-acetylneuraminate lyase", CARBOHYDRATE RES., vol. 306, 1998, pages 575 - 578, XP004127022 *
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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1987137A1 (fr) * 2006-02-09 2008-11-05 Medicago Inc. Synthèse d'acide sialique dans des végétaux
EP1987137A4 (fr) * 2006-02-09 2009-09-16 Medicago Inc Synthèse d'acide sialique dans des végétaux
US9212372B2 (en) 2006-02-09 2015-12-15 Medicago Inc. Synthesis of sialic acid in plants
US9657305B2 (en) 2006-02-09 2017-05-23 Medicago Inc. Synthesis of sialic acid in plants
JP2008005794A (ja) * 2006-06-30 2008-01-17 Kyowa Hakko Kogyo Co Ltd シチジン‐５´‐一リン酸‐ｎ‐アセチルノイラミン酸およびｎ‐アセチルノイラミン酸含有糖質の製造法
US10378034B2 (en) 2014-05-27 2019-08-13 Universitetet I Tromsø—Norges Arktiske Universitet Use of a N-acetylneuraminate lyase derived from the bacterium Aliivibrio salmonicida in the production of neuraminic acid and derivatives thereof

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