WO2001077314A1 - Gene $g(a)2,3-sialyltransferase modifie, son procede de production et saccharide complexe contenant de l'acide sialique - Google Patents

Gene $g(a)2,3-sialyltransferase modifie, son procede de production et saccharide complexe contenant de l'acide sialique Download PDF

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WO2001077314A1
WO2001077314A1 PCT/JP2001/003110 JP0103110W WO0177314A1 WO 2001077314 A1 WO2001077314 A1 WO 2001077314A1 JP 0103110 W JP0103110 W JP 0103110W WO 0177314 A1 WO0177314 A1 WO 0177314A1
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dna
protein
genus
sialic acid
sialyltransferase
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PCT/JP2001/003110
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English (en)
Japanese (ja)
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Tetsuo Endo
Satoshi Koizumi
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Kyowa Hakko Kogyo Co., Ltd.
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Priority to AU2001248749A priority Critical patent/AU2001248749A1/en
Publication of WO2001077314A1 publication Critical patent/WO2001077314A1/fr

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    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1081—Glycosyltransferases (2.4) transferring other glycosyl groups (2.4.99)
    • 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 modified ⁇ 2,3-sialyltransferase gene, and a method for producing a 2,3-sialyltransferase and a sialic acid-containing glycoconjugate using the gene.
  • 2,3-sialyltransferase gene a gene derived from an animal [J. Biol. Chem., 267, 21011 (1992), J. Biol. Chem., 268, 22782 (1993), Eur. J. Biochem. ., 216, 377 (1993), Biochem. Biophys. Res. Co., 194, 375 (1993), J. Biol. Chem., 269, 1394 (1994), J. Biol. Chem., 269, 10028 (1994), Glycobiology, 5, 319 (1995)]. It has been reported that the enzyme gene is expressed as an active protein in microorganisms such as Escherichia coli (JP-A-11-253163), but its activity is weak.
  • Sialic acid-containing glycoconjugates are known to be involved in cell adhesion and canceration of cells.Essentials of Glycobiolgy, Cold Spring Harbor Laboratory Press (1999)], pharmaceuticals such as cancer vaccines and reperfusion injury The application as is expected.
  • oligosaccharides containing sialic acid which are abundant in human milk, are known to function as receptors for influenza virus. Virology, 232, 19 (1997)], and are considered to be promising candidates for safe infection preventives.
  • an extraction method from human milk CAnal.
  • An object of the present invention is to provide an industrial method for producing 2,3-sialyltransferase and sialic acid-containing glycoconjugates.
  • the present inventors have conducted intensive studies to solve the above-mentioned problems, and have described a novel 2,3-sialyltransferase which has not been expressed in large amounts as an active protein in microorganisms such as Escherichia coli.
  • the inventors succeeded in mass production using Escherichia coli and established an industrial method for producing sialic acid-containing glycoconjugates using the enzyme.
  • the present invention has been completed.
  • the present invention relates to the following (1) and (20).
  • a culture solution of a transformant expressing a protein having 2,3-sialyltransferase activity derived from a microorganism belonging to the genus Hemophilus or a processed product of the culture solution is used as an enzyme source.
  • Complex glycoconjugates and cytidine monophosphate N-acetylneuraminic acid (hereinafter abbreviated as CMP—NeuAc) are allowed to exist in an aqueous medium, and the sialic acid is converted to the receptor via the 2,3 bond in the aqueous medium.
  • a method for producing a sialic acid-containing glycoconjugate comprising producing and accumulating a sialic acid-containing glycoconjugate by transferring to a glycoconjugate, and collecting the sialic acid-containing glycoconjugate from the aqueous medium.
  • a transformant expressing a protein having a 2,3 -sialyltransferase activity derived from a microorganism belonging to the genus Hemophilus is cultured in a medium, and the 2,3 -sialyltransferase activity is added to the culture.
  • microorganism is a microorganism belonging to the genus Escherichia.
  • the recombinant DNA is a recombinant DNA containing DNA encoding a protein having 2,3-sialyltransferase activity derived from a microorganism belonging to the genus Hemofilus. Production method as described.
  • a DNA encoding a protein having a 2,3-sialyltransferase activity derived from a microorganism belonging to the genus Hemophilus is selected from any one of the following [1] to [3]: The production method according to (6).
  • One or more codons in a DNA encoding a protein having an activity of 2,3-sialyltransferase derived from a microorganism belonging to the genus Hemophilus are frequently used in a host cell expressing the DNA.
  • the processed product of the culture solution is a concentrate of the culture solution, a dried product of the culture solution, cells obtained by centrifuging the culture solution, a dried product of the cells, a lyophilized product of the cells, Detergent treated cells, ultrasonically treated cells, mechanically milled cells, solvent-treated cells, enzyme-treated cells, protein of the cells.
  • the method for producing a sialic acid-containing glycoconjugate according to (1) which is a fraction, an immobilized product of the cells, or an enzyme preparation obtained by extraction from the cells. .
  • the DNA of the present invention is a DNA obtained by modifying a DNA encoding a protein having a 2,3-sialyltransferase activity derived from a microorganism belonging to the genus Hemofilus, such as a DNA derived from Hemophilus' Duclay.
  • a DNA encoding a protein having 2,3-sialyltransferase activity having a base sequence obtained by modifying one or more codons to codons frequently used in a host organism expressing the DNA; It encodes DNA and a protein comprising a base sequence in which at least one base among the bases of the DNA comprising the base sequence has been deleted, substituted or added, and which has a 2,3-sialyltransferase activity DNA and the like.
  • the host organism is not particularly limited as long as it can express the DNA of the present invention, and may be an individual organism, tissue of an organism, cultured cells of an organism, microbial cells, or the like as the host organism. Can be.
  • Preferred examples of the host organism include microorganisms, more preferably microorganisms belonging to the genus Escherichia, and particularly preferably Escherichia coli.
  • a frequently used codon is a codon in which the codon corresponding to each amino acid is at least 10% or more in the host cell, preferably 15% or more, more preferably 20% or more. Is a codon.
  • Amino acid deletion or substitution occurring in the protein encoded by the mutation-introduced DNA is not particularly limited, but is, for example, 1 to 20, preferably 1 to 15, and more preferably 1 to 5.
  • the mutated DNA in order for the DNA of the present invention to encode a protein having 2,3-sialyltransferase activity, the mutated DNA must have at least the amino acid sequence of SEQ ID NO: 2 and at least BLAST CJ. Mol. Biol. 215, 403 (1990)) and FASTA (Methods in Enzymology, 183, 63-98 (1990)), etc., have a homology of at least 60% or more, usually 80% or more, especially 95% or more. It is preferable to have
  • DNA of the present invention using DNA encoding a protein having a 2,3-sialyltransferase activity derived from Hemophilus duclay, for example, Molecular 'cloning second edition, Current 'Protocols in Molecular Biology, Nucleic Acids Research, 10, 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 (1985), Natl. Acad. Sci. USA, 82, 488 (1985), Nucleic Acids Research, 13, 4431 (1985), and the like.
  • Methods using synthetic DNA include, for example,
  • DNA encoding the protein produced by the above method may be prepared according to the method described in Molecular Cloning, Second Edition. Recombinant DNA was prepared by ligating with DNA
  • a host organism is transformed with the recombinant DNA,
  • the obtained transformant is cultured in an appropriate medium in which the transformant can grow, the protein is accumulated in the culture solution, and the protein is obtained from the culture solution.
  • a method of solubilizing the protein, isolating and purifying the protein by ion exchange, gel filtration, one method such as hydrophobic chromatography, or a combination of the chromatography methods is used. can give.
  • the sialic acid-containing glycoconjugate of the present invention can be obtained by using a culture solution of a transformant producing the protein, a processed product of the culture solution, or the purified protein as an enzyme source, a receptor glycoconjugate, CMP-Neu It can be obtained by allowing Ac to coexist in an aqueous solvent.
  • the receptor complex carbohydrate is not particularly limited as long as it becomes a substrate for a protein having a 2,3-sialyltransferase activity encoded by the DNA of the present invention, but preferably, galactose is present at the non-reducing end.
  • a more preferred substrate has a lactose, N-acetyl-lactosamine, lactate N-tetraose, lactate N-neotetraose, Lewis X, or Lewis a structure at the non-reducing end.
  • Sugars can be given. '
  • the generated sialic acid-containing glycoconjugate can be obtained by a usual chromatography method using activated carbon, an ion exchange resin, or the like.
  • the DNA of the present invention can be prepared by the following method. First, the amino acid sequence of 2,3-sialyltransferase is selected. As the amino acid sequence of the enzyme, any amino acid sequence having the enzyme activity can be used. For example, Hemophilus registered in a database such as Genebank represented by SEQ ID NO: 2 can be used. ⁇ Of 2,3-sialyltransferase derived from Duclay Amino acid sequences and the like. Next, a DNA encoding a protein having the enzymatic activity is designed using codons frequently used in host cells expressing the enzymatic activity.
  • the amino acid sequence of the selected enzyme is used as the frequency of codon usage found in the nucleotide sequence of the Escherichia coli gene (Codon Usage aataDase at kazusa (http: // www. In consideration of kazusa.or. jp / codon /)], the DNA of the present invention can be designed by converting it into a DNA sequence so as to have the most frequently used codon.
  • Examples of the DNA designed as described above include DNA having a base sequence of SEQ ID NO: 1 designed based on a 2,3-sialyltransferase gene derived from Hemophilus duclay. .
  • the adjacent synthetic DNAs have an overlapping sequence of 10 to L00 bases with each other, and the 5′-ends are arranged so that they alternate with the sense strand and the antisense strand.
  • Synthetic DNA having a length of 40 to 150 bases from the side is synthesized using an automatic DNA synthesizer (Model 8905 DNA synthesizer manufactured by Perceptive Biosystems). Examples of such synthetic DNAs include DNAs having the nucleotide sequences of SEQ ID NOs: 3 to 16 designed based on the DNA consisting of the nucleotide sequence of SEQ ID NO: 1.
  • the DNA of the present invention is artificially synthesized by PCR according to a conventional method (eg, PCR Protocols, Humana Press (1993), etc.).
  • the PCR conditions are limited as long as a PCR reaction using the synthetic DNA gives an amplified DNA fragment having the same length as the DNA of the present invention, which was the basis for designing the synthetic DNA.
  • a cycle of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 74 ° C for 60 seconds is one cycle. Conditions for performing 30 cycles can be given.
  • an appropriate restriction enzyme recognition sequence is placed at the 5 'end of the synthetic DNA located at both ends.
  • the DNA of the present invention can be easily cloned into a vector.
  • a synthetic DNA for example, a DNA having the base sequence described in SEQ ID NOS: 3 to 16 was used: The base sequence described in SEQ ID NO: 17 and SEQ ID NO: 18 that can be used in PCR It is possible to obtain a primer set of DNA having
  • the DNA of the present invention prepared in (1) above is used as it is or after being cut with an appropriate restriction enzyme or the like, and then ligated to a vector by a conventional method.
  • any vector such as a phage vector or a plasmid vector can be used as long as it is an autonomously replicable vector in the Escherichia coli K12 strain.
  • ZAP Express [Stratagene, Strategies, 5, 58 (1992)], pBluescript II SK (+) CNucleic Acids Research, 17, 9494 (1989)], human zap II (Stratagene), AgtlO , Agtll CDNA Cloning, A Practical Approach, 1, 9 (1985)], ⁇ TriplEx (manufactured by Clonetech), BlueMid
  • Escherichia coli used as a host for a recombinant DNA obtained by ligating the DNA of the present invention obtained in (1) to this vector can be used as long as it is a microorganism belonging to Escherichia coli.
  • Escherichia coli Y1088 Science, 222 , 778 (1983)
  • Escherichia coli Y109Q Science, 222, 778 (1983)
  • Escherichia coli NM522 J. Mol.
  • Any method for introducing the recombinant DNA can be used as long as it is a method for introducing the DNA into the host cells described above. For example, a method using calcium ions
  • the recombinant DNA is extracted from the transformant obtained as described above, and the nucleotide sequence of the DNA of the present invention contained in the recombinant DNA can be determined.
  • a commonly used nucleotide sequence analysis method for example, the dideoxy method !; Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)] or 373A-DNA sequencer
  • the DNA of the present invention obtained in (1) is a DNA having the same nucleotide sequence as the DNA designed as a group for synthesizing the DNA.
  • Examples of the transformant containing the recombinant DNA obtained as described above include Escherichia coli NM522 / pHE5, which has a plasmid DNA having the nucleotide sequence represented by SEQ ID NO: 1. .
  • the protein having 2,3-sialyltransferase activity which is encoded by the DNA of the present invention is described in Molecular Cloning Second Edition, Current Protocols, Molecular Biology, etc.
  • the DNA of the present invention can be expressed in host cells and produced by the following methods, for example. That is, based on the DNA of the present invention, a DNA fragment of an appropriate length containing a portion encoding the protein is prepared, if necessary, and the DNA fragment is inserted downstream of a promoter of an appropriate expression vector. A recombinant DNA is prepared. The recombinant DN
  • a into a host cell suitable for the expression vector to obtain a transformant It can be produced by culturing the transformant in a medium and accumulating a protein having ⁇ 2,3-sialyltransferase activity in the culture solution.
  • any cell that can express the target gene such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
  • An expression vector that is capable of autonomous replication in the host cell or that can be integrated into chromosomal DNA and that contains a promoter at a position where the DNA encoding the protein 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 has a promoter, a ribosome binding sequence, It is preferable that the vector comprises the DNA of the present invention and a transcription termination sequence. A gene that controls a promoter may be included.
  • expression vectors include, for example, pBTrp2, pBTacls pBTac2 (all commercially available from Berlin-Mannheim), PKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-l (Promega) ), QE-8 (manufactured by QIAGEN), XYPIO (Japanese Unexamined Patent Publication No. 58- ⁇ 0600), KYP200 CAgric. Biol. Chem., 48, 669 (1984)], pLSAl CAgric. Biol. Chem., 53, 277 (1989) )), PGELl [Proc. Natl. Acad. Sci.
  • trp promoter Isseki one P trp
  • lac promoter Isseki one P L promoter Isseki one
  • P B promoter evening one such as T7 promoter, raising the promo one evening one derived from Escherichia coli or phage, etc.
  • P trp x 2 two series P trp
  • tac promoter tac promoter
  • lacT7 promoter Isseki the let I promoter Isseki be used promoter evening one like that are artificially designed and modified as one it can.
  • a plasmid in which the distance between the Shine-Dalgarno sequence, which is a ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to 18 bases).
  • the transcription termination sequence is not necessarily required for the expression of the DNA of the present invention, but it is preferable to arrange the transcription termination sequence immediately below the structural gene.
  • the host cell Eshierihia, Serratia, Bacillus, Purebipaku Teriumu genus Corynebacterium, the genus Microbacterium, microorganisms belonging to Shiyudomonasu genus like, for example, Escherichia coli XU-Blue, Escherichia coli XL2-Blue s Escherichia coli DH1 S Escherichia coli MC1000, Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli No.49, Escherichia coli W3110, Escherichia coli NY49, Escherichia coli GI698, Escherichia coli TB1, Serratia ficaria s Serratia fonticola s Serratia liquefaciens s Serratia marcescens Bacillus subtilis, Bacill
  • Any method for introducing the recombinant DNA can be used as long as it is a method for introducing the DNA into the above host cells.
  • a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], the protoplast method (JP-A-63-248394), or the method described in Gene, 17, 107 (1982) or Mol. Gen. Genet., 168, 111 (1979).
  • examples of expression vectors include YEP13 (ATCC37115), YEp24 (ATCC37051), YCp50 (ATCC37419), pHS19, and pHS15.
  • Any promoter may be used as long as it functions in a yeast strain.
  • promoters for glycolytic genes such as hexose-kinase, etc .; PH05 Promoters, PGK Promos, GAP Promos, ADH Promos, al 1 Promos, gal 10 Promos, Heat Shock Polypeptide Promoters, MF al Promoters, CUP 1 Promos, etc.
  • PH05 Promoters, PGK Promos, GAP Promos, ADH Promos, al 1 Promos, gal 10 Promos, Heat Shock Polypeptide Promoters, MF al Promoters, CUP 1 Promos, etc. Can be raised.
  • fe king cells include Saccharomyces, Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichi, Candida and other microorganisms.
  • Saccharomyces Schizosaccharomyces, Kluyveromyces, Trichosporon, Schwanniomyces, Pichi, Candida and other microorganisms.
  • any method can be used as long as it is a method for introducing DNA into yeast, and examples thereof include an electoporation method (Methods EnzymoL, 194, 182 (1990)) and a spheroplast method [ Natl. Acad. Sci: USA, 75, 1929 (1978)], lithium acetate method [J. Bacteriol., 153, 163 (1983)] ', Proc. Natl. Acad. Sci. USA, 75, 1929 ( 1978).
  • an electoporation method Metals EnzymoL, 194, 182 (1990)
  • a spheroplast method [ Natl. Acad. Sci: USA, 75, 1929 (1978)]
  • lithium acetate method J. Bacteriol., 153, 163 (1983)
  • an expression vector for example, cDNAI , PcDM8 (manufactured by Funakoshi), pAGE107 (JP-A-3-22979, Cytotechnology, 3, 133 (1990)), pAS3-3 (JP-A-2-227075), pCDM8 (Nature, 329,840 (1987)), pcDNAI / Amp (manufactured by Invitrogen), pREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem., 101, 1307 (1987)], pAGE210 and the like.
  • any promoter can be used as long as it functions in animal cells.
  • the promoter of the immediate early (IE) gene of cytomegalovirus (CMV) the early promoter of SV40, the retro promoter of SV40, etc.
  • IE immediate early
  • CMV cytomegalovirus
  • the enhancer of the IE gene of human CMV may be used together with the promoter.
  • Examples of the host cells include Namalwa cells, which are human cells, COS cells, which are monkey cells, CH0 cells, which are Chinese hamster cells, and HBT5637 (Japanese Patent Publication No. 63-299).
  • any method for introducing DNA into animal cells can be used.
  • electoporation method [; Cytotechnology, 3, 133 (1990)] Calcium phosphate method (Japanese Unexamined Patent Publication No. 2-227075), Lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], Virology, 52, 456 (1973).
  • the recombinant gene transfer vector and the baculovirus are co-introduced into insect cells to obtain a recombinant virus in the culture supernatant of insect cells, and then the recombinant virus is transmitted to the insect cells to produce proteins. Can be done.
  • the gene transfer vector used in the method includes, for example, pVL1392, pVL1393, pBlueBacIII (both manufactured by Invitorogen) and the like.
  • baculoviruses include, but are not limited to, autographa californica macear polyhedrosis virus, which is a virus that infects night moth insects, such as Atographa, California, Nuclea, Polyhedrosis, etc. 5 Yes.
  • insect cells examples include Spod9, Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company (1992)) which are ovarian cells of Spodoptera f rugiperda, and High 5 (manufactured by Invitrogen) which is an ovarian cell of Trichoplusia ni. Can be used.
  • Methods for co-transferring the above recombinant gene into insect cells and the above baculovirus to prepare a recombinant virus include the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the ribofusion method, and the like. [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].
  • examples of the expression vector include a Ti plasmid and a tobacco mosaic virus vector.
  • Any promoter may be used as long as it functions in plant cells.
  • Examples of the host cell include plant cells of tobacco, potato, tomato, carrot, soybean, abrana, alfa alfa, rice, wheat, wheat, and the like.
  • Any method for introducing the recombinant DNA can be used as long as it is a method for introducing DNA into plant cells.
  • Agrobacterium Agrobacterium
  • electro volatilization method JP-A-60-251887
  • method using particle gun Gene gun
  • Patent No. 2606856, Patent No. 2517813 By culturing the transformant of the present invention obtained as described above in a medium, producing and accumulating a protein having 2,3-sialyltransferase activity in the culture, and collecting from the culture, The protein can be produced.
  • 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.
  • the transformant of the present invention is a transformant obtained using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host
  • the transformant can be used as a medium for culturing the transformant.
  • Either a natural medium or a synthetic medium may be used as long as it contains a carbon source, a nitrogen source, inorganic salts, and the like, and can efficiently culture the transformant.
  • Any carbon source may be used as long as the transformant can be assimilated, such as glucose, fructose, sucrose, molasses containing these, carbohydrates such as starch or starch hydrolysate, acetic acid, propionic acid, etc. Alcohols such as organic acids, ethanol, and propanol can be used.
  • nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate, and other ammonium-free salts of ammonium or organic acids, and other nitrogen-containing compounds, as well as peptone, meat extract, yeast extract, and copper extract.
  • Plyka, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
  • potassium monophosphate potassium monophosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.
  • the culture is performed under aerobic conditions such as shaking culture or deep aeration stirring culture.
  • the culturing temperature is preferably 15 to 40 ° C, and the culturing time is usually 16 hours to 7 days. It is preferable to maintain the pH during culturing between 3.0 and 9.0.
  • the pH is adjusted using an inorganic or organic acid, alkaline solution, urea, calcium carbonate, ammonia, etc. Do it.
  • an antibiotic such as ampicillin, tetracycline or chloramphenicol may be added to the medium during the culture.
  • the culture medium When culturing a microorganism transformed with a recombinant DNA using an inducible promoter as a promoter, the culture medium may be supplemented with an inductor if necessary.
  • an inductor For example, when culturing a microorganism transformed with a recombinant DNA using a promoter, isopropyl 1 /?-1 D-thiogalactopyranoside and the like are transformed with a recombinant DNA using a promoter.
  • indole acrylic acid or the like may be added to the medium.
  • RPMI 1640 medium J. Am. Med. Assoc., 199, 519 (1967)
  • Eagle's MEM medium Science , 122, 501 (1952)
  • Dulbecco's modified MEM medium Virology, 8, 396 (1959)
  • 199 medium Proc. Soc. Biol. Med., 73, 1 (1950)
  • a medium containing bovine fetal serum or the like can be used.
  • Culture is carried out usually p H 6 ⁇ 8, 3 0 ⁇ 4 0 ° C, 5% C 0 2 under the conditions such as the presence 1-7 days.
  • antibiotics such as kanamycin and penicillin may be added to the medium during the culture.
  • TNM-FH medium As a medium for culturing a transformant obtained by using an insect cell as a host, generally used TNM-FH medium (Pharmingen), Sf-900II SFM medium (Life Technologies), ExCell400, ExCe 11405 (All manufactured by JRH Biosciences), Grace's Insect Medium (Nature, 195, 788 (1962)), etc. can be used.
  • the cultivation is usually carried out for 1 to 5 days under conditions of ⁇ 6 to 7, 25 to 30 ° C and the like. If necessary, 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 cell organ of a plant.
  • the culture medium for culturing the transformant may be a commonly used Murashige and Skull (MS) medium, a white (White) medium, or a medium supplemented with a plant hormone such as auxin or cytokinin. Media or the like can be used.
  • the cultivation is usually performed at pH 5 to 9, 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 a DNA encoding a protein having 2,3-sialyltransferase activity is incorporated can be cultured in a normal culture.
  • the protein can be produced by culturing according to the method, producing and accumulating the protein, and collecting the protein from the culture.
  • Methods for producing a protein having 2,3-sialyltransferase activity include a method of producing the protein in a host cell, a method of secreting the protein out of the host cell, and a method of producing the protein on the host cell outer membrane. The method can be selected by changing the structure of the host cell or the protein to be produced.
  • a shellfish can be produced by adding a signal peptide in front of a polypeptide containing the active site of a protein having 2,3-sialyltransferase activity. Actively secretes proteins out of host cells Can be .
  • the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.
  • the transgenic animal or plant cells are redifferentiated to create transgenic non-human animals or transgenic plants (transgenic plants) into which the gene has been introduced.
  • a protein having 2,3-sialyltransferase activity can also be produced by using E. coli.
  • the protein is produced by breeding or cultivating according to a usual method to produce and accumulate the protein, and collecting the protein from the animal or plant individual. can do.
  • a transgenic non-human animal into which DNA encoding a protein having 2,3-sialyltransferase activity has been introduced is bred, and the protein is produced and accumulated in the animal.
  • the protein can be produced by collecting the protein from the animal. Examples of the place of production and accumulation in the animal include milk (JP-A-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.
  • the zein promoter, casein promoter, and lactate which are mammary cell-specific promoters, can be used.
  • Globulin promoter, whey acid protein promoter, and the like are preferably used.
  • Methods for producing a protein having 2,3-sialyltransferase activity using plant individuals include, for example, transfection into which DNA encoding the protein has been introduced.
  • Nick plants are cultivated according to known methods [tissue culture, 20 (1994), tissue culture, 21 (1995), Trends BiotechnoL, 15, 45 (1997)], and the protein is produced and accumulated in the plants.
  • a method for producing the protein by collecting the protein from the plant.
  • a conventional enzyme isolation and purification method can be used.
  • the protein of the present invention when expressed in a dissolved state in cells, the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, and Mentongaurinho. Crush cells with a mogenizer, dynomill, etc. to obtain a cell-free extract.
  • an ordinary enzyme isolation and purification method can be used, that is, a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Tylaminoethyl (DEAE)-Sepharose, anion exchange chromatography using a resin such as DIAI0N HPA-75 (manufactured by Mitsubishi Kasei), and cation using a resin such as S-Sepharose FF (manufactured by Pharmacia).
  • Ion exchange chromatography hydrophobic chromatography using resins such as butyl sepharose, phenylsepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, etc.
  • a purified sample can be obtained using techniques such as electrophoresis, such as electrofocusing, alone or in combination.
  • the cell When the protein forms an insoluble substance in the cell, the cell is similarly recovered, crushed, and centrifuged to collect the insoluble protein protein as a precipitate fraction.
  • the insoluble form of the recovered protein is solubilized with a protein denaturant.
  • the protein is returned to a normal three-dimensional structure by diluting or dialyzing the solubilized solution and reducing the concentration of the protein denaturing agent in the solubilized solution. After this operation, a purified sample of the protein can be obtained by the same isolation and purification method as described above.
  • a protein produced by the above method, or a derivative such as a protein having a sugar chain added to the protein is secreted extracellularly, the protein or the derivative is added to the culture supernatant.
  • the derivative of the protein can be recovered. That is, a culture supernatant is obtained by treating the culture by a technique such as centrifugation as described above, and purified from the culture supernatant by using the same isolation and purification method as described above. You can get a sample. Examples of the protein thus obtained include a protein having the amino acid sequence of SEQ ID NO: 2.
  • Proteins having 2,3-sialyltransferase activity can be obtained by chemical synthesis methods such as Fmoc method (fluorenylmethyloxycarbonyl method) and tB0c method (t-butyloxycarbonyl method). Can also be manufactured. Chemical synthesis can also be carried out using a peptide synthesizer such as Advanced ChemTech, Parkin Elmer, Pharmacia, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu.
  • Fmoc method fluorenylmethyloxycarbonyl method
  • tB0c method t-butyloxycarbonyl method
  • Examples of the processed product of the culture solution include a concentrate of the culture solution, a dried product of the culture solution, cells obtained by centrifuging the culture solution, a dried product of the cells, a freeze-dried product of the cells, and 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, enzyme preparations obtained by extraction from the cells, and the like.
  • the enzyme source used in the production of sialic acid-containing glycoconjugates has an activity capable of producing 1 / mol of sialic acid-containing glycoconjugates per minute at 37 ° C. as 1 unit (U). It is used at a concentration of lmU / L to 100,000 U / L, preferably lmU / L to I, 000 UZL.
  • Aqueous media used in the production of sialic acid-containing glycoconjugates include buffers such as water, citrate, carbonate, acetate, borate, citrate, and tris, and meta- Examples thereof include alcohols such as phenol and ethanol, 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 odecyl decylamine (for example, Nymein S-215, manufactured by NOF Corporation), cetyltrimethylammonium, promide, alkyldimethyl, pendiammonium and the like.
  • Cationic surfactants such as muchloride (eg, cation F2-40E, manufactured by NOF Corporation), anionic surfactants such as lauroyl sarcosinate, and alkyldimethylamine (eg, tertiary amine FB, manufactured by NOF Corporation) Any of tertiary amines, such as tertiary amines, which promote the production of sialic acid-containing glycoconjugates may be used, and one or more of them may be used in combination.
  • Surfactants are usually used at a concentration of 0.1 to 50 g / L.
  • the organic solvent include xylene, toluene, aliphatic alcohol, acetone, and ethyl acetate, which are usually used at a concentration of 0.1 to 5 OmlZL.
  • CMP-NeuAc which is a sugar nucleotide substrate used in the production of sialic acid-containing glycoconjugates, is not only a commercially available product, but also a reaction solution generated by utilizing the activity of microorganisms. A purified product can be used.
  • the sugar nucleotide substrate is used at a concentration of 0.1 to 50 Ommo1 / L.
  • the receptor glycoconjugate used in the production of the sialic acid-containing glycoconjugate any one can be used as long as it serves as a substrate for glycosyltransferase.
  • lactose, N-acetyl-lactosamine And an oligosaccharide having not more than 10 sugars having a galactose, lactose, N-acetyllactosamine, lacto-N-tetraose, lacto-N-neotetraose, Lewis X, or Lewis a structure at the non-reducing end Glucose and the like can be exemplified.
  • the receptor glycoconjugate is used at a concentration of 0.1 to 50 Ommo 1 / L.
  • inorganic salts MnC 1 2 or the like if necessary, ⁇ - Merukapute Yunoichiru like can be added.
  • the reaction for producing the sialic acid-containing glycoconjugate is carried out in an aqueous medium at pH 5 to 10, preferably pH 6 to 8, and 20 to 50 ° C for 1 to 96 hours.
  • Quantification of sialic acid-containing glycoconjugates formed in an aqueous medium can be performed according to a known method [Chemical and Industrial, 953 (1990)].
  • the sialic acid-containing glycoconjugate formed in the reaction solution can be collected by a usual method using activated carbon or ion-exchange resin, for example, the method described in Anal.Biochem., 85, 602 (1978). It can be performed according to.
  • FIG. 1 is a diagram showing the structure of a plasmid ⁇ E5 expressing 2,3-sialic acid fe transferase gene.
  • Amp r Ampicillin resistance gene
  • Amino acid sequence of Hemophilus 'Haemophilus ducreyi' represented by the amino acid sequence of SEQ ID NO: 2 as an amino acid sequence of 2,3-sialyltransferase Select (GenBank: AF101047) and enter the Codon Usage Database at kazusa
  • the resulting DNA sequence was converted into a DNA sequence, and a DNA sequence encoding a 2,3-sialyltransferase shown in SEQ ID NO: 1 was designed. Based on the designed base sequence, the DNAs described in SEQ ID NO: 3 to SEQ ID NO: 16 so that adjacent synthetic DNAs have an overlapping sequence of 20 bases with each other and alternate with the sense strand and antisense strand was synthesized using a Perceptive Biosystems 8905 DNA synthesizer.
  • SEQ ID NOS: 17 and 18 were synthesized.
  • Buffer solution consisting of hydrochloric acid (pH 8.0), 0.1% Triton X-100, 0.001% BSA, 200 zmo 1 / L dNTPs, and 2.5 units of KOD DNA polymerase (Toyobo)
  • the total volume is reduced to 50 ⁇ 1, covered with 50/1 mineral oil, set on a DNA thermocycler (PJ480, manufactured by PERK IN ELMER) for 30 seconds at 94 ° C and 30 seconds at 50 ° C. Perform 30 cycles of a reaction at 74 ° C for 60 seconds per cycle. Approximately 0.93 ⁇ 4: 10? The CR product was obtained.
  • PCR product After 0.5 ⁇ g of the PCR product was cleaved with restriction enzymes C1aI and BamHI, it was ligated together with 0.2 ⁇ g of pTrS3ODNA cut with restriction enzymes C1aI and BamHI.
  • the ligation reaction was carried out at 16 ° C for 16 hours using a gate kit (Takara Shuzo).
  • Escherichia coli NM522 strain was transformed according to the above-mentioned known method, and the transformant was transformed into an LB agar medium containing 50 zg / ml of ampicillin [Pactotripton (manufactured by Difco). After application to 10 g / L, yeast extract (manufactured by Difco) 5 g / L, NaCl 5 / L (pH 7.2), agar 15 g / L], the mixture was cultured overnight at 30 ° C. Plasmids were extracted from the thus grown colonies of the transformant according to a conventional method, and the structure of the plasmid was analyzed.
  • the strain is a strain expressing the 2,3-sialyltransferase gene.
  • the Escherichia coli NM522 / pHE5 strain obtained in Example 1 was placed in a large test tube containing 8 ml of LB medium containing 50 ⁇ g / ml of ampicillin, and cultured at 28 ° C. for 17 hours.
  • the culture solution was inoculated at 1% into a large test tube containing 8 ml of LB medium containing 50 ⁇ g / ml of ampicillin and cultured at 37 ° C. for 5 hours.
  • the culture broth 0.1 lm 1 was centrifuged to obtain wet cells.
  • the wet cells could be stored at 120 ° C if necessary, and could be thawed before use.
  • the wet cells (for 0.1 ml), 50 mmol / L citrate buffer (pH 7.0), 10 mmol / L MnCl 2 , 10 mmol / L lactose, 1 mmol / L CMP-NeuAc, 0.
  • a 0.1 ml reaction solution composed of 4% Nimin S-215 was prepared and reacted at 37 ° C for 16 hours.
  • the Escherichia coli NM522 / pHE5 strain obtained in Example 1 was inoculated into a large test tube containing 8 ml of LB medium containing 50 / g / ml of ampicillin, and cultured at 28 ° C. for 17 hours.
  • the culture was inoculated at 1% into a large test tube containing 8 ml of LB medium containing 50 ⁇ g / ml of ampicillin, and cultured at 37 ° C. for 5 hours.
  • a 0.1 ml portion of the culture was centrifuged to obtain wet cells.
  • the wet cells could be stored at 120 ° C if necessary, and could be thawed before use.
  • Wet cells (0. 1 ml min), 50mmo; i / L Kuen acid buffer one (. P H 7 0), 1 Ommo 1 / L MnC 1 2, 1 Ommo 1 / L CMP-NeuAc s 0. 4%
  • a 0.1 ml reaction solution containing Nimin S-215 and 1 Ommo 1 / L lacto-N-neotetraose was prepared and reacted at 37 ° C for 16 hours.
  • reaction product was analyzed using a sugar analyzer (DX-500, manufactured by Dionex) (same as in Example 2), and 0.15 mmo 1 / L (15 Omg / L) of sialyllactol N —Confirmed that neotetraose was generated and accumulated.
  • DX-500 manufactured by Dionex
  • sialic acid-containing glycoconjugates can be efficiently produced by using the enzyme.
  • SEQ ID NO: 3 Description of artificial sequence: Synthetic DNA SEQ ID NO: 4 Description of artificial sequence: Synthetic DNA SEQ ID NO: 5—Description of artificial sequence: Synthetic DNA SEQ ID NO: 6—Description of artificial sequence: Synthetic DNA SEQ ID NO: 7—Description of artificial sequence: Synthetic DNA SEQ ID NO: 8—Artificial sequence Description: Synthetic DNA SEQ ID NO: 9 Artificial Sequence Description: Synthetic DNA SEQ ID NO: 10—Artificial Sequence Description: Synthetic DNA SEQ ID NO: 11 Artificial Sequence Description: Synthetic DNA SEQ ID NO: 12—Artificial Sequence Description: Synthetic DNA Sequence No. 13—Description of Artificial Sequence: Synthetic DNA SEQ ID No.

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Abstract

Selon l'invention, l'utilisation d'un micro-organisme ayant un gène obtenu par modification de la séquence de bases du gène α2,3-sialyltransférase originaire d'un micro-organisme appartenant au genre ∫i⊃Haemophilus∫/i⊃, permet de produire de la α2,3-sialyltransférase en grande quantité. De plus, un saccharide complexe contenant de l'acide sialique peut être produit de façon économique et en grande quantité par l'apport d'un micro-organisme produisant l'enzyme ci-dessus avec CMP-NeuAc et un saccharide complexe récepteur dans un milieu aqueux.
PCT/JP2001/003110 2000-04-11 2001-04-11 Gene $g(a)2,3-sialyltransferase modifie, son procede de production et saccharide complexe contenant de l'acide sialique WO2001077314A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006112040A1 (fr) * 2005-04-15 2006-10-26 Japan Tobacco Inc. NOUVELLE β-GALACTOSIDE-α-2,3-SIALYLTRANSFÉRASE, GÈNE CODANT POUR CELLE-CI ET PROCÉDÉ DE PRODUCTION DE CELLE-CI
WO2006112253A1 (fr) * 2005-04-15 2006-10-26 Japan Tobacco Inc. NOUVELLE β-GALACTOSIDE-α2,3-SIALYLTRANSFERASE, GENE LA CODANT, ET SON PROCEDE DE PRODUCTION
WO2008126993A1 (fr) * 2007-04-13 2008-10-23 Gene Chem Inc Nouvelle 2, 3-sialyltransférase et utilisation de cette dernière
JP4856636B2 (ja) * 2005-04-15 2012-01-18 日本たばこ産業株式会社 新規なβ−ガラクトシド−α２，３−シアル酸転移酵素、それをコードする遺伝子およびその製造方法
US20210087599A1 (en) * 2017-07-26 2021-03-25 Jennewein Biotechnologie Gmbh Sialyltransferases and their use in producing sialylated oligosaccharides

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JPH11253163A (ja) * 1998-03-11 1999-09-21 Toyobo Co Ltd シアル酸転移酵素の製造法

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ENDO T. ET AL.: "Large-scale production of CMP-NeuAc and sialylated oligosaccharides through bacterial coupling", APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, vol. 53, no. 3, March 2000 (2000-03-01), pages 257 - 261, XP002941556 *
JOEL A. BOZUE ET AL.: "Haemophilus ducreyi produces a novel sialyltransferase", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 274, no. 7, 1999, pages 4106 - 4114, XP002941555 *
TAKASHI YASUKAWA ET AL.: "Kouzou kaiseki no tameno tanpakushitsu tairyou san seikei", JIKKEN IGAKU, vol. 13, no. 17, 1995, pages 2051 - 2055, XP002941558 *
YASUKAZU NAKAMURA ET AL.: "Codon usage tabulated from international DNA sequence databases: status for the year 2000", NUCLEIC ACIDS RESEARCH, vol. 28, no. 1, January 2000 (2000-01-01), pages 292, XP002941557 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006112040A1 (fr) * 2005-04-15 2006-10-26 Japan Tobacco Inc. NOUVELLE β-GALACTOSIDE-α-2,3-SIALYLTRANSFÉRASE, GÈNE CODANT POUR CELLE-CI ET PROCÉDÉ DE PRODUCTION DE CELLE-CI
WO2006112253A1 (fr) * 2005-04-15 2006-10-26 Japan Tobacco Inc. NOUVELLE β-GALACTOSIDE-α2,3-SIALYLTRANSFERASE, GENE LA CODANT, ET SON PROCEDE DE PRODUCTION
WO2006112025A1 (fr) * 2005-04-15 2006-10-26 Japan Tobacco Inc. NOUVELLE β-GALACTOSIDE-α-2,3-SIALYLTRANSFÉRASE, GÈNE CODANT POUR CELLE-CI ET PROCÉDÉ SERVANT À PRODUIRE CELLE-CI
US8030043B2 (en) 2005-04-15 2011-10-04 Japan Tobacco Inc. β-galactoside-α2,3-sialyltransferase, a gene encoding thereof, and a method for producing thereof
JP4856636B2 (ja) * 2005-04-15 2012-01-18 日本たばこ産業株式会社 新規なβ−ガラクトシド−α２，３−シアル酸転移酵素、それをコードする遺伝子およびその製造方法
EP2434018A2 (fr) 2005-04-15 2012-03-28 Japan Tobacco Inc. Nouvelle beta-galactoside-alpha2,3-sialyltransférase, gène la codant, et son procédé de production
EP2434018A3 (fr) * 2005-04-15 2012-04-25 Japan Tobacco Inc. Nouvelle beta-galactoside-alpha2,3-sialyltransférase, gène la codant, et son procédé de production
WO2008126993A1 (fr) * 2007-04-13 2008-10-23 Gene Chem Inc Nouvelle 2, 3-sialyltransférase et utilisation de cette dernière
US20210087599A1 (en) * 2017-07-26 2021-03-25 Jennewein Biotechnologie Gmbh Sialyltransferases and their use in producing sialylated oligosaccharides

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