WO2002033070A1

WO2002033070A1 - N-acetylneuraminic acid synthase and dna encoding this enzyme

Info

Publication number: WO2002033070A1
Application number: PCT/JP2001/008935
Authority: WO
Inventors: Satoshi Koizumi; Hiroshi Mizoguchi; Mari Ishikawa; Tetsuo Endo
Original assignee: Kyowa Hakko Kogyo Co., Ltd.
Priority date: 2000-10-16
Filing date: 2001-10-11
Publication date: 2002-04-25
Also published as: AU2001295917A1; JPWO2002033070A1

Abstract

A novel protein having an N-acetylneuraminic acid synthase activity; a DNA encoding this protein; a recombinant DNA containing the above DNA; a transformant carrying this recombinant DNA; and a process for producing the above-described protein or N-acetylneuraminic acid by using this transformant.

Description

m

N-Acetylneuraminic acid synthase and DNA encoding the enzyme

The present invention relates to a protein having N-acetyl neuraminic acid synthase activity, a DNA encoding the protein, a recombinant DNA containing the DNA, a transformant having the recombinant DNA, The present invention relates to a method for producing a protein having N-acetyl neuraminic acid synthase activity using a transformant, and a method for producing N-acetyl neuraminic acid using the transformant. Background surgery

As for N-acetylneuraminic acid synthase, an enzyme derived from a microorganism belonging to the genus Escherichia [Glycobiology, 1, 697 (1997) Biosci. Biotech. Biochem., Si, 2046 (1997)] has been obtained.

As for the N-acetylneuraminic acid synthase gene, a gene derived from a microorganism belonging to the genus Escherichia [J. Bacteriol., M, 312 (1995)], a gene derived from a microorganism belonging to the genus Streptococcus [J. Bacteriol., 181, 5176 (1999)], a gene derived from a microorganism belonging to the genus Campylopa [Mol. Microbiol., 35, 1120 (2000)], a gene derived from a microorganism belonging to the genus Legionella [Int. J. Med. Microbiol., 37 (2000)], but no gene derived from a microorganism belonging to the cyanobacteria or a gene belonging to the genus Nostock is known, and even reports suggesting the presence of the gene are known. Absent.

N-Acetylneuraminic acid has been shown to be involved in intercellular recognition at the non-reducing end of sugar chains in vivo [Essentials of Glycobiology, Gold Spring Harbor Laboratory Press ( 1999), Virology,, 19 (1997)], and application as pharmaceuticals are expected.

However, regarding the production of N-acetyl neuraminic acid, a method of decomposing colominic acid, which is an N-acetyl neuraminic acid polymer [J. Biochem., 82, 1425 (1977)], a method using an enzyme [ J. Am. Chem. Soc, iiil, 6481 (1988), J. Am. Chem. Soc ϋϋ, 7159 (1988), U.S. Pat.No. 5,665,574, Carbohydrate Res., 306, 575 (1998), Kai 10- 4961 Glycobiology, 1, 697 (1997)], however, all have problems in terms of cost and productivity, and an industrial production method has not yet been established.

In MosiOfi punctiforme ATCC29133, a species of cyanobacteria, the nucleotide sequence of its genomic DNA is under progress (http: 〃spider.jgi-psf.org / JGI_microbial / htna /), but N-acetylnoy Laminate synthase gene has not been identified, and no reports suggest its existence. Censor of the invention

An object of the present invention is to provide a protein having N-acetyl neuraminic acid synthase activity, a DNA encoding the protein, a recombinant DNA containing the DNA, and a transformant having the recombinant: DNA. Another object of the present invention is to provide a method for producing a protein having N-acetyl neuraminic acid synthase activity using the transformant, and a method for producing N-acetyl neuraminic acid using the transformant.

The present inventors have conducted intensive studies to solve the above-mentioned problems, and have used the sequence information of Nosiflfi punctiforme ATCC 29133, whose nucleotide sequence of genomic DNA is in progress, to obtain an N-acetylneuron derived from Escherichia coli. As a result of searching for a sequence homologous to the amino acid sequence of laminate synthase, a DNA encoding a novel N-acetylneuraminic acid synthase, which had not been identified, was found. As a result, the present invention has been completed.

That is, the present invention relates to the following (1) to (14).

(1) A protein having an amino acid sequence represented by SEQ ID NO: 1.

(2) a protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1 and having an N-acetyl neuraminic acid synthase activity .

(3) DNA encoding the protein of (1) or (2) above.

(4) A DNA having the nucleotide sequence represented by SEQ ID NO: 2.

(5) Under stringent conditions with DNA having the base sequence of SEQ ID NO: 2 DNA that hybridizes and encodes a protein having N-acetyl neuraminic acid synthase activity.

(6) The DNA according to any one of the above (3) to (5), wherein the DNA is derived from a microorganism belonging to the cyanobacterium (talQilkr).

(7) The DNA according to (6), wherein the DNA derived from a microorganism belonging to the cyanobacterium is a DNA derived from a beef belonging to the genus Nostoc.

(8) The DNA according to (7) above, wherein the microorganism belonging to the genus Nostock is Nostoc punci forme.

(9) A recombinant DNA obtained by incorporating the DNA of any one of the above (3) to (8) into a vector.

(10) A transformant obtained by introducing the recombinant DNA of (9) into a host cell.

(11) The transformant according to the above (10), wherein the host cell is Escherichia coli.

(12) culturing the transformant of the above (10) or (11) in a medium, producing and accumulating a protein having N-acetyl neuraminic acid synthase activity in the culture; A method for producing a protein having N-acetyl neuraminic acid synthase activity.

(13) A culture of the transformant of the above (10) or (11) or a processed product of the culture is used as an enzyme source, and the enzyme source, N-acetyl mannosamine and phosphoenolpyruvate are added to an aqueous solution. N-acetylneuraminic acid, wherein N-acetylneuraminic acid is produced and accumulated in the aqueous medium, and N-acetylneuraminic acid is collected from the aqueous medium. Method of producing acid.

(14) The processed product of the culture is a concentrate of the culture, a dried product of the culture, a cell obtained by centrifuging the culture, a dried product of the cell, a lyophilized product of the cell, Cell treated with a surfactant, ultrasonically treated cell, mechanically milled cell, solvent-treated cell, enzyme-treated cell, cell-treated enzyme (13) The method according to (13) above, which is a protein fraction, an immobilized product of the cells, or an enzyme preparation obtained by extraction from the cells.

The protein of the present invention includes N-acetyl having the amino acid sequence represented by SEQ ID NO: 1. A protein having a laminic acid synthase activity, or an amino acid sequence represented by SEQ ID NO: 1 comprising an amino acid sequence in which one or more amino acids have been deleted, substituted, or added; and N-acetylneuraminic acid A protein having a synthase activity can be mentioned. The deletion, substitution or addition of the above amino acids can be performed by the methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Gold Spring Harbor Laboratory Press (1989) (hereinafter abbreviated as Molecular Cloning 2nd Edition), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997) (hereinafter abbreviated as current 'protocols' in molecular 'biology'), Nucleic Acids Res., IQ, 6487 (1982), Proc. Natl. Acad. Sci. USA, 11, 6409 (1982), Gene,, 315 (1985), Nucleic Acids Res., 11, 4431 (1985), Proc. Natl. Acad. Sci. USA, 82, 488 (1985). It can be carried out by introducing a site-specific mutation into a DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 1 using a mutagenesis method.

The number of amino acids to be deleted, substituted or added is not particularly limited. , Preferably:! -20, more preferably 1-10, and even more preferably 1-5.

In addition, by using a PCR [Gene, 71, 51 (1989)] using a pair of PCR primers each having a sequence into which the desired mutation (deletion, substitution, addition) has been introduced at the 5 'end thereof, Mutations can be introduced into DNA encoding a protein consisting of the amino acid sequence represented by 1. That is, first, a sense primer corresponding to the 5 'end of the DNA and an antisense primer corresponding to the sequence immediately before (5' side) the mutation introduction site having a sequence complementary to the mutation sequence at the 5 'end. PCR is carried out using the DNA as type III, and a fragment A (3, having a mutation introduced at the end) from the 5th end to the mutation introduction site of the DNA is amplified. Then, the DNA is ligated with a sense primer corresponding to the sequence immediately after (3 'side) having the mutation sequence at the 5' end and an antisense primer corresponding to the 3 'end of the DNA. The PCR is performed, and a fragment B from the mutation-introduced site of the DNA having a mutation introduced at the 5 ′ end to the 3 ′ end is amplified. If these amplified fragments are purified and mixed together and PCR is performed without adding Type I primers, Since the sense strand of fragment A and the antisense strand of amplified fragment B have a common mutation-introducing site, they are hybridized, and the PCR reaction proceeds as a primer / gun type, whereby the DNA into which the mutation has been introduced is amplified.

Further, the deletion, substitution, insertion or addition of one or more amino acid residues in the amino acid sequence represented by SEQ ID NO: 1 means that any one or more amino acid residues in the same sequence Means that there is a deletion, substitution, insertion or addition of one or more amino acid residues at the position, the deletion, substitution, insertion or addition may occur at the same time, and the amino acid to be substituted, inserted or added Residues may be natural or non-natural. Natural amino acid remnants include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glucamic acid, glycine, L-histidine, L-isoleucine, L-leucine, and L-lysine. , L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, L-cysteine and the like.

The following are examples of amino acid residues that can be substituted for each other. Amino acid residues included in the same group can be substituted for each other.

Group A: leucine, isoleucine, norleucine, norin, norparin, alanine, 2-aminobutanoic acid, methionine, 〇-methylserine, t-butylglycine, t-butylylalanine, cyclohexylalanine

Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid

. Group: Asparagine, Guru Yu Min

Group D: lysine, arginine, orditin, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid

Group E: Proline, 3-hydroxyproline, 4-hydroxyproline

Group F: serine, threonine, homoserine

Group G: fenirualanine, tyrosine

In order for the protein of the present invention to have N-acetyl neuraminic acid synthase activity, it must have at least 60%, usually at least 80%, and especially at least 95% identity with the amino acid sequence represented by SEQ ID NO: 1. It is preferable to have _n The amino acid sequence and nucleotide sequence identity can be determined by the algorithm BLAST [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)] and FASTA [Methods Enzymol., 183, 63 (1990)] by Karlin and Altschul. Can be determined. Based on this algorithm BLAST, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When a nucleotide sequence is analyzed by BLASTN based on BLAST, parameters are, for example, Score = 100 and wordlength = 12, for example. In the case of analyzing an amino acid sequence by BLASTX based on BLAST, each parameter is, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, use the default parameters of each program. Specific methods of these analysis methods are known (http: //www.ncbi.n1m.nih.ov.).

The DNA of the present invention includes, under stringent conditions, DNA encoding the protein of the present invention, DNA having the nucleotide sequence represented by SEQ ID NO: 2, or DNA having the nucleotide sequence represented by SEQ ID NO: 2. Examples of the DNA include a DNA which hybridizes and encodes a protein having N-acetyl neuraminic acid synthase activity.

The DNA hybridizing under the above-mentioned stringent conditions refers to a colony hybridization method using, for example, a DNA fragment of the present invention such as a DNA having the nucleotide sequence of SEQ ID NO: 2 or a partial DNA fragment thereof as a probe. Means the DNA obtained by using the plaque hybridization method or the Southern plate hybridization method.Specifically, the DNA is obtained by using a filter in which DNA derived from colony or plaque is immobilized. After performing hybridization at 65 ° C in the presence of OHIO 1 / L sodium chloride, 0.1 to 2 times concentration of SSC solution (composition of 1 times concentration of SSC solution) Can be identified by washing the filter at 65 ° C using 15 Ommo 1 / L sodium chloride and 15 mmo 1 / L sodium citrate). Kill. Hybridization is described in Molecular 'Cloning 2nd Edition, Current'Protocols' in 'Molecular' Biology, DN A Clonin II: Core Techniques, A Practical Approach, Second Edition, Oxford University (1995) It can be performed according to the method that is being performed. Hybridy Specific examples of the DNA that can be used have a homology of at least 60% or more with the nucleotide sequence represented by SEQ ID NO: 2 when calculated based on the above algorithm BLAST.] DNA, DNAs having a homology of 70% or more, more preferably 80% or more, still more preferably 90% or more, particularly preferably 95% or more, and most preferably 98% or more can be mentioned.

Hereinafter, the present invention will be described in detail.

[1] Preparation of DNA of the present invention

(1) Identification of N-acetylneuraminic acid synthase gene using genome DNA database

Nostoc uncti forme ATCC29133 is currently undergoing nucleotide sequencing for its genomic DNA,

http: 〃 spider, jgi-psf.org/JGI_microbial/html/, etc. Using a genome DNA database that can be browsed by accessing the website, it has homology to known N-acetylneuraminic acid synthase. Encoding protein: You can search for DNA.

As the amino acid sequence of the known N-acetyl neuraminic acid synthase used in the homology search, any amino acid sequence of a protein having N-acetyl neuraminic acid synthase activity can be used. However, specific examples include the amino acid sequence of N-acetylneuraminic acid synthase derived from Escherichia coli [J. Bacteriol., 117, 312 (1995)].

Although any method can be used as the search method, a method of performing a homology search on the database using a program such as the above BLAST or FASTA can be exemplified.

However, it is expected that the homology of N-acetyl neuraminic acid synthase is low among different kinds of organisms, and thus the sequence selected above is a protein that actually has N-acetyl neuraminic acid synthase activity. You need to make sure it is.

(2) Preparation of DNA of the present invention The DNA of the present invention can be prepared from a microorganism belonging to the cyanobacterium. Examples of the microorganism belonging to the cyanobacterium include, for example, microorganisms belonging to Nostock.Specifically, the microorganisms belonging to the cyanobacterium include, for example, Nostoc uctifornte ATCC29133. Microbiology, Cliff, 3233 (1994)].

After culturing, the chromosomal DNA of the microorganism is isolated and purified by a known method (for example, current 'protocols' in' molecular virology).

The fragment containing the DNA of the present invention can be obtained by preparing a primer based on the nucleotide sequence of the genome specified in the above (1), using the genomic DNA as a type II, PCR method [PCR Protocols, Academic Press ( 1990)].

Also, the target DNA can be obtained by a hybridization method using synthetic DNA designed as a probe based on the base sequence of the genome.

The obtained DNA is used as it is, or after digestion with an appropriate restriction enzyme or the like, incorporated into a vector by a conventional method, and a commonly used nucleotide sequence analysis method, for example, the dideoxy method [Proc. Natl. Acad. Sci. USA, 74: , 5463 (1977)] or 373A. DNA sequencer (Perkin-Elma Inc.) or the like can be used to determine the nucleotide sequence of the DNA.

Examples of the vector incorporating the DNA include pBluescript KS (+) (Stratagene), pDIRECT [Nucleic Acids Res., 18, 6069 (1990)], pCR-Script Amp SK (+) (Stratagene) PT7Blue (Novadin), pCR11 (Invitrogen) and pCR-TRAP

(Made by Gene Hunter).

Further, based on the determined nucleotide sequence of DNA, the desired DNA can be prepared by chemical synthesis using Perceptive's 8905-type DNA synthesizer manufactured by Biosystems.

Examples of the DNA having the novel nucleotide sequence obtained as described above include DNA having the nucleotide sequence represented by SEQ ID NO: 2, and the like. Examples of Escherichia coli having a plasmid having a DNA having a nucleotide sequence represented by SEQ ID NO: 2 include Miaiiklik coli banded 522 / pNPl described below.

Examples of host E. coli harboring the recombinant DNA include, for example, Escherichia coli XLl-Blue, Escherichia coli XL2-Blue, Escherichia coli DHl, Rscherichia coli MC1000, Escherichia coli KY3276, Escherichia ooli W1485, Kscherichia.ooli JM109, EscherOKaHB Escherichia coli No. 49, Escherichia coli W311Q, Rscherichia coli NY49, Rscherichia coli MP347, Rscherichia coli NM522 and the like can be mentioned.

As a method for introducing the recombinant DNA, any method can be used as long as it is a method for introducing DNA into the above host cells. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, 69, 2110 (1972)], protoplast method (JP-A-63-248394), electroporation method [Nucleic Acids Res., I £, 6127 (1988)] and the like.

[2] Preparation of the protein of the present invention

The protein of the present invention can be obtained by the method described in the above [1], for example, by the method described in Molecular 'Clothing Second Edition, Current' Protocols' in 'Molecular' Biology, etc. Can be produced by expressing the DNA of the present invention obtained by the method described above in a host cell.

Based on the DNA of the present invention, if necessary, a DNA fragment having an appropriate length containing a portion encoding the protein is prepared. Further, by substituting the base so that the nucleotide sequence of the portion encoding the protein has an optimal codon for expression in the host, the production rate of the protein can be improved.

The recombinant DNA is prepared by inserting the DNA fragment downstream of the promoter of an appropriate expression vector.

By introducing the recombinant DNA into a host cell suitable for the expression vector, a transformant producing the protein of the present invention can be obtained.

As the host cell, any cells that can express the gene of interest, such as bacteria, yeast, animal cells, insect cells, and plant cells, can be used.

Expression: As DNA, it is capable of autonomous replication in the above-mentioned host cell or is assembled into a chromosome. A promoter containing a promoter at a position where the DNA of the present invention can be transcribed is used.

When a prokaryote such as a bacterium is used as a host cell, the recombinant DNA containing the DNA encoding the protein of the present invention is capable of autonomous replication in the prokaryote, and at the same time, promotes ribosome binding. It is preferably a recombinant DNA comprising the sequence, the DNA of the present invention, a transcription termination sequence. A gene that controls the promoter may be included. PHelixl (manufactured by Roche Diagnostics), PKK233-2

(Amersham 'Pharmacia' Biotech), pSE280 (Invitrogen), pGEMEX-1 (Promega), pQE-8 (Qiagen), pET-3 (Novadin), ρΚΥΡΙΟ

(JP-A-58-U0600), pKYP200 [Agric. Biol. Chem., 48, 669 (1984)], pLSAl [Agric. Biol. Ghem., 53, 277 (1989)], pGELl [Proc. Natl. Acad. USA, 82, 4306 (1985)], pBluescriptl SK (+) pBluescript II KS (-) (Stratagene), TrS30 [prepared from E. coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [ E. coli JM109 / pTrS32 (FERM BP-5408)], pPAC31 (W098 / 12343), pUC19 [Gene, 33, 103 (1985)], pSTV28 (Takara Shuzo), PUC118 (Takara Shuzo), pPAl (special 63-233798).

Any promoter may be used as long as it functions in a host cell such as Escherichia coli. For example, tra promoter (Pine), lac promoter Isseki one (Plaa), P _L promoter Isseki one, P _R promoter and P _SE promoter, promoter derived from E. coli and file one di-, etc., SP 0 1 promoter , SP02 Promo, pen P promoter and the like. The two series are not the promoter Ptrp (Pke x 2), ia flop port motor, i _AcT7 promoters artificially designed and modified promoters like _{l et} I promoter, etc. can also be used.

It is preferable to use a plasmid in which the distance between the Shine-Dalgarno sequence, which is the ribosome binding sequence, and the initiation codon is adjusted to an appropriate distance (for example, 6 to ^ bases).

In the recombinant of the present invention! NA, a transcription termination sequence is always required for expression of the DNA of the present invention. Although not necessary, it is preferable to arrange a transcription termination sequence immediately below the structural gene. Prokaryotes include microorganisms belonging to the genus Escherichia, Serratia, Bacillus, Brevipacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., for example, Kscherichi coli XLl-Blue, Esoheri nhi col i XL2-Blue, Escheri chi col i DH1, Escherichia coli MC1000, Escherichia noli KY3276, Escheri ctik mli W1485, Escherichia coli JM109, Escherichia coli HBi01, Escherichia col i No.49, Escherichia coli W3ll0s Esicaria rata , Serratia fonticola, Serratia liguefacienss Serratia marcescens.Baci l lus subti l is.

Corynebacterium glutamicum ATCC14067, Corynebacterium glutamicum ATCC13869, Corynebacterium acetoacidophi lum ATCC 丄 3870, Microbacterium am bandit iaphi lum

ATCG15354, Pse domonas sp. D-OUO and the like.

As a method for introducing the recombinant DNA, any method for introducing the DNA into the above host cells can be used. For example, a method using calcium ions [Proc. Natl. Acad. Sci. USA, M, 2110 (1972)], protoplast method (JP-A-63-248394), and electroporation method [Nucleic Acids Res., Ifi, 6127 (1988)]. When a yeast strain is used as a host cell, for example, YEpl3 (ATGG37U5), YEp24 (ATCC37051), YCp50 (ATGC37419), pHS19, pHS15 and the like can be used as expression vectors.

Any promoter may be used as long as it functions in the yeast strain.Examples include PH05 promoter, PGK promoter, GAP promoter, ADH promoter, gal1, promoter, gal10 promoter, heat Promoters such as the shock polypeptide promoter, the Wai promoter, and the CUP1 promoter. Examples of the host cell include yeast strains belonging to the genus Saccharomyces, Schizosaccharomyces, Kluybaceae, Trichosporon, Schizinomyces, Pichia, Candida, etc. Saccharomvces cerevi si ae.

U Schi zosaccharomyces pombe, Kluyveromyces lacti ss Tri chosporon pul lulans, Schwanni omvces alluvi us, Pi ohi pastori Cild a lltiLLa and the like. As a method for introducing the recombinant DNA, any method can be used as long as it is a method for introducing the DNA into yeast. For example, the elect opening method [Methods Enzymol., M, 182 (1990)], the spheroplast method USA, Si, 4889 (1984)], the lithium acetate method [J. Bacteriol., 153, 163 (1983)], and the like.

When an animal cell is used as a host, an expression vector, for example, pcDNAI, pcDM8 (commercially available from Funakoshi), AGE107 (JP-3- 22979), _P AS3-3 (JP 2-227075), pCDM8 [Nature , 840 (1987)], pcDNAI / Amp (manufactured by Invitrogen), PREP4 (manufactured by Invitrogen), pAGE103 [J. Biochem, Dish, 1307 (1987)], pAGE2lO, pAMo, pAMoA and the like can be used. .

Any promoter can be used as long as it functions in animal cells. For example, the promoter of the immediate early (IE) gene of cytomegalovirus (CMV), the early promoter of SV40 or the meta promoter Examples include a thionine promoter, a retrovirus promoter, a heat shock promoter, and an SR promoter. Alternatively, the enhancer of the IE gene of human CMV may be used together with the promoter.

Host cells include mouse myeloma cells, rat 'myeloma cells', mouse 'hybridoma cells', human Namalwa cells or Namalwa KJM-1 cells, human fetal kidney cells, human leukemia Cell, African green monkey kidney cell, CH0 cell which is a Chinese-hamster cell, HBT5637 (Japanese Patent Application Laid-Open No. 63-299), and the like. Mouse / myeloma cells include SP2 / 0, NS0, etc., rat's myeloma cells include YB2 / 0, etc., human fetal kidney cells include HEK293 (ATCC: CRL-1573), etc., and human leukemia cells include BALL-1. African green monkey kidney cells include COS-1 and COS-7.

As a method for introducing a recombinant DNA, any method can be used as long as it is a method for introducing DNA into animal cells. For example, an electroporation method [Cyto-7 echnology, 3, 133 (1990)], the calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), the lipofusion method [Proc. Natl. Acad. Sci. USA, M, 7413 (1987)], and the method described in Virology, 52, 456 (1973). Can be raised. -When insect cells are used as a host, for example, Baculoviras Expression Vectors, A Laboratory Manual, WH Freeman and Company, New York (1992), current-protocolezin molecular 'no ^; Proteins can be expressed by the method described in Biology, A Laboratory Manual, Bio / Technology, 6, 47 (1988) and the like.

That is, the recombinant gene transfer vector and baculovirus are co-transfected into insect cells to obtain recombinant virus in the insect cell culture supernatant, and then the recombinant virus is infected into insect cells to express the protein. Can be done.

Examples of the gene transfer vector used in the method include pVL1392, pVL1393, pBlueBacII (all manufactured by Invitrogen) and the like.

The baculovirus, e.g., monkey in be used such as out publicly available, power Nuclear Doroshisu 'virus to single-poly is a virus that infects burglary Gaka insects (Autographa californica nuclear polyhedrosis virus) ₀

As insect cells, single cells of eggs of Spofioptera. Fn] giperria, ovary cells of Ikhopliiaiaiii, cultured cells derived from silkworm ovary, and the like can be used.

Spodoptera fn] giDer (Sf9, Sf2l (Baculovirus 'Expression Vectors', 'Laboratory' Manual) for k-cell single cells, etc.), etc., for Trichoplusi iii ovarian cells, High 5, BTI-TN-5B1-4 ( Cultured cells derived from silkworm ovaries, such as Invitrogen) can be exemplified by mhyx moi.

Examples of the method for co-transferring the above-described baculovirus and the above-described recombinant gene transfer vector into insect cells for preparing a recombinant virus include a calcium phosphate method (Japanese Patent Laid-Open No. 2-227075), a lipofection method [Proc. Natl. Acad. Sci. USA, 84, 7413 (1987)].

When a plant cell is used as a host cell, as an expression vector, for example, Ti Bra Sumid, tobacco mosaic virus vector and the like can be mentioned.

Any promoter may be used as long as it functions in a plant cell, and examples thereof include the cauliflower mosaic virus (CaMV) 35S promoter and the geneactin 1 promoter.

Examples of the host cell include plant cells of tobacco, potato, tomato, carrot, soybean, rape, alfalfa, rice, wheat, wheat, and the like.

As a method for introducing a recombinant vector, any method can be used as long as it is a method for introducing DNA into plant cells. For example, agropacterium (toh kriM) (JP-A-59-140885; 60-78080, W094 / 00977), electro-volatilization method (JP-A-60-251887), method using a particle gun (gene gun) (Patent No. 2606856, Patent No. 2517813) and the like. .

When a gene is expressed using yeast, animal cells, or insect cells as a host, a sugar or sugar chain-added protein can be obtained.

The protein of the present invention can be produced by culturing the transformant obtained as described above in a medium, producing and accumulating the protein of the present invention in the culture, and collecting from the culture.

The method for culturing the transformant of the present invention in a medium can be performed according to a usual method used for culturing a host.

A culture medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host contains a carbon source, a nitrogen source, inorganic salts, and the like which can be used by the organism. Either a natural medium or a synthetic medium may be used as long as the medium can be efficiently cultured.

The carbon source may be any one that can be assimilated by the organism, such as glucose, fructose, sucrose, carbohydrates such as rice nectar, starch or starch hydrolysate, and organic acids such as acetic acid and propionic acid. Acids, alcohols such as ethanol and propanol, and the like can be used.

Nitrogen sources include ammonia, ammonium chloride, ammonium sulfate, and ammonium acetate. Ammonia, ammonium salts of inorganic or organic acids such as ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn starch, casein hydrolyzate, soybean meal and soybean meal Hydrolysates, various fermentation cells, and digests thereof can be used.

As the inorganic salt, potassium (II) phosphate, potassium (II) phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate, and the like can be used.

The cultivation is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is

The temperature is preferably 15 to 40 ° C, and the culture time is usually 5 hours to 7 days. During the culture, maintain the pH at 3.0 to 9.0. The pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like.

If necessary, an antibiotic such as ampicillin-tetracycline may be added to the medium during the culture.

When culturing a transformed microorganism in an expression vector using an inducible promoter as a promoter, an inducer may be added to the medium, if necessary. For example, when culturing a microorganism transformed with an expression vector using the i promoter, isopropanol ??! )-When culturing a microorganism transformed with thiogalactoviranoside or the like in an expression vector using a trc promoter, indoleacrylic acid or the like may be added to the medium.

As a medium for culturing transformants obtained using animal cells as a host, commonly used RPMI 1640 medium [J. Am. Med. Assoc., Dist., 519 (1967)], Eagle's MEM medium [Science , 501 (1952)], DMEM medium [Virology, S, 396 (1959)], 199 medium [Proc. Soc. Biol. Med., ΊΆ, 1 (1950)], or fetal calf serum or the like was added to these mediums. A medium or the like can be used.

Culture is usually pH 6-8, it performed 1-7曰間under conditions such as 25~40 ° C, 5% C 0 2 presence.

If necessary, an antibacterial substance such as kanamycin, penicillin or streptomycin may be added to the medium during the culturing. Commonly used culture media for transformants obtained using insect cells as a host include T 丽 -FH media (Pharmingen) and Sf-900 II SFM media (Life's Technologies). ExCell400, ExCell140 (all manufactured by JRH Biosciences), Grace's Insect Medium [Nature, 195, 788 (1962)] and the like can be used.

Cultivation is usually carried out for 1 to 5 days under conditions of pH 6 to 7 and 25 to 30 ° C.

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 plant cell or organ. As 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. An added medium or the like can be used.

The cultivation is usually performed at pH 5 to 9 and 20 to 40 ° C for 3 to 60 days.

If necessary, antibiotics such as kanamycin and hygromycin may be added to the medium during the culture.

As described above, a transformant derived from a microorganism, animal cell, or plant cell having a recombinant DNA into which the DNA encoding the protein of the present invention has been incorporated is cultured according to a conventional culture method, and the protein is purified. By producing and accumulating the protein, and collecting the protein from the culture, the protein can be produced.

The method for producing the protein of the present invention includes a method of producing the protein in a host cell, a method of secreting the protein outside the host cell, and a method of producing the protein on the host cell outer membrane.

When the protein of the present invention is produced in the host cell or on the host cell outer membrane, the method of Paulson et al. [J. Biol. Chem., 264, 17619 (1989)] and the method of Lowe et al. [Proc. Natl. Acad. USA, 86, 8227 (1989), Genes Develop., 4, 1288 (1990)] or JP 05-336963, W094 / 23021, etc. It can be actively secreted outside.

That is, the protein of the present invention is expressed by adding a signal peptide in front of the protein containing the active site of the protein of the present invention using a gene recombination technique, whereby the protein of the present invention can be used as a host. It can be secreted extracellularly.

Further, according to the method described in Japanese Patent Application Laid-Open No. 2-227075, the production amount can be increased using a gene amplification system using a dihydrofolate reductase gene or the like.

Furthermore, by redifferentiating the animal or plant cells into which the gene has been introduced, an individual animal (transgenic non-human animal) or individual plant (transgenic plant) into which the gene has been introduced is created. The protein of the present invention can be produced using an individual. When the transformant is an animal or plant individual, the protein is produced or cultivated according to a conventional method to produce and accumulate the protein, and the protein is collected from the animal or plant individual to obtain the protein. Can be manufactured.

As a method for producing the protein of the present invention using animal individuals, for example, known methods [Am. J. Clin. Nutr.,, 639S (1996), Am. J. Clin. Nutr., 03, 627S ( 1996), Bio / Technology, 9, 830 (1991)], and a method for producing the protein of the present invention in an animal constructed by introducing a gene.

In the case of an animal individual, for example, breeding a transgenic non-human animal into which DNA encoding the protein of the present invention has been introduced, producing and accumulating the protein in the animal, and collecting the protein from the animal Thus, the protein can be produced. Examples of the production-accumulation site in the animal include milk (Japanese Patent Application No. 63-309192), eggs, and the like of the animal. Any promoter that can be used in this case can be used as long as it functions in animals. Examples of such promoters include casein promoters, casein promoters, lactoglobulin promoters, and whey acid, which are breast cell-specific promoters. Sexual protein promoters and the like are preferably used.

Examples of the method for producing the protein of the present invention using plant individuals include, for example, transgenic plants into which DNA encoding the protein of the present invention has been introduced [Tissue culture, 0 (1994), Tissue culture, 21 (1995) , Pp. 45 (1997)], producing and accumulating the protein in the plant Φ, and collecting the protein from the plant to produce the protein. can give.

The protein produced by the transformant of the present invention is isolated and purified by a conventional method. Enzyme isolation and purification methods can be used.

For example, when the protein of the present invention is expressed in a dissolved state in a cell, the cells are collected by centrifugation after completion of the culture, suspended in an aqueous buffer, and then sonicated with a sonicator, French press, or the like. The cells are disrupted using a Manton Gaurin homogenizer, Dynomill, etc. to obtain a cell-free extract.

From the supernatant obtained by centrifuging the cell-free extract, a normal enzyme isolation / purification method, that is, a solvent extraction method, a salting-out method using ammonium sulfate, a desalting method, a precipitation method using an organic solvent, Luminoethyl (DEAE) —Anion exchange chromatography using resins such as Sepharose and DIAION HPA-75 (Mitsubishi Kasei), and cation exchange chromatography using resins such as S-Sepharose FF (Pharmacia) Electrophoresis such as chromatography, hydrophobic chromatography using resins such as butyl sepharose and phenylsepharose, gel filtration using molecular sieve, affinity chromatography, chromatofocusing, isoelectric focusing, etc. A purified sample can be obtained using techniques such as electrophoresis alone or in combination. When the protein is expressed in an insoluble form in the cells, the cells are similarly recovered, crushed, and separated from the precipitate fraction obtained by performing eccentric separation. After recovery, the insoluble form of the protein is solubilized with a protein denaturant.

After diluting or solubilizing the solubilized solution containing no protein denaturing agent or diluting the protein denaturing agent to such a concentration that the protein is not denatured, the protein is formed into a normal three-dimensional structure. A purified sample can be obtained by the same isolation and purification method as described above.

When the protein of the present invention or its derivative such as a modified sugar is secreted extracellularly, the protein or its derivative such as a sugar chain adduct can be recovered in the culture supernatant.

That is, a soluble fraction is obtained by treating the culture by a technique such as centrifugation as described above, and a purified sample is obtained from the soluble fraction by using the same isolation and purification method as described above. be able to.

Examples of the protein obtained in this manner include a protein having the amino acid sequence shown in SEQ ID NO: 1.

Further, the polypeptide of the present invention is produced as a fusion protein with another protein, Purification can also be performed using affinity chromatography using a substance having an affinity for the combined protein. For example, the method of Lowe et al. [Proc. Natl. Acad. Sci. USA, 2 £, 8227 (1989), Genes Develop., I, 1288 (1990)], JP-A-5-336963, W094 / 23021. According to the method, the polypeptide of the present invention can be produced as a fusion protein with protein A and purified by affinity chromatography using imnoglobulin G.

In addition, the polypeptide of the present invention can be produced as a fusion protein with an F1ag peptide and purified by affinity chromatography using an anti-F1ag antibody [Proc. Natl. Acad. Sci. USA , S £, 8227 (1989), Genes Develop., 4, 1288 (1990)]. Furthermore, the polypeptide can be purified by affinity chromatography using an antibody against the polypeptide itself.

Based on the amino acid information of the protein obtained above, the chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) and the Seven Boc method (71-butyloxycarbonyl method) The protein of the invention can be produced. Advanced ChemTech, Parkin

• Chemical synthesis can also be performed using peptide synthesizers such as Elma, Pharmacia, Protein Technology Instrumen, Synthecell-Vega, PerSeptive, and Shimadzu.

[3] Preparation of N-acetylneuraminic acid

A culture of the transformant obtained by the culture according to the above [2] and a processed product of the culture are used as an enzyme source, and the enzyme source, N-acetyl mannosamine and phosphoenolpyruvate are added to an aqueous medium. N-acetylneuraminic acid can be produced in the aqueous medium.

Examples of the processed product of the culture include a concentrate of the culture, a dried product of the culture, cells obtained by centrifuging the culture, a dried product of the cells, a freeze-dried product of the cells, the cells Surfactant treated product, ultrasonically treated product of the cells, mechanically milled product of the cells, solvent-processed product of the cells, enzyme-treated product of the cells, protein fractionation of the cells Products, immobilized products of the cells or enzyme preparations obtained by extraction from the cells.

The enzyme source used in the production of N-acetylneuraminic acid is l〃mol / min at 37 ° C per minute. The activity capable of producing N-acetylneuraminic acid as 1 unit (U) is 1 mU / 1 to 1,000 U / 1, preferably a concentration of 10 mU / l to 100 U / 1. Used in

Aqueous media used in the production of N-acetylneuraminic acid include water, phosphates, carbonates, acetates, buffers such as borate, citrate and tris, and alcohols such as methanol and ethanol. And esters such as ethyl acetate, ketones such as acetone, and amides such as acetoamide. Further, a culture solution of the microorganism used as the enzyme source can be used as an aqueous medium.

In the production of N-acetyl neuraminic acid, a surfactant or an organic solvent may be added as necessary. Examples of the surfactant include nonionic surfactants such as polyoxyethylene octadecylamine (for example, Nimeen S-215, manufactured by NOF Corporation), cetyltrimethylammonium bromide alkyldimethylbenzylbenzyl chloride, and the like. (For example, cationic surfactants such as cation F2-40E, manufactured by NOF Corporation), anionic surfactants such as lauroyl'zalcosinate, and alkyldimethylamine (for example, tertiary amine FB, manufactured by NOF Corporation) Any of the tertiary amines that promote the production of N-acetyl neuraminic acid, such as the above tertiary amines, may be used alone or in combination of one or more. The surfactant is usually used at a concentration of 0.1 to 50 g / 1. Examples of the organic solvent include xylene, toluene, aliphatic alcohol, acetone, and ethyl acetate, which are usually used at a concentration of 0.1 to 50 ml / l.

The reaction for producing N-acetyl neuraminic acid is carried out in an aqueous medium at pH 5 to 10, preferably pH 6 to 8, and 20 to 50 ° G for 1 to 96 hours. In the production reaction, it is possible to add inorganic salts such as Mn C 1 ₂ if necessary.

The quantification of N-acetyl neuraminic acid formed in the aqueous medium can be performed using a sugar analyzer manufactured by Dionex or the like [Anal. Biochem., 189, 151 (1990)].

The N-acetylneuraminic acid generated in the reaction solution can be collected by a usual method using activated carbon, an ion exchange resin, or the like. Simple theory FIG. 1 is a diagram showing a process for constructing a plasmid pNP1 expressing N-acetyl neuraminic acid synthase.

The symbols in the drawings represent the following meanings.

A mp ^r : Ampicillin resistance gene

P _lrp : T rp Promo Overnight

n e u B: N-Acetylneuraminic acid synthase gene

Examples of the present invention will be described below, but the present invention is not limited to these examples. Example 1 Homology Search Using Genome D Ν Α Sequence Database

Nostoc punctiforme ATCC29133 Genome Sequence Database

(http: 〃 spider. jgi-psf.org/JGI—microbial/html/) and the amino acid sequence of N-acetylneuraminic acid synthase derived from Escherichia coli [J. Bacteriol., ill, 312 (1995 )] Was searched using BLAST.

As a result, an amino acid sequence represented by SEQ ID NO: 1 was obtained as an amino acid sequence having high homology to the amino acid sequence of N-acetylneuraminic acid synthase derived from Escherichia coli, and a DNA encoding the amino acid sequence was obtained. DNA having the nucleotide sequence represented by SEQ ID NO: 2 was obtained.

Example 2 Construction of a strain expressing a gene derived from a microorganism belonging to the genus Nostock

Nostoc punctiforme ATCC29133 was cultured by the method described in Microbiology, 140, 3233 (1994).

After the culture, the chromosome DNA of the microorganism was isolated and purified by the method described in Current “Protocols” in “Molecular Biology”.

Using a DNA having the nucleotide sequence represented by SEQ ID NO: 3 or 4 synthesized using a Perceptive Biosystems Model 8905 DNA synthesizer, the DNA fragment containing the gene selected in Example 1 was used. The width was increased by the following method.

Using the above synthetic DNA as a primer set, staining of Nostoc punctiforme ATCC29133 PCR was performed using chromosomal DNA as type I. PCR is a reaction containing 0.1 mg of chromosomal DNA, 0.5 mmol / U of each primer, 2.5 units of Pfu DNA polymerase (manufactured by Stratagene), 4 ml of x10 buffer for Pfu DNA polymerase, and 200 imnol / 1 of deoxyNTP. Using 40 ml of the liquid, the process was repeated 30 times at 9.4 ° C for 1 minute, at 42 ° C for 2 minutes, and at 72 ° C for 3 minutes.

After 1/10 volume of the reaction mixture was subjected to agarose gel electrophoresis to confirm that the target fragment had been amplified, an equal amount of TE [10 iMol / 1 Tris-HCK 1 mmol / 1 EDTA (pH 8.0)] Saturated phenol / chloroform (1 vol / 1 vol) was added and mixed.

After the mixture is centrifuged, 2 volumes of cold ethanol is added to the obtained upper layer, mixed, and left at -80 ° C for 30 minutes. The liquid was centrifuged to obtain a precipitate of DNA.

The DNA precipitate was dissolved in 20 ml of TE.

Using 5 ml of the lysate, the DNA was digested with restriction enzymes and £ _ajHI, DNA fragments were separated by agarose gel electrophoresis, and a 1.0 kb: DNA fragment was purified using GeneClean II kit (Funakoshi). Collected.

After cutting 0.21 1 of pTr S 30 1) with the restriction enzyme _01 ^ 1 and 3_201111, the DNA fragment was separated by agarose gel electrophoresis: a 4.2 kb DNA fragment was recovered in the same manner. The ligation fragments of l.Okb and 4.2kb were subjected to ligation reaction at 16 ° for 16 hours using a ligation kit (Takara Shuzo).

Using the ligation reaction solution, Escherichia coli 522522 was transformed according to the above-mentioned known method, and the transformant was LB agar medium containing 50 mg / ml of ampicillin [Pactotripton (Difco) iOg / Yeast extract] (Manufactured by Difco), 10 g / l, sodium chloride 5 g / l, agarose 15 g], and cultured overnight at 28 ° C.

Plasmid was extracted from the thus grown transformant colonies according to the above-mentioned known method to obtain pNP1, an expression plasmid. Fig. 1 shows the construction procedure and structure of the plasmid.

Example 3 Production of N-acetylneuraminic acid

The Escherichia, coli 丽 522 / pNPl strain obtained in Example 2 was transformed with L containing 50 mg / ml of ampicillin. A large test tube containing 8 ml of B medium was inoculated and cultured at 28 ° C for 17 hours. The culture solution was placed at 1 ° / に in a large test tube containing 8 ml of LB medium containing 50 mg / ml of ampicillin. After inoculation and culturing at 28 ° C for 5 hours, 0.4 ml of the culture was centrifuged to obtain wet cells.

The wet cells could be stored at -20 ° C if necessary, and could be thawed before use.

丽 522 / pNPl strain wet cells obtained above, 100 t ol / l Tris-HCl (pH 7.5), 8.3 mmol / l MnGl ₂ , 91 t ol / l N-acetyl mannosamine, 42 maraudal ol The reaction was carried out at 28 ° C. for 30 minutes in a 0.1 ml reaction solution consisting of / l phosphoenolpyruvic acid and 4 g / l Nimeen S-215.

After completion of the reaction, the reaction product was analyzed under the following analysis conditions using a sugar analyzer (DX-500, manufactured by Dionex), and 0.48 ol / l (150 mg / l) of N-acetylethyl It was confirmed that laminic acid was generated and accumulated.

Analysis conditions:

Column: CarboPAC PA10

Eluent: Eluent A: 0, Eluent B: 500 ol / l NaOH

Gradient: Change from a composition consisting of 8% of eluent B at 0 minutes to a composition consisting of 100% of eluent B linearly over 30 minutes.

Detector: pulsed dornometry detector possible under the hood

According to the present invention, N-acetyl neuraminic acid synthase can be produced in large quantities. In addition, N-acetylneuraminic acid can be efficiently produced by using the enzyme.

"Sequence List Free Text"

SEQ ID NO: 3—Description of artificial sequence: Synthetic DNA

SEQ ID NO: 4 Description of artificial sequence: Synthetic! ) N A

Claims

The scope of the claims

1. A protein having the amino acid sequence represented by SEQ ID NO: 1.

2. A protein comprising an amino acid sequence in which one or more amino acids have been deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 1, and having N-acetyl neuraminic acid synthase activity.

3. A DNA encoding the protein according to claim 1 or 2.

4. Has the nucleotide sequence represented by SEQ ID NO: 2: DNA.

5. A DNA that hybridizes with a DNA having the base sequence of SEQ ID NO: 2 under stringent conditions and encodes a protein having N-acetyl neuraminic acid synthase activity.

6. The DNA according to any one of claims 3 to 5, wherein the DNA is derived from a microorganism belonging to the cyanobacterium (fiyanQkkiki).

7. The DNA of claim 6, wherein the DNA is derived from a microorganism belonging to the genus Cyanobacteria: The DNA is a DNA derived from a microorganism belonging to the genus Nostiin.

8. The DNA according to claim 7, wherein the microorganism belonging to the genus Nostock (si) is Nostoc puncti forme.

9. A recombinant DNA obtained by incorporating the DNA according to any one of claims 3 to 8 into a vector.

10. The recombinant according to claim 9, a transformant obtained by introducing DNA into a host cell.

11. The transformant according to claim 10, wherein the host cell is Escherichia coli.

12. The transformant according to claim 10 or 11, wherein the transformant is cultured in a medium, a protein having N-acetyl neuraminic acid synthase activity is produced and cultured in the culture, and the protein is converted from the culture. A method for producing a protein having N-acetyl neuraminic acid synthase activity.

13. A culture of the transformant according to claim 10 or 11 or a processed product of the culture is used as an enzyme source, and the enzyme source, N-acetyl mannosamine and phosphoenolpyruvate are contained in an aqueous medium. To form N-acetyl neuraminic acid in the aqueous medium. Collecting N-acetyl neuraminic acid from the aqueous medium. A method for producing N-acetyl neuraminic acid.

14. The processed product of the culture is a concentrate of the culture, a dried product of the culture, a cell obtained by centrifuging the culture, a dried product of the cell, a lyophilized product of the cell, Treated body with a surfactant, ultrasonically treated cell, mechanically milled cell, solvent-treated cell, enzyme-treated cell, protein of the cell 14. The method according to claim 13, wherein the product is a fraction, an immobilized product of the cells, or an enzyme preparation obtained by extraction from the cells.