WO2001046400A1 - MODIFIED α-1,2-FUCOSYLTRANSFERASE GENE AND PROCESS FOR PRODUCING α-1,2-FUCOSYLTRANSFERASE AND FUCOSE-CONTAINING SUGAR CHAIN - Google Patents
MODIFIED α-1,2-FUCOSYLTRANSFERASE GENE AND PROCESS FOR PRODUCING α-1,2-FUCOSYLTRANSFERASE AND FUCOSE-CONTAINING SUGAR CHAIN Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- 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
- C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
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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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/52—Genes encoding for enzymes or proenzymes
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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)
- C12N9/1051—Hexosyltransferases (2.4.1)
Definitions
- the present invention relates to a modified 1,2-fucose transferase gene, a method for producing a 1,2-fucose transferase using the gene, and a method for producing a fucose-containing saccharide.
- Fucose-containing sugar chains were known as blood group antigen sugar chains.In recent years, it has been revealed that their structure changes as cells become cancerous [Anal. Biochem., 251, 89 (1997)]. It is expected to be applied to tumors and pharmaceuticals. Human milk is rich in oligosaccharides, and fucose-containing sugar chains (fucosyllactose is one of the main components) account for more than 70% of all oligosaccharides [Glycobiology, 8, 615 (1998)]. Since a sugar chain having a Fucal-2Gal structure contained in the oligosaccharide is known to inhibit infection of Candida albicans [Infect. Immun., 59. 1650 (1991)], a fucose-containing sugar chain Is considered a potential candidate for safe infection control drugs.
- An object of the present invention is to provide an industrial method for producing a fucose-containing saccharide.
- the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and have proposed a 1,2-fucose transferase which has been difficult to express as a large amount as an active protein in microorganisms such as Escherichia coli.
- a 1,2-fucose transferase which has been difficult to express as a large amount as an active protein in microorganisms such as Escherichia coli.
- the present invention relates to the following (1) to (29).
- the D.NA according to (3) which is a modification in one or more base sequences selected from base sequences selected from the group consisting of:
- the DNA represented by the nucleotide sequence of SEQ ID NO: 1 has a nucleotide sequence in which one or more bases are deleted, substituted or added, and has a 1,2-fucose transferase activity.
- a recombinant DNA obtained by ligating a DNA selected from the DNAs according to any one of (1) to (7) in a vector.
- a transformant selected from the transformants according to any one of (9) to (12) is cultured in a medium, and the transformant has 1,2-fucose transferase activity in the culture.
- a method for producing a protein having al, 2-fucose transferase activity comprising producing and accumulating a protein, and collecting the protein from the culture.
- a culture solution of a transformant selected from the transformants according to any one of (9) to (12) or a processed product of the culture solution is used as an enzyme source, and the enzyme source and the receptor are used.
- Carbohydrate and fucose guanosine diphosphate (hereinafter abbreviated as GDP-fucos) are made to exist in an aqueous medium, and fucose is transferred to an acceptor carbohydrate by ⁇ 1,2 bond in the aqueous medium.
- a method for producing a fucose-containing saccharide comprising producing and accumulating a saccharide containing saccharide, and collecting a saccharide containing fucose from the aqueous medium.
- GTP guanosine-5'-triphosphate
- the product is used as an enzyme source, the enzyme source, the precursor, the saccharide, and the acceptor saccharide are allowed to exist in an aqueous medium, and fucose-containing saccharide is produced and accumulated in the aqueous medium.
- 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, Cell treated with surfactant, ultrasonically treated cell, mechanically milled cell, solvent-treated cell, enzyme-treated cell, cell-treated enzyme
- a fucose-containing saccharide according to (14) or (15) which is a protein fraction, an immobilized product of the cells, or an enzyme preparation obtained by extraction from the cells.
- the precursor is guanine, xanthine, hypoxanthine, guanosine, xanthosine, inosine, guanosine-5′-phosphate, xanthosine-5′-monophosphate or inosine-5′-phosphate (15 ).
- GDP from sugars and GTP-Microorganisms that have the ability to produce fucose include glucokinase, phosphomannome enzyme, mannose-l-monophosphate guanylyltransferase, phosphoglucome enzyme, phosphofructokinase, GDP-
- Microorganisms include genes encoding glucokinase (hereinafter abbreviated as gIk gene), genes encoding phosphomannom yuichi (hereinafter abbreviated as manB gene), mannose-11-guanylyl phosphate Gene encoding ferulase (hereinafter abbreviated as manC gene), gene encoding phosphoglucose enzyme (hereinafter abbreviated as pgm gene), gene encoding phosphofructokinase (hereinafter abbreviated as pfk Gene), a gene encoding GDP-mannose 4,6-dehydratase (hereinafter abbreviated as gmd gene) and a gene encoding GKDM epimerase / reductase (hereinafter abbreviated as wcaG gene)
- the DNA of the present invention is a DN ⁇ modified from a DN DN encoding a protein having a 1,2-fucosetransferase activity derived from Helicobacter pylori, for example,
- (6) a base sequence in which at least one base among the bases of the DNA having the base sequence according to any one of (1) to (5) is deleted, substituted or added; and DNA encoding a protein having 1,2-fucose transferase activity, such as DNA having a base sequence different from the base sequence of the 1,2-fucose transferase gene derived from Helicobacter pylori. can give.
- the poly C sequence described in (a) above is a base sequence having at least eight or more consecutive Cs
- the TAA-like repeat sequence described in (b) is a TAA, GA A, AAA, TAG
- it is a nucleotide sequence in which a sequence selected from the TGA sequence exists at least three times or more consecutively in an arbitrary order.
- any cells capable of expressing the DNA of the present invention such as bacteria, yeast, animals, 'insect cells, plant cells, etc., are used. be able to.
- a frequently used codon is a codon in which the codon corresponding to each amino acid is at least 10% or more, preferably 20% or more, in the host cell.
- the number of deletions, substitutions, or additions of amino acids occurring in the protein encoded by the mutation-introduced DNA is not particularly limited, but deletion, substitution, or addition can be performed by a known method such as the site-directed mutation described above.
- the number is, for example, 1 to 20, preferably 1 to 15, and more preferably 1 to 5.
- the DNA of the present invention comprises a protein having 1,2-fucose transferase activity.
- BLAST J. Mol. Biol., 215, 403 ( ⁇ )] or analysis software such as FASTA (Method in Enzymology, 183, 63-69)
- FASTA Method in Enzymology, 183, 63-69
- DNA encoding a protein having a 1,2-fucosetransferase activity derived from Helicobacter pylori is used, for example, Molecular 'cloning second edition, Current Protocols 'In' Molecular Biology, Nucl. Acids Res., 1 ⁇ , 6487 (1982), Proc. Natl. Acad. Sci. USA, 79, 6409 (1982), Gene, 34, 315 ( 1985), Nucl. Acids Res., 13, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985), etc. I can give it.
- Methods using synthetic DNA include, for example, (1) artificially synthesizing DNA by PCI removal according to a conventional method (PGR Protocols, Humana Press, (1993), etc.); A synthetic DNA of about 10 Obp is prepared for both the sense strand and the antisense strand so as to cover the entire length of the DNA, and after annealing, the DNA is ligated with DNA ligase.
- DNA encoding the protein produced by the above method is described in Molecular Cloning, Second Edition.
- a recombinant DNA is prepared by ligating with the vector DNA according to the method described in (1) above, and cells are transformed with the recombinant DNA according to the method described in Molecula cloning second edition, A method of culturing in a suitable medium in which transformed cells can grow, accumulating the protein in the culture, and obtaining the protein from the culture.
- the protein may be solubilized and then isolated and purified by ion exchange, gel filtration, hydrophobic chromatography, or the like, or a combination of the chromatography methods.
- the fucose-containing saccharide of the present invention can be obtained by treating a culture solution of a transformant producing the protein or a processed product of a culture solution, or using the purified protein as an enzyme source, and converting the receptor saccharide and GDP-fucose into an aqueous solution. It can be obtained by coexisting in a medium.
- the fucose-containing saccharide of the present invention comprises a culture solution of a microorganism capable of producing GTP from a precursor or a processed product of a culture solution, and one or more saccharides and GTP capable of producing GDP-fucose from GTP. It can also be obtained using GDP-fucos obtained by coexisting a culture solution of a microorganism composed of microorganisms or a processed product of the culture solution in an aqueous medium.
- Precursor here it is not particularly limited as long as it is converted to GTP by microorganisms, preferably Guanin, Kisanchi down, hypoxanthine, guanosine, xanthosine, inosine, guanosine one 5 5 - monophosphate, xanthosine one 5, monophosphate, inosine-5'-phosphate.
- the sugar is not particularly limited as long as it can be converted to GDP-fucose, but is preferably a sugar selected from glucose, fructose and mannose.
- the receptor saccharide is not particularly limited as long as it can be a substrate for a protein having 1,2-fucose transferase activity to which the DNA of the present invention is co-ligated, but galactose is preferably added at the non-reducing end.
- galactose is preferably added at the non-reducing end.
- substrates include lactose, N-acetyllactosamine, Lewis X and Lewis a.
- the microorganism having the ability to produce GTP from the precursor is not limited as long as it is a microorganism having the ability, but is preferably a microorganism belonging to the genus Corynebacterium, more preferably Corynebacterium ammonia ammonia.
- Can be Microorganisms capable of producing GDP-fucose from sugars and GTP include glucokinase, phosphomannomase, mannose-l-guanylyl phosphate monotransferase, phosphok-solekom ylose, and phosphofructokina.
- a microorganism comprising one or more microorganisms having a recombinant DNA of a DNA fragment and a DNA fragment containing one or more types of enzymes selected from enzymes, more preferably a microorganism derived from Escherichia coli.
- One or more types of recombinant Escherichia coli carrying the glk gene, m.anB gene, manC gene, pgm gene, pfk gene, gmd gene and wcaG gene can be mentioned.
- the generated fucose-containing saccharide can be obtained by ordinary chromatography 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 the 1,2-fucose transferase is selected. As the amino acid sequence of the enzyme, any amino acid sequence having the enzyme activity can be used. For example, the amino acid sequence registered in a database such as GenBank represented by SEQ ID NO: 2 can be used. Amino acid sequence of ⁇ 1,2-fucosyltransferase derived from Helicobacter pylori. 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 codon usage database at kazusa (http: // ⁇ .kazusa.or.jp /
- the DNA of the present invention can be designed by converting to a DNA sequence so as to have the most frequently used codons.
- the amino acid sequence of the selected 1,2-fucose transferase is derived from Helicobacter pylori, it is found in the DNA encoding the amino acid sequence.
- the above substitution involves converting a codon in the nucleotide sequence selected from (i), (ii) and (iii) into a codon that is most frequently used in a host cell that expresses the DNA of the present invention.
- the substitution with the nucleotide sequence represented by nucleotide numbers 397 to 408 and 411 to 434 in the nucleotide sequence of SEQ ID NO: 1 can be mentioned. .
- DNA designed as described above for example, a DNA having the nucleotide sequence of SEQ ID NO: 1 designed based on the gene for the 1,2-fucosetransferase derived from Helicopaque pulp and UA 802 should be mentioned. Can be.
- adjacent synthetic DNAs have an overlapping sequence of 10 to 100 bases with each other, and are alternately arranged with the sense strand and the antisense strand.
- the synthetic DNA having a length of 40 to 150 bases is synthesized using an automatic DNA synthesizer (Model 8905 DNA synthesizer manufactured by Perceptive Biosystems).
- Examples of such a synthetic DNA include DNAs having the nucleotide sequences of SEQ ID NOs: 3 to 16 designed based on the DNA having the nucleotide sequence of SEQ ID NO: 1.
- a PCR method according to a conventional method (for example, PCR Protocols, Humana Press, (1993), etc.) Synthesizes DNA artificially.
- the PCR conditions are such that when a PCR reaction is performed using the synthetic DNA, an amplified fragment having the same length as the DNA of the present invention, which is the basis of the design of the synthetic DNA, is obtained.
- a DNA having the nucleotide sequence of SEQ ID NOs: 3 to 16 is used, 30 cycles of 94 ° C for 30 seconds, 50 ° C for 30 seconds, and 74 ° C for 60 seconds are performed.
- the conditions to be performed can be raised.
- an appropriate restriction enzyme recognition sequence into 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 DNA having the nucleotide sequence of SEQ ID NO: 17 or 18 which can be used in PCR using the DNA having the nucleotide sequence of SEQ ID NO: 3 to 16 I can give you a set.
- the DNA of the present invention prepared in the above (1) is directly or after digestion with an appropriate restriction enzyme or the like, and ligated to a vector by an ordinary method.
- any vector such as a phage vector or a plasmid vector can be used as long as it is a vector that can replicate autonomously in Escherichia coli K12 strain.
- ZAP Express (Stratagene) , Strategies, 5, 58 (1992)
- Bluescript II SK (+) (Nucleic Acids Research, 17, 9494 (1989))
- ⁇ zap II (Stratagene)
- gtl0, gtll ⁇ Cloning, A Practical Approach, 1, 49 (1985)]
- TriplEx (Clontech), ⁇ BlueMid (Clontech), LExCell (Pharmacia), PT7T318U (Pharmacia), pcD2 CMol. Cell. Biol., 3, 280 (1983)]
- pUC18 [Gfene, 33, 103 (1985)].
- Escherichia coli used as a host for the recombinant DNA obtained by ligating the DNA of the present invention obtained in (1) in the above step (1) can be any microorganism belonging to Escherichia coli. Specifically, Escherichia coli XLl-Blue MRF 5 CStratagene , Strategies, 5, 81 (1992)), Escherichia coli C600 (Genetics, 39, 440 (1954)), Escherichia coli Y1Q88 (Science, 222, 778 (1983)), Escherichia coli Y1090 (Science, 222, 778 (1983)), Escherichia coli NM522 (J. Mol. Biol., 166, 1 (1983)) ⁇ Escherichia coli K802 (J. Mol. Biol., 16, 118 (1966)), Escherichia coli JM105 (Gene, 38, 275 (1985)].
- 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 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.
- the nucleotide sequence is determined by a commonly used nucleotide sequence analysis method, for example, the dideoxy method (Proc. Natl. Acad. Sci. USA, 74, 5463 (1977)) or the 373A DNA sequencer.
- the DNA of the present invention obtained in (1) is the same as the DNA designed as a group for synthesizing the DNA.
- Examples of the transformant containing the recombinant DNA obtained as described above include, for example, Escherichia coli NM522 / pGT35, which contains a plasmid DNA having the nucleotide sequence represented by SEQ ID NO: 1. it can.
- the protein having a 1,2-fucosyltransferase activity encoded by the DNA of the present invention can be obtained by the methods described in Molecular Cloning, Second Edition, Current Protocols, Molecular Biology, and the like. Use, for example, According to the method, the DNA of the present invention can be produced by expressing it in a host cell. That is, based on the DNA of the present invention, a DNA fragment of an appropriate length containing a portion encoding the protein was prepared as necessary, and the DNA fragment was inserted downstream of the promoter of an appropriate expression vector. Make recombinant DNA.
- the recombinant DNA is introduced into a host cell suitable for the expression vector to obtain a transformant, the transformant is cultured in a medium, and 1,2-fucose transferase activity is added to the culture solution. It can be produced by accumulating proteins having the same.
- any cell that can express the target gene such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.
- a promoter capable of autonomously replicating or integrating into a chromosomal DNA in the above-described host cell and capable of transcribing a DNA encoding the protein of the present invention can be used. 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 and a ribosome binding sequence.
- the DNA comprises the DNA of the present invention and a transcription termination sequence. It may contain a gene that controls the promoter every night.
- expression vectors include pBTrp2, pBTacl, and pBTac2 (all sold by Boehringer Mannheim), pKK233-2 (Pharmacia), pSE280 (Invitrogen), pGEMEX-1 (Promega) PQE-8 (manufactured by QIAGEN), KYPIO (58-110600), pKYP200 CAgric. Biol. Chem., 48, 669 (1984)], pLSAl CAgric. Biol.
- pTrs32 prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)
- pGHA2 prepared from CEscherichia coli IGHA2 (FERM B-400), JP-A-60-221091), pGKA2 CEscherichia coli IGKA2 (FERM BP-6798), JP-A-60-221091), Term2 (US4686191, US4939094, US5160735), pSupex, pUBHO, pTP5, pC194, pEG400 (J. Bacteriol., 172, 2392 (1990)), pGEX (Pharmacia), pET system (Novagen) and the like.
- promoter evening one (P trp), promoter evening one, can be mentioned P L promoter evening one, promoter evening one derived from the P R promoter evening one, one such T7 promoter evening, Escherichia coli, phage and the like.
- the promoter evening one obtained by two series of P trp (P trp x 2) , tac promoter, may also be used lacT7 promoter Isseki one, the let I promoter evening one like that are artificially designed and modified as promoter .
- a plasmid in which the distance between the Shine-Dalgarao 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.
- Examples of the host cell include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevipacterium, Corynepacterium, Microbacterium, Pseudomonas, etc., for example, Escherichia coli XL1-Blue Escherichia coli XL2-Blue, Escherichia coli DH1, Escherichia coli MC1000 N Escherichia coli KY3276, Escherichia coli W1485, Escherichia coli JM109 N Escherichia coli thigh 01, Escherichia coli No.49, Escherichia coli W3110 Escherichia coli NY49, Escherichia coli GI6, Escherichia coli GI6 , Serratia fonticola ⁇ Serratia liquefaciens, Serratia larcescens ⁇ Bacillus subtilis, Bacillus amyloliquefacine
- 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 for example, a method using calcium ions
- yeast When yeast is used as a host cell, as an expression vector, for example, YEP13
- promoters for glycolytic genes such as hexose kinase, PH05 promoter, PH05 promoter, etc. PGK promoter, GAP promoter, ADH promoter, gal 1 promoter, gal 10 promoter, heat shock polypeptide promoter, MF al promoter, CUP 1 promoter, etc. .
- Host cells include microorganisms belonging to the genera Saccharomyces Ss Schizosaccharomyces, luyveromyces, Trichosporon, Schwanniomyces, Pichia, Candida, etc.
- Kluyveromyces lactis, Trichosporon pullulans, Schwanniomyces alluvius, Candida utilis and the like can be mentioned.
- Any method for introducing the recombinant DNA can be used as long as it is a method for introducing DNA into yeast.
- the electroporation method [Methods EnzymoL, 194, 182 (1990)), Spheroplast method (Proc. Natl. Acad. Sci. USA, 75, 1929 (1978)), lithium acetate method (J. Bacteriol., 153, 163 (1983)), Natl. Acad. Sci. USA, 75, 1929 (1978).
- examples of expression vectors include pcDNAI, pcDM8 (manufactured by Funakoshi), PAGE107 (Japanese Unexamined Patent Publication (Kokai) No. 3-22979, Cytotechnology, Sa, 133 (1990)), pAS3-3 (Special Kaihei 2-227075), pCDM8 (Nature, 329, 840 (1987)), pcDNAI / Amp (Invitrogen), pREP4 (Invitrogen), pAGE103 (J. Biochem., 101, 1307 (1987)), pAGE210 Etc. can be given.
- any promoter can be used as long as it functions in animal cells.
- the promoter of the IE (immediate early) gene of cytomegalovirus (CMV) the early promoter of SV40, and the retro promoter Virus promoters, meta-oral thionein promoters, heat shock promoters, SRa promoters, etc.
- 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 'hams cells, and HBT5637 (Japanese Patent Publication No. 63-299). it can.
- any method for introducing DNA into animal cells can be used.
- electoral poration method CCytotechnology, 3, 133 (1990) The calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the ribofusion method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)], and Virology, 52, 456 (1973).
- polypeptide When an insect cell is used as a host, for example, the current protocol, a molecular weight, a non-f-year-old D-, Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), The polypeptide can be expressed by the method described in Bio / Technology, 6, 47 (1988) and the like.
- the recombinant virus is further infected into insect cells to express the protein.
- Examples of the gene transfer vector used in the method include, for example, pVL1392, pVL1393, pBlueBacI II (all manufactured by Invitorogen) and the like.
- the baculovirus for example, Atographa californica virus, which is a virus that infects night roth moth insects, Nuclear monopolypolyhedosis virus, and the like (Autographa californica nuclear polyhedrosis virus) can be used. .
- Insect cells include Sf9 and Sf21 (Baculovirus Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992)) which are ovarian cells of Spodoptera frugiperda, and High 5 (manufactured by Invitrogen) which is an ovarian cell of Trichoplusia. Can be used.
- Methods for co-transfection of the above-described recombinant gene and the baculovirus into insect cells for preparing a recombinant virus include the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), Lipofexion, and the like. Natl. Acad. Sci. USA, 84, 7413 (1987)].
- the expression vector may be, for example, Ti plasmid, a tobacco mosaic virus vector, or the like. Any promoter can be used as long as it can be expressed in plant cells.
- cauliflower mosaic virus (CaMV) 35S promoter, inineactin 1 promoter, etc. be able to.
- Host cells include plant cells such as tobacco, potato, tomato, carrot, soybean, abrana, alfa alfa, rice, wheat, oats, etc. Can be.
- any method can be used as long as it is a method for introducing DNA into plant cells.
- Agrobacterium Agrobacterium
- an electroporation method Japanese Patent Application 60-251887
- a method using a particle gun Gene gun
- Patent No. 2606856, Patent No. 2517813 Patent No. 2606856, Patent No. 2517813
- the transformant of the present invention obtained as described above is cultured in a medium, the protein of the present invention is produced and accumulated in the culture, and the protein of the present invention is produced by collecting from the culture. it can.
- 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 the medium contains the obtained carbon source, nitrogen source, inorganic salts and the like and can efficiently culture the transformant.
- the carbon source may be any as long as the transformant can be assimilated, such as glucose, fructose, sucrose, molasses containing them, carbohydrates such as starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Alcohols such as ethanol, propanol and the like can be used.
- Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and other inorganic or organic acid ammonium salts, other nitrogen-containing compounds, and peptone, meat extract, yeast extract, cone steep Liquor, casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digests thereof can be used.
- Inorganic salts include potassium (II) phosphate, potassium (II) phosphate, and magnesium phosphate It is possible to use shim, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like.
- the culture is performed under aerobic conditions such as shaking culture or deep aeration and stirring culture.
- the culture temperature is preferably 15 to 40 ° C, and the culture time is usually 16 hours to 7 days.
- the pH during the culture is preferably maintained at 3.0 to 9.0.
- the pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia and the like.
- an antibiotic such as ampicillin, tetracycline or chloramphenicol may be added to the medium during the culture.
- the medium may be supplemented with the culture medium if necessary.
- a microorganism transformed with a recombinant DNA using a promoter such as isopropyl-1- / D-thiogalactopyranoside is used.
- 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 obtained by adding fetal calf serum or the like to these mediums can be used.
- Culture is carried out usually pH 6 ⁇ 8, 30 ⁇ 40 ° 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.
- Cultivation is usually carried out at pH 6-7, 25-30 ° C, etc. for 1-5 days.
- 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.
- 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
- auxin or cytokinin is added to these mediums.
- An added medium or the like can be used.
- Cultivation is usually carried out at pH 5-9 and 20-40 ° C for 3-60 days.
- an antibiotic such as kanamycin or hygromycin may be added to the culture during the culture.
- a transformant derived from a microorganism, animal cell, or plant cell having a recombinant DNA into which a DNA encoding the polypeptide 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.
- a method for expressing a gene in addition to direct expression, secretory production, fusion protein expression, and the like can be performed according to the method described in Molecular Cloning, Second Edition, and the like.
- 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 out of the host cell, and a method of producing the protein on the outer membrane of the host cell.
- the method can be selected by changing the structure.
- the protein of the present invention When the protein of the present invention is produced in a host cell or on a host cell outer membrane, the method of Paulson et al. [J. Biol. Chem. 5 264, 17619 (1989)] and the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)], or the methods described in JP-A-5-336963, JP-A-6-823021, etc. Thus, the protein can be actively secreted out of the host cell.
- the protein of the present invention is expressed in a form in which a signal peptide is added in front of the polypeptide containing the active site, whereby the protein of the present invention can be positively extracellularly expressed outside the host cells. Can be secreted. .
- the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like. Furthermore, by redifferentiating the cells of the transgenic animal or plant, an individual animal (transgenic non-human animal) or 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 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 the protein of the present invention has been introduced is bred, the protein is produced and accumulated in the animal, and the protein is collected from the animal. By doing so, the protein can be produced.
- the place of production and accumulation in the animal include milk (eg, JP-A-63-309192) and eggs of the animal.
- Any promoter that can be used in this case can be used as long as it can be expressed in animals.
- the casein promoter which is a breast cell-specific promoter, An overnight promoter, a lactoglobulin promoter, a folic acid ⁇ 1 raw protein promoter and the like are preferably used.
- a transgenic plant into which a DNA encoding the protein of the present invention has been introduced can be prepared by a known method [tissue culture, 20 (1994), tissue culture, 21 ( 1995), Trends Biotechnol., 15, 45 (1997)], producing and accumulating the protein in the plant, and collecting the protein from the plant to produce the protein. There is a way to do it.
- an ordinary enzyme isolation / purification method can be used.
- the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, Mentongau. Crush cells with a mogenizer, dynomill, etc. to obtain a cell-free extract.
- a normal enzyme isolation and purification method i.e., a solvent extraction method, a salting out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Getylaminoethyl (DEAE)-Sepharose, anion exchange chromatography using resin such as DIAI0N HPA-75 (Mitsubishi Kasei), and resin such as S-Sepharose FF (Pharmacia) Cation exchange chromatography method, hydrophobic chromatography method using resins such as butyl cepharose-phenyl cephalosporose, gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, A purified sample can be obtained by using techniques such as electrophoresis such as isoelectric focusing alone or in combination.
- the cells When the protein is expressed by forming an insoluble form in the cells, the cells are similarly collected, crushed, and centrifuged to collect the insoluble form of the 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, the same isolation and purification method as above Thus, a purified sample of the protein can be obtained.
- the protein of the present invention or a derivative such as a protein to which a saccharide is added to the protein is secreted extracellularly
- the protein or a derivative of the protein can be recovered in the culture supernatant. That is, a culture supernatant is obtained by treating the culture by a method such as centrifugation as described above, and a purified sample is obtained from the culture supernatant by using the same isolation purification method as described above. Can be obtained.
- Examples of the protein thus obtained include a protein having the amino acid sequence of SEQ ID NO: 2.
- the protein of the present invention can also be produced by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). be able to. Chemical synthesis can also be carried out using a peptide synthesizer such as Advanced ChemTech, Parkinmar, Pharmacia, Protein Technology Instrument, Synthecel Vega, PerSeptive, Shimadzu.
- a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the tBoc method (t-butyloxycarbonyl method). be able to.
- Chemical synthesis can also be carried out using a peptide synthesizer such as Advanced ChemTech, Parkinmar, Pharmacia, Protein Technology Instrument, Synthecel Vega, PerSeptive, Shimadzu.
- a fucose-containing saccharide is produced in an aqueous medium by using, as an enzyme source, a culture solution of the transformant obtained by the culture according to the above [2] and a treated product of a culture solution obtained by variously treating the culture solution. be able to.
- 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 fucose-containing saccharides is 0.1 mU / L or less, where 1 unit (U) is an activity capable of producing 1 mol of fucose-containing saccharide per minute at 37 ° C. , 000 U / L, preferably from 1 mU / L to!, 000 U / L.
- Aqueous media used in the production of fucose-containing saccharides include water, phosphates, carbonates, acetates, borates, citrates, buffers such as tris, alcohols such as methanol and ethanol, and ethyl acetate. And the like, esters such as acetone, ketones such as acetone, and amides such as acetoamide.
- 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 non-ionic surfactants such as polyoxyethylene octadecylamine (for example, Nymein S-215, manufactured by NOF Corporation), cetyltrimethylammonium, propylamide, dimethylammonium, and benzylammonium.
- Cationic surfactants such as muchloride (eg, cation F2-40E, manufactured by NOF Corporation), anionic surfactants such as lauroyl sarcosinate, alkyldimethylamines (eg, tertiary amine FB, NOF Corporation) Any of the tertiary amines such as those produced by Fucose can be used as long as it promotes the production of fucose-containing saccharides, and one or more of them can 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 50 ml / L.
- GDP-Fuc Guanosine diphosphate fucose
- a product purified from the reaction solution can be used.
- the sugar nucleotide substrate is used at a concentration of 0.1 to 500 mmol / X.
- any one can be used as long as it serves as a substrate for a glycosyltransferase.
- lactose, N-acetyllactosamine, Lewis X, Lewis a, Gal ⁇ l-4 [ Fuc al 3 3] other GlcNAc? l-3Gal ⁇ l- 4Glc (LNFP III) the non-reducing terminal galactose Ichisu or N- Oligosaccharides having no more than 10 saccharides having an acetyl lactosamine structure can be exemplified.
- the acceptor saccharide is used at a concentration of 0.1 to 500 mmol / L.
- inorganic salts MnC 1 2 or the like if necessary, - Merukapute Tano Ichiru like can be added.
- the production reaction of the fucose-containing saccharide 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.
- the quantification of fucose-containing saccharides formed in the aqueous medium can be performed according to a known method [Chemical and Industrial, 953 (1990)].
- the fucose-containing saccharide formed in the reaction solution can be collected by a usual method using an activated carbon ion exchange resin.
- an activated carbon ion exchange resin for example, in the case of 2,1 fucosyllactose, J. Org. Chem., 47, It can be carried out according to the method described in 5416 (1982).
- fucose-containing saccharide examples include fucosyl lactos, fucosyl N-acetyl lactosamine, Lewis Y, Lewis b and the like.
- Fig. 1 shows the structure of pGT35, a plasmid expressing 1,2-fucose transferase gene.
- Amp r is an Anbishirin resistance gene
- the P trn is t rp promoter evening one
- ⁇ 1 Ding 3 Wahi 1 represents a 2-fucosyltransferase gene.
- Fig. 2 shows the construction process of plasmid pNK11 expressing glk, manB and manC.
- Amp r is an ampicillin resistant gene
- the P L is P L promoter Isseki one
- the CI857 is cI857 Riburessa
- the g lk is Gurukokina Ichize gene
- Man'B the phospho Man'nomu evening Ichize gene
- manC Represents the mannose-11-guanylyltransferase gene.
- FIG. 3 shows the steps for constructing the gmd expression plasmid pGE19.
- Amp r is Ampicillin resistance gene, P rn 3 ⁇ 4t rp Promoter overnight, P la .
- the l ac flop Romo - shows the the evening one, g m d is GDP- mannose 4, 6 Dehidora the evening over synthase gene Figure 4 wc aG expression plasmid pGE 8 of reclamation process.
- Amp r is an ampicillin resistant gene
- the P L is P L promoter evening one
- 01857 is a 0,185 7 Riburedzusa one
- a wc aG is GKDM Epimera Ichize / Redaku evening Ichize gene
- Example 1 Construction of a strain expressing a l, 2-fucose transferase
- amino acid sequence of 1,2-fucose transferase As the amino acid sequence of 1,2-fucose transferase, the amino acid sequence of Helicobacter pylori (UA802) represented by SEQ ID NO: 2 was selected, and the amino acid sequence of 1,2-fucose transferase (GenBank: AF076779) was selected. Considering the codon usage frequency of Escherichia coli [Codon Usage database at kazusa (http://www.kazusa.or.jp/codon/)], it is converted to a DNA sequence consisting of the most frequently used codons. The DNA sequence shown in SEQ ID NO: 1 was designed.
- the adjacent synthetic DNAs have an overlapping sequence of 20 bases with each other, and are alternated with the sense strand and the antisense strand, and are described in SEQ ID NO: 3 to SEQ ID NO: 16.
- DNA was synthesized using a Perceptive Biosystems 8905 DNA synthesizer.
- DNAs described in SEQ ID NOS: 17 and 18 each containing a restriction enzyme recognition sequence for cloning at the end; DNA was also synthesized.
- -A buffer consisting of hydrochloric acid (pH 8.0), 0.1% Triton X-100 ⁇ 0.001% BSA ⁇ 200 mol / L dNTPs, and 2.5 units of 10 »DNA polymerase (Toyobo)
- a 60 second cycle was performed for 30 cycles to obtain a PCR product of about 0.9 kb.
- PCR product 0.5 g was cleaved with restriction enzymes C1aI and HindIII, and ligated together with 0.2 g of pBluescriptll SK (+) DNA (Stratagene) cut with restriction enzymes C1aI and HindIII.
- a kit manufactured by Takara Shuzo
- the ligation reaction was performed at 16 ° C for 16 hours.
- Escherichia coli strain 522 was transformed according to the known method described above, and the transformant was transformed into an LB agar medium containing 50 ⁇ g / ml ampicillin [Pactotrypton (manufactured by Difco Co.). ) made 10 GZL, yeast extract (Difco) 5 g / L s NaC 1 5 g / L (P H 7.2), after application to agar 15 g / LD, overnight culture. nourishing at 30 ° C.
- Plasmids were extracted from the thus grown colonies of the transformant according to a conventional method to obtain plasmid pGT32.
- the obtained plasmid pGT320.5 was cleaved with restriction enzymes ClaI and S, and the approximately 0.9 kb C1 ⁇ I—SacI DNA fragment separated by agarose gel electrophoresis was converted to the restriction enzyme C1aI.
- the ligation reaction was performed at 16 ° C. for 16 hours using a Leigession kit (Takara Shuzo) together with pTrS30 0.2 zg (Takara Shuzo) cut with SacI.
- 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 ⁇ g / ml of ampicillin [Pactotripton (manufactured by Difco). After application to 10 g / L, 5 g / L of yeast extract (manufactured by Difco), 5 g / L of NaCl (pH 7.2), and 15 g / L of agar), the whole was cultured at 30 ° C.
- Plasmids were extracted from the grown transformant colonies according to a conventional method to obtain plasmid pGT35.
- the plasmid had a structure as shown in FIG. 1 in which a 0.9 kb totally synthesized DNA fragment was inserted into the C1aI and SacI sites of the pTrS30mid.
- Escherichia coli thigh 522 was transformed according to a known method, and the transformant was spread on an LB agar medium containing 50 g / ml of ambicilin, and then cultured overnight at 30C. .
- Escherichia coli 522 / pGT35 a strain expressing the 1,2-1, fucose transferase gene, was obtained. '
- Example 2 Construction of glk, manB, manC, pgm, and pf kB gene expression strains, ' ⁇
- the DNA primer of SEQ ID NO: 19 and the DNA of SEQ ID NO: 20 are perceptive' Biosystems 8905 It was synthesized using a type DNA synthesizer. Using the above synthetic DNA as a primer set, PCR was performed using plasmid pNT46 (W098 / 12343) DNA containing glk gene as type III.
- PCR is 1 ng primer for each NT ng of pNT46 DNA 0.5] nol / L s Pfu DNA polymerase-2.5 units, xlO buffer for Pfu DNA polymerase (Stratagene) 4 ⁇ 1, deoxy NTP each Using 40 il of a reaction solution containing 200 mol / L, the process was repeated 30 times at 94 ° C; I minute, 42 ° C for 2 minutes, and 72 ° C for 3 minutes 30 times.
- ManB and manC expression plasmids pNK7 (W098 / 12343) O.Zj g, were digested with restriction enzymes BamHI and Sail, DNA fragments were separated by agarose gel electrophoresis, and a 8.2 kb fragment was recovered similarly. .
- the 1.3 kb and 8.2 kb fragments were ligated at 16 ° C. for 16 hours using a ligation kit.
- Escherichia coli NM522 strain was transformed according to the above-mentioned known method, and the transformant was applied to LB agar medium containing 50 g / ml of ampicillin, and then cultured overnight at 30 ° C. .
- a plasmid was extracted from the grown transformant colonies in accordance with the above-mentioned known method to obtain glk, manB, and manC gene expression plasmid pNK11.
- the structure of the plasmid was confirmed by restriction enzyme digestion (FIG. 2).
- Escherichia coli NM522 / pNT55 (W098 / 12343) was transformed according to a known method, and the transformant was transformed into ampicillin. After coating on LB agar medium containing chloramphenicol and 10 ⁇ g / ml, the cells were cultured overnight at 30 ° C.
- Escherichia coli 522 / ⁇ 11 / ⁇ 55 which is a co-expressing strain of the glk, manB, manC ;, pgm, and pfkB genes, was obtained by selecting the grown transformants.
- PCR was carried out according to the method described in Example 1 using chromosomal DNA 0.1 / g of Escherichia coli W3110 (ATCC27325) strain as type III.
- reaction solution was subjected to agarose gel electrophoresis, and after confirming that the target fragment was amplified, an equal amount of TE-saturated phenol Z was added to the remaining reaction solution, followed by mixing.
- pBluescriptll SK + 0.2 ⁇ g was digested with restriction enzymes EcoRI and HindIII, DNA fragments were separated by agarose gel electrophoresis, and a 3.0 kb DNA fragment was recovered in the same manner.
- the 1.1 kb, 0.4 kb and 3.0 kb fragments were subjected to ligation reaction at 16 ° C for 16 hours using a Ligation kit.
- Escherichia coli NM522 strain was transformed using the ligation reaction solution according to the above-mentioned known method, and the transformant was applied to LB agar medium containing 50 ⁇ g / L of ampicillin, and then applied at 30 ° C. Culture was performed once.
- Plasmid was extracted from the thus grown colonies of the transformant in accordance with the above-mentioned known method to obtain an expression plasmid pGE19.
- the structure of the plasmid was confirmed by restriction enzyme digestion (FIG. 3).
- Chromosomal DNA of Escherichia coli W3110 was synthesized using the DNAs described in SEQ ID NOS: 25 and 26 synthesized using a Perceptive Biosystems 8905 DNA synthesizer as primer sets. PCR was performed according to the method described in Example 1 as a template. '
- DNA fragments were separated by agarose gel electrophoresis, and a 5.2 kb DNA fragment was recovered in the same manner.
- the 1.0 kb and 5.2 kb fragments were ligated at 16 ° C and 16 ° C using a ligation kit.
- the ligation reaction was performed for a time.
- Escherictiia coli NM522 strain was transfected according to the known method described above, and the transformant was applied to an LB agar medium containing 50 ⁇ g / L of ampicillin, and then 30 ° C. Overnight.
- Plasmid was extracted from the grown transformants in accordance with the above-mentioned known method to obtain an expression plasmid pGE8.
- the structure of the plasmid was confirmed by restriction enzyme digestion (FIG. 4).
- the Escherichia coli NM522 / pGT35 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.
- 0.1 ml of the culture was centrifuged to obtain wet cells.
- the wet cells could be stored at ⁇ 20 ° C. if necessary, and thawed before use.
- the wet cells (for 0.1 ml), 50 mM citrate buffer (pH 7.0), 10 mmol / L MnCl 2 , 10 mmol / L lactose, 10 mmol / L GDP-Fuc, 0.4% Namin
- the reaction was carried out at 37 ° C. for 24 hours in a 0.1 ml reaction solution composed of S-215.
- reaction product was analyzed using a Dionex Sugar Analyzer (DX-500), and 0.82 mmol / L (400 mg / L) of 2, -fucodyl lactose was generated and accumulated in the reaction solution. Confirmed that.
- DX-500 Dionex Sugar Analyzer
- the Escherichia coli NM522 / pGT35 strain obtained in Example 1 was inoculated into a 1-L baffled Erlenmeyer flask containing 125 ml of LB medium containing 50 ⁇ g / ml of ampicillin, and incubated at 28 ° C. and 220 rpm for 17 hours. Cultured. 125 ml of the culture was inoculated into a 5 L culture tank containing 2.5 L of M9 medium containing 50 fflg / L of ampicillin, and cultured at 37 ° C for 6 hours at 600 rpm and aeration rate of 2.5 L / min. Was.
- the pH of the culture was maintained at 7.0 using, and glucose was added as needed.
- the culture was centrifuged to obtain wet cells.
- the wet cells could be stored at ⁇ 20 ° C. if necessary, and thawed before use.
- Escherichia coli ⁇ 522 / ⁇ 11 / ⁇ 55 obtained in Example 2 was LB medium containing 50 g / inl ampicillin and 10 ⁇ ⁇ g / ml chloramphenicol [Bactotripton (manufactured by Difco). 10. g / L, yeast Extract (manufactured by Difco) 5 g / L, NaCI 5 g / L (pH 7.3)] Inoculate into a 1 L Erlenmeyer flask with baffle containing 125 ml, and culture at 220 rpm at 28 ° C for 17 hours did.
- 125 ml of the culture was added to a TB medium containing 50 mg / L of ambivisillin and 10 mg / L of chloramphenicol [Darcose 10 g / L, pactotryptone (Difco) 12 g / L, yeast extract (D I: co Ltd.) 24 g / L, KH 2 P 0 4 2.3 g / L, K 2 HP 0 4 12.5 g / L (pH unadjusted) 2.5 was inoculated into 5 L fermentor containing the L, After culturing at 30 ° C for 4 hours under the conditions of 600 rpm and an aeration amount of 2.5 LZ, culturing was performed at 40 ° C for 3 hours.
- the pH of the culture solution was maintained at 7.0 using 28% ammonia water, and glucose was added as necessary.
- the culture was centrifuged to obtain wet cells.
- the wet cells could be stored at 120 ° C. if necessary, and thawed before use.
- Escherichia coli NM522 / pGE19 strain obtained in Example 3 was ampicillin
- the culture was inoculated into a 1 L Erlenmeyer flask with a baffle containing 125 ml of LB medium containing 1 ml / ml, and cultured at 28 ° C. at 220 rpm for 17 hours.
- 125 ml of the culture was inoculated into a 5 L culture tank containing 2.5 L of TB medium containing 50 ⁇ g / ml of ampicillin, and cultured at 37 ° C for 6 hours at 600 rpm and aeration rate of 2.5 L / min. was done.
- the pH of the culture solution was maintained at 7.0 using 28% aqueous ammonia, and glucose was added as needed.
- the culture was centrifuged to obtain wet cells.
- the wet cells could be stored at ⁇ 20 ° C. if necessary, and thawed before use.
- the Escherichia coli NM522 / pGE8 strain obtained in Example 4 was inoculated into a 1 L baffled Erlenmeyer flask containing 125 ml of LB medium containing 50 g of ampicillin, and The cells were cultured at 28 ° C. and 220 rpm for 17 hours. 125 ml of the culture was inoculated into a 5 L culture tank containing 2.5 L of TB medium containing 50 ⁇ g / ml of ampicillin, and cultured at 30 ° C. for 4 hours under the conditions of 600 rpm and an aeration of 2.5 LZ. Culture was performed at 40 ° C for 3 hours. During the cultivation, the pH of the culture was maintained at 7.0 using 28% aqueous ammonia, and glucose was added as needed. The culture was centrifuged to obtain wet cells. '
- the culture was centrifuged to obtain wet cells.
- the wet cells could be stored at ⁇ 20 ° C. if necessary, and thawed before use.
- Escherichia coli ⁇ 522 / ⁇ 11 / ⁇ 55 strain wet cell 25 g / L Escherichia coli NM522 / pGE19 strain wet cell 15 g / L Escherichia coli NM522 / pGE8 strain wet cell 15 g / L Escherichia coli strain 522 / pGT35 strain
- Body 50 g / L Gorynebacterium niagenes ATCC21170 strain Wet cells 150 g / L, flatatose 100 g / L, mannose 30 g / L, lactose 100 g / L GMP 30 g / L KH 2 P 0 4 25 g / L MgS0 4 - 7H 2 0 5 g / L, phytic acid 5 g / L, Naimi one emission S- 21 5 4 g / L, the reaction solution 30 ml of 200 ml having a composition of xy
- reaction product was analyzed using HP LC, and it was confirmed that 13.4 g / L of 2,1-fucosyllactose was generated and accumulated in the reaction solution.
- the Escherichia coli NM522 / pGT35 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 solution was inoculated at 1% into a large test tube containing 8 ml of LB medium containing 50 ⁇ g / ml of ambicillin, and cultured at 37 ° C. for 5 hours.
- the culture solution was centrifuged at 0.1 ml for 1 minute to obtain wet cells.
- the wet cells could be stored at -20 ° C if necessary, and could be thawed before use.
- the wet cells (for 0.1 ml), SOmmolZL citrate buffer (pH 7.0) 10 mol / L MnCl 2 10 mmol / L GDP-Fuc, 0.4% Nymein S-215 and lactone-N-neofcopenose III
- the reaction was performed at 37 ° C for 24 hours in 0.1 ml of a reaction solution containing (LNFPIII) at 10 mmol / L.
- the reaction product is analyzed using a Dionex Sugar Analyzer (DX-500), and 2.9 mmol / L (1.4 g / L) of lactate N-neodifucohexaose I is produced in the reaction solution. I confirmed that it was accumulating.
- ⁇ , 2 fucose transferase can be produced in large quantities by a gene recombination technique.
- fucose-containing saccharides such as 2′-fucosyllactose can be efficiently produced.
- SEQ ID NO: 4 Description of artificial sequence: Synthetic DNA '
- SEQ ID NO: 12 Description of artificial sequence: synthetic DNA
- SEQ ID NO: 17 Description of artificial sequence: synthetic DNA
- SEQ ID NO: 18 Description of artificial sequence: synthetic DNA SEQ ID No. 19—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 20—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 21—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 22—Description of Artificial Sequence: Synthetic DNA SEQ ID No. 23—Artificial Sequence Description: Synthetic DNA SEQ ID NO: 24—Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 25—Description of Artificial Sequence: Synthetic DNA SEQ ID NO: 26—Description of Artificial Sequence: Synthetic DNA
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CA002395076A CA2395076A1 (en) | 1999-12-21 | 2000-12-20 | Modified .alpha.-1,2-fucosyltransferase gene and process for producing .alpha.-1,2-fucosyltransferase and fucose-containing sugar chain |
EP00985799A EP1243647A1 (en) | 1999-12-21 | 2000-12-20 | Modified alpha-1,2-fucosyltransferase gene and process for producing alpha-1,2-fucosyltransferase and fucose-containing sugar chain |
AU22216/01A AU2221601A (en) | 1999-12-21 | 2000-12-20 | Modified alpha-1,2-fucosyltransferase gene and process for producing alpha-1,2-fucosyltransferase and fucose-containing sugar chain |
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JP36224399A JP2001546898A (en) | 1999-12-21 | 1999-12-21 | MODIFIED alpha -1,2-FUCOSYLTRANSFERASE GENE AND PROCESS FOR PRODUCING alpha -1,2-FUCOSYLTRANSFERASE AND FUCOSE-CONTAINING SUGAR CHAIN |
JP11/362243 | 1999-12-21 |
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WO2003018794A1 (en) | 2001-08-24 | 2003-03-06 | Kyowa Hakko Kogyo Co., Ltd. | α-1,2-FUCOSYL TRANSFERASE AND DNA ENCODING THE SAME |
JP2018515118A (ja) * | 2016-04-25 | 2018-06-14 | ソウル ナショナル ユニバーシティ アールアンドディービー ファウンデーション | コリネバクテリウムグルタミクムを用いた2’−フコシルラクトースの生産方法 |
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DK1137789T3 (da) | 1998-12-09 | 2010-11-08 | Phyton Holdings Llc | Fremgangsmåde til fremstilling af et glycoprotein med glycosylering af human type |
FR2796082B1 (fr) | 1999-07-07 | 2003-06-27 | Centre Nat Rech Scient | Procede de production d'oligosaccharides |
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EP1959018B1 (en) | 2002-03-19 | 2011-05-11 | Stichting Dienst Landbouwkundig Onderzoek | Optimizing glycan processing in plants |
AR042145A1 (es) | 2002-11-27 | 2005-06-08 | Dow Agrociences Llc | Produccion de inmunoglobulinas en plantas con una fucocilacion reducida |
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WO2012007481A2 (en) | 2010-07-12 | 2012-01-19 | Universiteit Gent | Metabolically engineered organisms for the production of added value bio-products |
DK2479263T3 (da) | 2011-01-20 | 2014-02-03 | Jennewein Biotechnologie Gmbh | Nye fucosyltransferaser og deres anvendelser |
WO2012127410A1 (en) * | 2011-03-18 | 2012-09-27 | Glycom A/S | Synthesis of new fucose-containing carbohydrate derivatives |
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- 2000-12-20 WO PCT/JP2000/009033 patent/WO2001046400A1/ja not_active Application Discontinuation
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WO2003018794A1 (en) | 2001-08-24 | 2003-03-06 | Kyowa Hakko Kogyo Co., Ltd. | α-1,2-FUCOSYL TRANSFERASE AND DNA ENCODING THE SAME |
EP1426441A1 (en) * | 2001-08-24 | 2004-06-09 | Kyowa Hakko Kogyo Co., Ltd. | Alpha-1,2-fucosyl transferase and dna encoding the same |
EP1426441A4 (en) * | 2001-08-24 | 2005-03-16 | Kyowa Hakko Kogyo Kk | ALPHA-1,2-FUCOSYL TRANSFERASE AND DNA ENCODING THE SAME |
US7214517B2 (en) | 2001-08-24 | 2007-05-08 | Kyowa Hakko Kogyo Co., Ltd. | α1,2-fucosyltransferase and DNA encoding the same |
JP2021121215A (ja) * | 2011-12-16 | 2021-08-26 | インビオース エヌ.ヴェ.Inbiose N.V. | コラン酸、マンノシル化及び/又はフコシル化オリゴ糖を合成するための変異体微生物 |
RU2746739C1 (ru) * | 2015-01-30 | 2021-04-20 | Йенневайн Биотехнологи Гмбх | Ферментативный способ производства моносахаридов в свободной форме из сахаров, активированных нуклеотидами |
JP2018515118A (ja) * | 2016-04-25 | 2018-06-14 | ソウル ナショナル ユニバーシティ アールアンドディービー ファウンデーション | コリネバクテリウムグルタミクムを用いた2’−フコシルラクトースの生産方法 |
JP2020022506A (ja) * | 2016-04-25 | 2020-02-13 | ソウル大学校産学協力団Seoul National University R&Db Foundation | コリネバクテリウムグルタミクムを用いた2’−フコシルラクトースの生産方法 |
US10570399B2 (en) | 2016-04-25 | 2020-02-25 | Seoul National University R&Db Foundation | Corynebacterium glutamicum for use in producing 2′-fucosyllactose |
US10876122B2 (en) | 2016-04-25 | 2020-12-29 | Seoul National University R&Db Foundation | Method of producing 2′-fucosyllactose using recombinant Corynebacterium glutamicum |
Also Published As
Publication number | Publication date |
---|---|
KR20020062361A (ko) | 2002-07-25 |
EP1243647A1 (en) | 2002-09-25 |
CA2395076A1 (en) | 2001-06-28 |
JP2001546898A (en) | 2003-06-10 |
AU2221601A (en) | 2001-07-03 |
CN1433467A (zh) | 2003-07-30 |
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