JP2005046031A - FUSED PROTEIN HAVING alpha1,3- FUCOSYL TRANSFERASE VI ACTIVITY AND METHOD FOR PRODUCING THE SAME - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To make it possible to easily efficiently and massively produce a fused protein having an α1,3-fucosyl transferase VI activity as a soluble protein, and to make it possible to easily obtain the 1,3-fucosyl transferase VI itself from the fused protein. <P>SOLUTION: The recombinant fused protein comprising a sugar-binding protein and a 1,3-fucosyl transferase VI. The method for producing the recombinant fused protein is characterized by comprising the following processes (1) to (3): (1) transforming Escherichia coli with an expression vector in which a DNA encoding a sugar-binding protein is connected to a DNA encoding the 1,3-fucosyl transferase VI under the control of a promoter capable of acting in the Escherichia coli to express the fused protein of both the proteins, (2) culturing the obtained transformant to produce the fused protein comprising the sugar-binding protein and the 1,3-fucosyl transferase VI, and (3) separating the fused protein from the obtained culture product. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【００９５】
【発明の効果】
本発明の方法では、α１，３−フコシルトランスフェラーゼＶＩ（ＦＵＴ６）活性を有する融合タンパク質を可溶性タンパク質として容易に効率よく大量に生産することができる。また、融合タンパク質からα１，３−フコシルトランスフェラーゼＶＩ（ＦＵＴ６）そのものも容易に得ることができる。
【００９６】
本発明によって得られるα１，３−フコシルトランスフェラーゼＶＩ（ＦＵＴ６）は、自体糖鎖工学研究用試薬として有用で有るだけでなく、得られたα１，３−フコシルトランスフェラーゼＶＩ（ＦＵＴ６）を利用することにより、研究用途或いは医薬用途として有用な種々のルイス構造糖鎖、或いはルイス構造糖鎖類似体、フコース酸含有糖鎖、フコース酸含有糖鎖を含む種々の化学物質等の糖鎖合成が容易になる。さらに、種々の有用タンパク質や種々の糖鎖含有化学物質の糖鎖修飾にも利用が可能となる。また、研究用途或いは医薬用途として有用なα１，３−フコシルトランスフェラーゼＶＩ（ＦＵＴ６）抗体を容易に得ることが可能となる。
【００９７】
【配列表】

【図面の簡単な説明】
【図１】本図は、ＦＵＴ６ が触媒する反応を示した図である。詳しくは、糖ヌクレオチドであるＧＤＰ−フコース（ＧＤＰ−Ｆｕｃ）をフコース供与体として、糖受容体であるＲ_１−Ｇａｌβ１−４ＧｌｃＮＡｃ−Ｒ_２にフコースをα１−３結合で転移し、Ｒ_１−Ｇａｌβ１−４（Ｆｕｃα１−３）ＧｌｃＮＡｃ−Ｒ_２を生成せしめる反応を示した図である。
【図２】本図は、大腸菌で発現させたＭＢＰ−ＦＵＴ６融合タンパク質のＦＵＴ６活性測定の結果を示した図である。
【図３】本図は、大腸菌で発現したＭＢＰ−ＦＵＴ６のＦＵＴ６活性に及ぼす反応液のｐＨの影響について示した図である。
【図４】本図は、大腸菌で発現したＭＢＰ−ＦＵＴ６のＦＵＴ６活性に及ぼすマンガン濃度の影響について示した図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for efficiently producing α1,3-fucosyltransferase VI, which is a kind of glycoprotein processing enzyme, at low cost. Specifically, the present invention relates to a fusion protein of maltose-binding protein and α1,3-fucosyltransferase VI protein and a method for producing the protein in Escherichia coli.
[0002]
[Prior art]
Sugar chains in which various monosaccharides are linked by glycosidic bonds exist in cells as intracellular organelle components, cell surface components, secreted glycoproteins, and the like. The structure of these sugar chains varies depending on the species and tissue, but also varies depending on the time of occurrence and disease within the same organism and tissue, so the previously proposed protein thermal stability and hydrophilicity In addition to functions such as imparting physical properties to proteins such as charge and protease resistance, it is clear that sugar chains are involved in intercellular recognition such as development / differentiation, nervous system, immune system, and metastasis of cancer. In recent years, much attention has been paid to its application to various fields such as pharmaceuticals.
[0003]
These sugar chains are synthesized by glycosyltransferase in vivo. Glycosyltransferase is an enzyme that uses a sugar nucleotide as a sugar donor to transfer sugar to a sugar chain as an acceptor and to extend the sugar chain, and the specificity of the sugar acceptor for the sugar chain structure is strict. In general, it is said that one glycosidic bond is formed by one glycosyltransferase. These glycosyltransferases are important enzymes in that they are used for sugar chain research, particularly for convenient synthesis of useful sugar chains and modification of natural sugar chains. However, the amount of naturally occurring glycosyltransferase is extremely small, and it was practically difficult to supply a large amount stably.
[0004]
α1,3-fucosyltransferase is an important enzyme involved in the formation of glycoprotein or glycolipid sugar chain, and R1-Galβ1- of glycoprotein or glycolipid which is a sugar acceptor using GDP-Fuc as a sugar donor. Activity of transferring fucose to N-acetylglucosamine of a sugar chain of 4GlcNAc-R2 structure with α1-3 bond to generate a sugar chain of R1-Galβ1-4 (Fucα1-3) GlcNAc-R2 structure (hereinafter referred to as α1-3FucT activity (In some cases, R1 represents OH, sialic acid, or fucose, and R2 represents various sugar chains). α1,3-fucosyltransferases are currently classified into six types (α1,3-fucosyltransferases III to VII, α1,3-fucosyltransferase IX) depending on the substrate specificity (for example, Non-patent Document 1 or Table- 1).
[0005]
α1,3-fucosyltransferase III (hereinafter sometimes abbreviated as FUT3) is an enzyme called Lewis enzyme, and Galβ1-4GlcNAc-R2 (hereinafter sometimes abbreviated as type II sugar chain) or α2- 3βGalβ1-4GlcNAc-R2 (hereinafter sometimes abbreviated as sialyl type II sugar chain) or Fucα1-2Galβ1-4GlcNAc-R2 (hereinafter also abbreviated as HII type sugar chain) as a substrate, and Galβ1-4 ( Fucα1-3) GlcNAc-R2 (hereinafter Le^XOr α2-3Galβ1-4 (Fucα1-3) GlcNAc-R2 (hereinafter referred to as sLe).^XOr Fucα1-2Galβ1-4 (Fucα1-3) GlcNAc-R2 (hereinafter referred to as Le)^YMay be omitted). Furthermore, FUT3 uses GDP-Fuc as a sugar donor to transfer fucose to N-acetylglucosamine of the sugar chain of R1-Galβ1-3GlcNAc-R2 structure by α1-4 linkage, and R1-Galβ1-3 (Fucα1-4) GlcNAc- It also has an activity to generate a sugar chain having an R2 structure (hereinafter sometimes abbreviated as α1-4FUcT activity). Only 6 types of FUT3 have clear α1-4FucT activity, and Galβ1-3 (Fucα1-4) GlcNAc-R2 (hereinafter referred to as Le) is obtained by this activity.^aOr α2-3Galβ1-3 (Fucα1-4) GlcNAc-R2 (hereinafter referred to as sLe).^aOr Fucα1-2Galβ1-3 (Fucα1-4) GlcNAc-R2 (hereinafter referred to as Le)^bMay be omitted) (see Non-Patent Document 1 or Table 1).
[0006]
α1,3-fucosyltransferase IV (hereinafter sometimes abbreviated as FUT4) is an enzyme called myeloid α1-3FucT, which uses a type II sugar chain, a sialyl type II sugar chain, or a type HII sugar chain as a substrate, Le^XSLe^XOr Le^YCan be synthesized. However, sLe^XThe synthetic activity is very weak (for example, see Non-Patent Documents 1 and 2 or Table 1).
[0007]
α1,3-fucosyltransferase V (hereinafter sometimes abbreviated as FUT5) is a type II sugar chain, sialyl type II sugar chain or HII type sugar chain as a substrate, and Le.^XSLe^XOr Le^YCan be synthesized. Moreover, although it is very weak, it exhibits α1-4FucT activity, and Le^aOr sLe^aOr Le^bCan be synthesized (for example, see Non-Patent Document 1 or 3 or Table 1).
[0008]
α1,3-fucosyltransferase VI (hereinafter sometimes abbreviated as FUT6) is an enzyme in the present invention and is also called plasma α1-3FUcT. FUT6 uses a type II sugar chain, sialyl type II sugar chain or HII type sugar chain as a substrate, and Le^XOr sLe^XOr Le^YThe specific activity is the strongest (see, for example, Non-Patent Documents 1 and 2 or Table 1). Moreover, even if a short oligosaccharide is used as a substrate, strong transfer activity can be exhibited (see Non-Patent Documents 1 and 2).
[0009]
α1,3-fucosyltransferase VII (hereinafter sometimes abbreviated as FUT7) has the strictest substrate specificity, and sialyl type II sugar chain sLe^XOnly the reaction that synthesizes the catalyst. FUT7 is sLe on leukocytes and lymphocytes^XIt is considered to be involved in the synthesis of sugar chains (for example, see Non-Patent Documents 1 and 2 or Non-Patent Documents 4 to 6 or Table 1).
[0010]
α1,3-fucosyltransferase IX (hereinafter sometimes abbreviated as FUT9) uses a type II sugar chain or HII type sugar chain as a substrate, and Le^XOr Le^YCan be synthesized, but sLe^XThere is no synthetic activity. FUT9 has an extremely strong activity of transferring fucose to N-acetylglucosamine of the lactosamine unit at the non-reducing end, while FUT3, FUT4, FUT5, and FUT6 have a strong activity of transferring to N-acetylglucosamine of the internal lactosamine unit. (See Patent Document 1 or 7).
[0011]
As described above, α1,3-fucosyltransferase is deeply involved in the synthesis of a Lewis structure sugar chain which is considered to be deeply involved in cancer metastasis and the like.^x, SLe^x, Le^YFUT6 was important as a sugar chain synthesis application in that it has a high synthesis ability for.
[0012]
For the above reasons, a stable supply of FUT 6 has been desired.
[0013]
Although this FUT6 is known to exist in various animal organs, it is difficult to obtain a large amount of stable organs, and it is very time-consuming to purify to a single protein. Therefore, production by genetic recombination is expected.
[0014]
The cDNA encoding the enzyme has already been derived from humans (for example, see Non-Patent Document 8 or 9), chimpanzee (for example, see Non-Patent Document 10), and bovine (for example, see Non-Patent Document 11). It has been isolated.
[0015]
In consideration of cost, mass production of a recombinant protein is advantageous by expressing the protein in microorganisms such as bacteria and yeast. However, in the expression system of microorganisms, many of the expressed proteins may exist as insoluble inclusion bodies (inclusion bodies), which requires a solubilization / regeneration process prior to purification. I couldn't say that. Furthermore, in order to obtain an enzyme expressed as a partly soluble protein with high purity, many purification steps including various chromatographic processes are required, and it takes a lot of time and cost. The expression of was not sufficient.
[0016]
On the other hand, as a method of expressing a target gene in a large amount and facilitating purification of an expression product, there is a method of expressing a target protein as a fusion protein with glutathione-S-transferase (GST), protein A or the like. . In the method, a fusion protein with GST. The protein can be easily purified by affinity column chromatography using glutathione as a ligand, and the fusion protein with protein A can be purified by affinity chromatography using IgG as a ligand. However, when this method is used, there is a high possibility of producing inclusion bodies as in the previous period. For these reasons, it has been practically difficult to stably supply a large amount of FUT6.
[0017]
[Non-Patent Document 1]
"Protein Nucleic Acid Enzyme December 1998 Special Issue Glycobiology From Glycan Information Transmission to Reception Mechanism", Kyoritsu Shuppan Co., Ltd., p77, 1998
[0018]
[Non-Patent Document 2]
“Biochemical and Biophysical Research Communications”, 273, p131, 1997
[0019]
[Non-Patent Document 3]
“The Journal of Biological Chemistry”, 272, p8764, 1997
[0020]
[Non-Patent Document 4]
“The Journal of Biological Chemistry”, 269, p14730, 1994
[0021]
[Non-Patent Document 5]
“The Journal of Biological Chemistry”, 269, p16789, 1994
[0022]
[Non-Patent Document 6]
"Cell", 86, p643, 1996
[0023]
[Non-Patent Document 7]
"FEBS Letter", 462, p289, 1999
[0024]
[Non-Patent Document 8]
“Biochemical and Biophysical Research Communications”, 187, p152, 1992
[0025]
[Non-patent document 9]
“The Journal of Biological Chemistry”, 267, p24575, 1992
[0026]
[Non-Patent Document 10]
“The Journal of Biological Chemistry”, 272, p29721, 1997
[0027]
[Non-Patent Document 11]
“The Journal of Biological Chemistry”, 272, p8764, 1997
[0028]
[Non-Patent Document 12]
“The Journal of Biological Chemistry”, 259, p10606, 1984
[0029]
[Non-Patent Document 13]
"Proceedings of the National Academy of the United States of America", Vol. 69, p. 2110, p. 1910, "Proceedings of the National Academy of Science of the United States of America".
[0030]
[Non-Patent Document 14]
"Molecular Genetics and Genomics", 168, p111, 1979
[0031]
[Non-Patent Document 15]
"Journal of Molecular Biology", 56, p209, 1971
[0032]
[Non-Patent Document 16]
"Cell engineering separate volume Experimental protocol series Glycobiology experimental protocol glycoprotein, glycolipid, proteoglycan", Shujunsha, 1996
[0033]
[Means for Solving the Problems]
As a result of intensive studies to solve the above problems, the present inventors have determined that FUT6 is genetically manipulated as a fusion protein with a sugar-binding protein such as maltose binding protein (hereinafter sometimes abbreviated as MBP). FUT6 can be obtained as a soluble protein, and the protein can be easily purified by affinity chromatography using the specific affinity of the sugar-binding protein, and the resulting fusion protein exhibits FUT6 activity. I have found it.
[0034]
Furthermore, the present inventors have found that FUT6 can also be obtained from the fusion protein by a protease that specifically cleaves the sequence of the fusion site between the sugar-binding protein and FUT6.
[0035]
That is, the present invention includes the following inventions.
[0036]
Item 1. A recombinant fusion protein of a sugar-binding protein and α1,3-fucosyltransferase VI.
Item 2. Item 2. The recombinant fusion protein according to Item 1, wherein α1,3-fucosyltransferase VI is derived from a human.
Item 3. The recombinant fusion protein according to claim 1 or 2, wherein α1,3-fucosyltransferase VI is the protein described in (a) or (b).
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2
(B) A protein having an α1,3-fucosyltransferase VI activity, comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in (a)
Item 4. Item 4. The protein according to Item 3, wherein α1,3-fucosyltransferase VI comprises at least the amino acid sequence represented by amino acid numbers 41 to 359 in the amino acid sequence represented by SEQ ID NO: 2.
Item 5. Item 5. The protein according to any one of Items 1 to 4, wherein α1,3-fucosyltransferase VI comprises an amino acid sequence in which amino acids corresponding to all or part of the transmembrane region of the protein are deleted.
Item 6. Item 6. The recombinant fusion protein according to any one of Items 1 to 5, comprising a protease recognition sequence between the sugar-binding protein and α1,3-fucosyltransferase VI.
Item 7. Item 6. The recombinant fusion protein according to any one of Items 1 to 5, wherein the sugar-binding protein is a maltose-binding protein.
Item 8. The recombinant fusion protein according to claim 6, wherein the protease is a protease selected from the group consisting of blood coagulation factor Xa, enterokinase, and genenase I.
Item 9. Item 9. A DNA encoding the recombinant fusion protein according to any one of Items 1 to 8.
Item 10. An expression vector comprising the DNA according to Item 9.
Item 11. Item 11. A transformant transformed with the expression vector according to Item 10.
Item 12. Item 9. The method for producing a recombinant fusion protein according to any one of Items 1 to 8, which comprises the following steps (1) to (3).
(1) Expression in which a DNA encoding a sugar-binding protein and a DNA encoding α1,3-fucosyltransferase VI are linked so as to be expressed as a fusion protein of both proteins under the control of a promoter capable of functioning in E. coli Using a vector, E. coli is transformed,
(2) culturing the obtained transformant to produce a fusion protein of a sugar-binding protein and α1,3-fucosyltransferase VI;
(3) Separating the fusion protein from the obtained culture
Item 13. Item 13. The method according to Item 12, wherein α1,3-fucosyltransferase VI is derived from a human.
Item 14. Item 14. The method according to Item 13, wherein the DNA encoding α1,3-fucosyltransferase VI comprises at least a base sequence encoding the amino acid sequence represented by amino acid numbers 41 to 359 in the amino acid sequence represented by SEQ ID NO: 2.
Item 15. Item 15. The DNA according to any one of Items 12 to 14, wherein the DNA encoding α1,3-fucosyltransferase VI comprises a base sequence encoding an amino acid sequence from which amino acids corresponding to all or part of the transmembrane site of the protein have been deleted. Method.
Item 16. Claims wherein the DNA encoding α1,3-fucosyltransferase VI comprises a base sequence obtained by deleting the base sequence encoding amino acids corresponding to all or part of the transmembrane site of the protein from the base sequence shown in SEQ ID NO: 1. Item 16. The method according to any one of Items 12 to 15.
Item 17. Item 17. The method according to any one of Items 12 to 16, wherein the sugar binding protein is a maltose binding protein.
Item 18. Item 18. The DNA according to Item 17, wherein the DNA encoding the maltose-binding protein is derived from a vector selected from the group consisting of pMAL p2, pMAL p2X, pMAL p2E, pMAL p2G, pMAL c2, pMAL c2X, pMAL c2E, and pMAL c2G. Method.
Item 19. Item 19. A method for producing α1,3-fucosyltransferase VI, which comprises the step of collecting α1,3-fucosyltransferase VI by removing the sugar-binding protein portion from the fusion protein obtained by the method according to any one of Items 12-18. .
Item 20. The DNA encoding a sugar binding protein contains a base sequence encoding a protease recognition sequence on the C-terminal side of the protein, and the sugar binding protein portion is removed from the fusion protein by the action of the protease. The method described.
Item 21. 21. The method according to claim 20, wherein the protease is a protease selected from the group consisting of blood coagulation factor Xa, enterokinase, and genenase I.
[0037]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is characterized in that the protein having FUT6 activity is a fusion protein of FUT6 and a sugar-binding protein, and the fusion protein is a soluble protein.
[0038]
Furthermore, the present invention allows FUT6 to be produced as a soluble fusion protein by expressing FUT6 in E. coli as a fusion protein with a sugar-binding protein, and utilizes the specific affinity possessed by the sugar-binding protein. Thus, the fusion protein and FUT6 can be purified easily and in large quantities.
[0039]
Fusion protein of the present invention
In the present invention, a fusion protein of a sugar-binding protein such as MBP and FUT6 (hereinafter, in the case of a fusion protein with a maltose-binding protein, it may be abbreviated as “MBP-FUT6”) has a sugar-binding protein. As long as it retains specific affinity, ie, high affinity for maltose and oligosaccharides and polysaccharides having maltose residues in the case of MBP, and FUT6 has the enzyme activity, it can be in any form. Good. Furthermore, as shown in FIG. 1, it is preferable that FUT6 obtained after removing at least the sugar-binding protein portion retains the original enzyme activity.
[0040]
Preferably, the fusion protein is one in which FUT6 is linked to the C-terminal side of the sugar-binding protein, and particularly preferably, the sugar-binding protein and FUT6 are easily cleaved at the fusion site by enzymatic or chemical means. It has the structure to obtain.
[0041]
A sugar-binding protein is a protein that retains high affinity for sugars and oligosaccharides and polysaccharides having sugar residues, and examples thereof include various lectins, maltose-binding proteins, cellulose-binding proteins, and chitin-binding proteins. . Preferred are maltose binding protein, cellulose binding protein, chitin binding protein and the like, and more preferred is maltose binding protein (hereinafter sometimes abbreviated as MBP).
[0042]
The sugar-binding protein may be derived from any biological species, but is preferably derived from a prokaryotic organism such as a bacterium, and more preferably derived from E. coli.
[0043]
In addition, as long as the MBP has a site having specific affinity for maltose or a oligosaccharide having a maltose residue (for example, maltotriose) or a polysaccharide (for example, amylose), the entire sequence is not necessarily present. It is not necessary to include it, and it may include a part of the sequence. Oligosaccharides (for example, maltotriose, etc.) or polysaccharides (for example, amylose, etc.) consisting of amino acids in which one or more amino acids are deleted, substituted or added in a known amino acid sequence and having maltose or maltose residues A protein having a site having specific affinity for can be exemplified.
[0044]
In the present invention, the gene encoding MBP is derived from pMAL p2, pMAL p2X, pMAL p2E, pMAL p2G, pMAL c2, pMAL c2X, pMAL c2E, or pMAL c2G (all manufactured by New England Biolabs). Can do.
[0045]
The sugar-binding protein of the present invention particularly preferably contains an amino acid sequence that is recognized and cleaved by a protease with high sequence specificity on the C-terminal side of the protein. Such proteases include factor Xa, enterokinase, genenase I, thrombin, renin, etc. Preferably, the protease is a factor Xa recognition sequence, ie, Ile-Glu-Gly-Arg (SEQ ID NO: 3) or Ile-Asp-Gly-Arg (SEQ ID NO: 4), enterokinase recognition sequence, ie Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 5), genenase I recognition sequence, ie His-Tyr (sequence) No. 6) or Tyr-His (SEQ ID NO: 7), more preferably a factor Xa recognition sequence or enterokinase recognition sequence. By introducing such a protease recognition sequence on the C-terminal side of the sugar binding protein, for example, the MBP moiety can be easily removed by allowing the protease to act after purification of MBP-FUT6.
[0046]
In a more preferred embodiment, the sugar binding protein includes a spacer sequence of about 5 to 15 amino acid residues between the sugar binding protein site and the protease recognition site. Due to the presence of the spacer sequence, the sugar binding protein moves away from FUT6, reducing the possibility of intramolecular interaction of the fusion protein, and in the case of MBP-ST3GalI fusion protein, the specific affinity of maltose and amylose for the MBP moiety is increased. To do. In the case of the present invention, pMAL p2 or pMAL p2X or pMAL p2E containing the spacer sequence of SEQ ID NO: 9 (Ser-Ser-Ser-Asn-Asn-Asn- Asn- Asn- Asn- Asn- Asn- Asn- Asn- Asn) Alternatively, pMAL p2G, pMAL c2, pMAL c2X, pMAL c2E, or pMAL c2G (all manufactured by New England Biolabs) can be used.
[0047]
FUT6 may be derived from any species, but is preferably derived from a mammal, and more preferably derived from a human. Furthermore, in the present invention, FUT6 does not need to contain the full-length FUT6 sequence as long as it retains FUT6 activity, and may contain a part of the sequence. For example, (a) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 or (b) an amino acid sequence in which one or several amino acids are deleted, substituted or added in (a) above, α1,3- It may be a protein having fucosyltransferase VI activity.
[0048]
In particular, since FUT6 is a transmembrane protein present in the Golgi apparatus, considering the generation of MBP-FUT6 as a soluble protein, one FUT6 excluding all or part of the highly hydrophobic transmembrane domain region is considered. The use of parts is also a preferred embodiment of the present invention. Specifically, it preferably includes an amino acid sequence represented by at least 41 to 359 of the amino acid sequence of SEQ ID NO: 2.
[0049]
Further, it may be a protein having one or more amino acids deleted, substituted or added in the amino acid sequence represented by at least 41 to 359 of the amino acid sequence of SEQ ID NO: 2 and having FUT6 activity.
[0050]
Method for producing the fusion protein of the present invention
In the present invention, the sugar-binding protein-FUT6 is such that the DNA encoding the sugar-binding protein and the DNA encoding FUT6 are eventually transcribed and translated as a fusion protein having the above-mentioned properties--in frame (in frame) —obtained by transforming E. coli with an expression vector containing the ligated chimeric DNA and culturing the resulting transformant in a suitable medium.
[0051]
In the present invention, the DNA encoding FUT6 may be derived from any biological species, but is preferably derived from a mammal, more preferably from a human. Furthermore, in the present invention, the DNA encoding FUT6 does not need to contain the entire coding amount II of the FUT6 gene as long as the transcription and translation product retains FUT6 activity, and may contain a part in the coding region. good. In particular, since FUT6 is a transmembrane protein present in the Golgi apparatus, the FUT6 coding sequence excluding the region encoding a highly hydrophobic transmembrane domain is considered in consideration of the production of sugar-binding protein-FUT6 as a soluble protein. The use of is also one of the preferred embodiments of the present invention.
[0052]
DNA encoding FUT6 can be prepared by any method known per se based on the sequence of a known FUT6 gene sequence (see, for example, Non-Patent Documents 8 to 11). For example, based on a known FUT6 gene sequence, a suitable oligonucleotide primer pair covering all or part of the coding region of FUT6 is synthesized, and total RNA or polyA extracted from cells or tissues expressing FUT6⁽⁺⁾It can be cloned by performing RT-PCR using RNA or chromosomal DNA as a template. At this time, in order to facilitate cloning into the vector behind it, an appropriate restriction enzyme recognition sequence can also be added to the end of the oligo primer to be used.
[0053]
Alternatively, an appropriate oligonucleotide probe is synthesized based on a known FUT6 gene sequence, and a cDNA library prepared from cells or tissues expressing FUT6 by a conventional method is screened by plaque (or colony) hybridization. Can also be cloned.
[0054]
In addition, DNA encoding FUT6 is prepared by preparing a conventional antibody from all or part of partially or completely purified FUT6 as an antigen, and using this to prepare a FUT6-expressing cell or tissue by a conventional method. The library can also be cloned by antibody screening.
[0055]
Alternatively, the DNA is synthesized using a DNA / RNA automatic synthesizer based on the known FUT6 gene sequence so that the partial sequence of the sense strand and the partial sequence of the antisense strand partially overlap, and the PCR method By repeating the operation of obtaining a longer partial sequence as a double-stranded DNA, a desired DNA sequence can be obtained.
[0056]
In a preferred embodiment of the present invention, it contains all or part of DNA encoding human-derived α1,3-fucosyltransferase VI (hereinafter sometimes abbreviated as hFUT6). For example, in the amino acid sequence shown in SEQ ID NO: 2, at least the base sequence encoding the amino acid sequence shown in amino acid numbers 41 to 359, preferably in the base sequence shown in SEQ ID NO: 1, is shown in at least base numbers 121 to 1077. DNA containing a base sequence. Alternatively, the DNA encoding hFUT6 has a nucleotide sequence encoding an amino acid sequence obtained by deleting the amino acid sequence corresponding to all or part of the transmembrane site of the protein, preferably all or part of the transmembrane site of the protein. DNA containing a base sequence from which the base sequence encoding the corresponding amino acid sequence has been deleted. The transmembrane domain of hFUT6 is presumed to be a part represented by amino acid numbers 15 to 34 in the amino acid sequence represented by SEQ ID NO: 2 from the results of hydrophobicity plot analysis. Therefore, the DNA encoding the domain is preferably a base sequence represented by base numbers 43 to 102 in the base sequence represented by SEQ ID NO: 1.
[0057]
Of course, the DNA encoding hFUT6 may be a DNA containing the base sequence encoding the entire amino acid sequence shown in SEQ ID NO: 2, preferably a DNA containing the entire base sequence shown in SEQ ID NO: 1.
[0058]
In the present invention, the DNA encoding a sugar binding protein is exemplified by the DNA encoding the above-mentioned various proteins, preferably a DNA encoding a maltose binding protein, a cellulose binding protein or a chitin binding protein, more preferably DNA encoding maltose binding protein.
[0059]
The DNA may be derived from any biological species, but is preferably derived from prokaryotes such as bacteria, and more preferably derived from E. coli.
[0060]
In addition, as long as the DNA encoding MBP encodes a translation product having specific affinity for a oligosaccharide having a maltose maltose residue (for example, maltotriose) or a polysaccharide (for example, amylose). However, it is not always necessary to include the entire code area, and a part of the code area may be included.
[0061]
MBP derived from E. coli is a secreted protein present in the periplasm, and the initial translation product contains a signal peptide at its N-terminus. The DNA encoding MBP of the present invention may or may not contain a base sequence (signal sequence) encoding a signal peptide that can function in host E. coli. Since Escherichia coli is a Gram-negative bacterium having an outer membrane on the outside of the cell wall, it is unlikely that the expressed MBP-FUT6 is secreted into the medium even if the DNA encoding MBP contains a signal sequence. . However, if MBP-FUT6 accumulates in the periplasmic space, the fusion protein can be recovered by spheroplasting without completely disrupting the cells, so that subsequent purification can be performed more easily. it can. However, in this case, it is particularly preferable to delete the transmembrane domain of FUT6. If the domain exists, MBP-FUT6 may stay in the inner membrane without reaching the periplasmic space, and the recovery of the fusion protein becomes complicated.
[0062]
It is particularly preferable that the DNA encoding MBP of the present invention includes a base sequence encoding an amino acid sequence that is recognized and cleaved by a protease having high sequence specificity on the C-terminal side of the protein. Such proteases include factor Xa, enterokinase, genenase I, thrombin, renin, etc. Preferably, the protease is a factor Xa recognition sequence, ie, Ile-Glu-Gly-Arg (SEQ ID NO: 3) or Ile-Asp-Gly-Arg (SEQ ID NO: 4), enterokinase recognition sequence, ie Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 5), genenase I recognition sequence, ie His-Tyr (sequence) No. 6) or Tyr-His (SEQ ID NO: 7), more preferably a factor Xa recognition sequence or enterokinase recognition sequence. By introducing such a protease recognition sequence on the C-terminal side of the sugar binding protein, for example, the MBP moiety can be easily removed by allowing the protease to act after purification of MBP-FUT6.
[0063]
In a more preferred embodiment, the DNA encoding a sugar binding protein includes a spacer sequence of about 5 to 15 amino acid residues between the sugar binding protein coding region and the protease recognition site. The presence of the spacer sequence reduces the possibility of intramolecular interaction of the fusion protein because the sugar binding protein moves away from FUT6, and in the case of the MBP-ST3GalI fusion protein, the specific affinity of the MBP moiety for maltose and amylose is increased. To do.
[0064]
The DNA encoding MBP can be cloned using a known MBP gene sequence [for example, based on the E. coli-derived MBP (Non-patent Document 12) using a per se known method similar to that exemplified for DNA encoding FUT6. In addition, the above protease recognition sequence and spacer sequence can be introduced into the C-terminal side of MBP by a conventional method. In the case of the present invention, the spacer sequence of SEQ ID NO: 8 is included.
[0065]
Further, DNA encoding E. coli-derived MBP is, for example, pMAL p2 (including signal sequence), pMAL p2X (including signal sequence), pMAL p2E (including signal sequence) or pMAL p2G (signal) manufactured by New England Biolabs. PMAL c2 (deleting the signal sequence) or pMAL c2X (deleting the signal sequence) or pMAL c2E (deleting the signal sequence) or pMAL c2G (deleting the signal sequence) From commercially available vectors.
[0066]
In order to facilitate the construction of a chimeric DNA encoding a sugar binding protein-FUT6 fusion protein, an appropriate restriction enzyme recognition site can be added to the end of the DNA encoding the sugar binding protein by a conventional method. When cloning a DNA encoding FUT6 into a vector, if a restriction enzyme recognition site is added to the N-terminal side of FUT6, the same restriction enzyme recognition site (or the same sticky end) is also added to the DNA encoding the sugar-binding protein. Another restriction enzyme recognition site to be generated may be introduced.
[0067]
The expression vector of the present invention is any expression vector in which the chimeric DNA encoding the sugar-binding protein-FUT6 is placed under the control of a promoter that can function in E. coli. The promoter region includes a -35 region and a -10 region, which are consensus sequences that determine the binding position of RNA polymerase. As a system for expressing a desired recombinant protein in large quantities, it is more desirable to use a promoter region of an inducible enzyme. Examples of such a promoter region include trp promoter, lac promoter, recA promoter, lpp promoter, tac promoter and the like. These promoter regions further include an operator to which the repressor protein binds. When an inducer (for example, lactose or IPTG for the lac promoter) is added, binding of the repressor protein to the operator is suppressed, and a large amount of the gene placed under the control of the promoter is expressed. The expression vector also contains a consensus Shine-Dalgarno (SD) sequence upstream of the translation initiation codon. Further, the expression vector contains a transcription termination signal, that is, a terminator region, downstream of the chimeric DNA encoding sugar binding protein-FUT6. As the terminator region, a commonly used natural or synthetic terminator can be used. In addition to the promoter region and terminator region described above, the expression vector of the present invention needs to contain a replication origin capable of autonomous replication in host E. coli. Examples of such a replication origin include ColE1 ori, M13 ori, and the like.
[0068]
The expression vector of the present invention preferably further contains a selection marker gene for selecting a transformant. As the selectable marker gene, resistance genes for various drugs such as tetracycline, ampicillin, kanamycin and the like can be used. When the host Escherichia coli is an auxotrophic mutant, a wild-type gene that complements the auxotrophy can be used as a selectable marker gene.
[0069]
The transformant of the present invention can be prepared by transforming host E. coli with the expression vector of the present invention. The host strain is not particularly limited, and examples thereof include commercially available XL1-Blue strain, BL-21 strain, JM107 strain, TB1 strain, JM109 strain, C600 strain, DH5α strain, HB101 strain and the like.
[0070]
Introduction of an expression vector into a host cell can be performed using a conventionally known method. Examples include the method of Cohen et al. (Calcium chloride method) (see non-patent document 13), the protoplast method (see non-patent document 14), the competent method (see non-patent document 15), the electroporation method, and the like.
[0071]
Sugar-binding protein-FUT6 is obtained by culturing a transformant expressing an expression vector containing the chimeric DNA encoding the above-mentioned sugar-binding protein-FUT6 in an appropriate medium. It can be obtained by collecting.
[0072]
The medium used contains a carbohydrate such as glucose, fructose, glycerol or starch as a carbon source. Further, it may contain an inorganic or organic nitrogen source (for example, ammonium sulfate, ammonium chloride, casein hydrolyzate, yeast extract, polypeptone, bactotryptone, beef extract, etc.). These carbon sources and nitrogen sources do not need to be used in pure form, and those having low purity are advantageous because they contain abundant amounts of growth factors and inorganic nutrients. Further, if desired, a variety of nutrient sources [eg, inorganic salts (eg, sodium or potassium diphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (eg, vitamin B1) Antibiotics (eg, ampicillin, kanamycin, etc.) may be added to the medium.
[0073]
The transformant is usually cultured at pH 5.5 to 8.5, preferably pH 6 to 8, usually 18 to 40 ° C., preferably 20 to 35 ° C., for 1 to 150 hours. It can be appropriately changed depending on the culture scale.
[0074]
When culturing in a large tank, in order to avoid growth delay in the production process of the target protein, after inoculating the microbial cells in a small amount of medium and pre-culturing for 1 to 24 hours, It is preferable to inoculate in a large tank.
[0075]
When the expression of sugar-binding protein-FUT6 is controlled by the promoter system of the inducible protein gene, the inducer may be added from the start of induction, but is more preferably added in the early logarithmic growth phase. Bacterial growth can be monitored by measuring the absorbance of the culture at 660 nm. For example, when lac promoter or tac promoter is used, isopropylthio-β-D-galactoside (hereinafter sometimes referred to as IPTG) may be used as an inducer when the absorbance at 660 nm becomes 0.4 to 0.6. For example, 0.1 to 1.0 mM. The timing and rate of addition of the inducer can be appropriately changed depending on the culture conditions, culture scale, and type of inducer.
[0076]
In the method of the present invention, the sugar-binding protein-FUT6 is purified by using an insoluble carrier in which a fraction containing the fusion protein is bound to a ligand containing various sugar residues that specifically bind to the sugar-binding protein. It can be achieved in one step by subjecting to affinity chromatography.
[0077]
For example, when the sugar-binding protein is MBP, when the DNA encoding MBP lacks the signal sequence, MBP-FUT6 is localized in the soluble fraction of the bacterial cells. The cells are collected by filtration or centrifugation, and the cells are disrupted by lysozyme treatment and surfactant treatment, ultrasonic treatment, osmotic shock, etc., and the resulting cell extract is used for affinity chromatography .
[0078]
On the other hand, when the DNA encoding MBP has a signal sequence, the expressed MBP-FUT6 is highly likely to accumulate in the secreted periplasmic space. Therefore, in that case, the bacterial cells are spheroplasted by lysozyme treatment or the like to release MBP-FUT6 into the solution, and after filtering or centrifuging to remove the bacterial cells, the resulting supernatant is used for affinity chromatography. .
[0079]
As an adsorbent for affinity chromatography, for example, in the case of MBP, amylose resin in which amylose is immobilized on agarose beads (for example, Amylose resin manufactured by New England Biolabs, etc.) can be used. Ligands for MBP and other insoluble matrix groups (eg, cellulose, dextran, synthetic polymers, etc.) may be used.
[0080]
Glucose-binding protein-FUT6 is prepared by adding the adsorbent to the fraction containing it and stirring it for an appropriate period of time, followed by filtration, washing the adsorbent, and an appropriate concentration of sugar-binding protein. An eluent containing a sugar that inhibits the binding of the adsorbent (for example, maltose in the case of MBP) is added, stirred for an appropriate time, and then filtered to purify the filtrate. Alternatively, the adsorbent is packed in a column, a fraction containing sugar-binding protein-FUT6 is applied, washed with an appropriate buffer, and then the eluent having an appropriate concentration is applied and adsorbed on the column. It can also be obtained by eluting FUT6.
[0081]
The MBP-FUT6 of the present invention thus obtained has FUT6 activity and can be used as an enzyme as it is.
[0082]
FUT6 Preparation of
As described above, in a preferred embodiment of the present invention, since the sugar-binding protein-FUT6 contains an amino acid sequence that is recognized and cleaved by a sequence-specific protease at its fusion site, the sugar-binding protein purified as described above. -When the protease is allowed to act on FUT6, the sugar-binding protein portion is removed to produce the desired FUT6. Preferably the protease is Factor Xa. In the present invention, pMAL p2, pMAL p2X, pMAL c2 or pMAL c2X (manufactured by New England Biolabs) containing the factor Xa recognition sequence of SEQ ID NO: 3 can be used. The reaction temperature, the pH of the solution, the reaction time, etc. when the protease is allowed to act on the sugar-binding protein-FUT6 can be appropriately selected depending on the type of protease used. For example, in the case of factor Xa, By reacting at 4 to 40 ° C. for 1 to 25 hours in a buffer solution, the sugar-binding protein and FUT6 can be cleaved. After completion of the reaction, the above adsorbent is added to the reaction solution and stirred for an appropriate time, so that only the released sugar-binding protein is adsorbed to the adsorbent, so that only FUT6 is purified by filtering the adsorbent. Can do. Alternatively, FUT6 can also be purified by applying the reaction solution to a column packed with the adsorbent and collecting the flow-through fraction.
[0083]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. However, these are merely examples, and the scope of the present invention is not limited to the examples.
[0084]
Example 1 PCR by hFUT6 cDNA Fragment acquisition and expression vector construction
(1) PCR primer design
Based on the base sequence of human α1,3-fucosyltransferase VI (FUT6) cDNA described in SEQ ID NO: 1, oligonucleotides shown in SEQ ID NOs: 10 and 11 were synthesized. The former is a base sequence represented by base numbers 121 to 134 in the base sequence represented by SEQ ID NO: 1, and the latter is a base sequence represented by base numbers 1065 to 1080 in the base sequence represented by SEQ ID NO: 1.
[0085]
(2) Reverse transcription reaction from RNA derived from human squamous cell A431 strain
Reverse transcription reaction by RT PCR high (RT PCR high: manufactured by Toyobo Co., Ltd.) using random primers with 5 μg of total RNA extracted from human squamous cell A431 strain by RNA extraction kit (RNeasy: product of QIAGEN) went. The obtained reaction solution was designated as A431 1ss cDNA solution.
[0086]
(3) PCR reaction
PCR reaction was performed using the MA A431 1ss cDNA and the synthetic oligonucleotide under the following conditions.
[A431 1ss cDNA 2.5 μl, 2 mM dNTPs (manufactured by Toyobo) 2.5 μl, X10KOD Buffer No. 1 (Toyobo Co., Ltd.) 2.5 μl, 25 mM MgCl₂(Manufactured by Toyobo Co., Ltd.) 1.0 μl, 10 pmoles / μl of the oligonucleotide described in SEQ ID NO: 10 1.0 μl, 10 pmoles / μl of the oligonucleotide described in SEQ ID NO: 11, 1.0 U / μl KOD DNA Polymerase ( Toyobo Co., Ltd.) 0.5 μl, reaction volume 25 μl, 98 ° C. (30 seconds) -56 ° C. (30 seconds) -74 ° C. (30 seconds) after 4 cycles, 98 ° C. (30 seconds) -62 ° C. (30 seconds) ) -74 ° C. (30 seconds) 31 cycles].
After the PCR reaction, the reaction solution was subjected to agarose gel electrophoresis, and a DNA fragment as a PCR product was confirmed.
[0087]
(4) Confirmation of base sequence and construction of expression vector
The DNA fragment is cleaved with restriction enzymes EcoRI and BamHI, and then ligated using pMAL c2 or pMAL p2 (manufactured by New England Biolabs) and Ligation High (manufactured by Toyobo Co., Ltd.) that have been cleaved with the restriction enzymes in advance. E. coli strain JM109 was transformed and selected on LB plate medium containing 50 μg / ml ampicillin.
As a result of preparing a plasmid holding the DNA fragment from E. coli and determining the nucleotide sequence of the DNA fragment according to a conventional method, it may match the base sequence represented by base numbers 121 to 1080 in the base sequence described in SEQ ID NO: 1. This confirmed that the desired hFUT6 cDNA fragment was obtained. Moreover, the vector constructed by ligating the PCR fragment to pMAL c2 was named pMF6-C101, and the vector constructed by ligating the PCR fragment to pMAL p2 was named pMF6-P101, and the construction of the expression vector was completed at the same time.
[0088]
Example 2 FUT6 Confirmation of expression
(1) Preparation of MBP-hFUT6 fusion protein using recombinant E. coli
E. coli JM109 strain transformed with the expression vector pMF6-C101 or pMF6-P101 obtained in Example 1 was placed in a test tube containing 5 ml of LB medium containing 50 μg / ml ampicillin and 0.2% glucose. Inoculated and cultured with shaking overnight at 37 ° C. 500 μl of the obtained culture solution was inoculated into a 500 ml culture flask containing 50 ml of the same medium. After carrying out the shaking culture at 37 ° C., when OD660 = 3.0 to 4.0 is reached, the culture temperature is lowered to 25 ° C., and the culture is continued at 25 ° C. for 30 minutes. IPTG at a concentration of 0.3 mM was added, and the culture was further continued at 25 ° C. for 16 hours. After culturing, the cells were collected by centrifugation, suspended in 20 ml of buffer solution 1 (20 mM HEPES pH 7.5 0.2 M NaCl 1 mM EDTA 1 mM Benzamidine HCl 10 mM 2 mercaptoethanol), and the cells were disrupted with an ultrasonic crusher. Thereafter, the supernatant obtained by centrifugation was used as a crude enzyme solution. The MBP-hFUT6 fusion protein was adsorbed by passing the crude enzyme solution through an amylose resin (New England Biolabs) column (5 ml) that had been equilibrated with buffer 1 in advance. After washing the column with buffer 1, MBP-hFUT6 fusion protein adsorbed with buffer 1 containing 10 mM maltose was eluted. The eluted fractions were collected and the maltose was removed with a desalting column PD-10 (Pharmacia) to obtain an MBP-hFUT6 fusion protein solution.
[0089]
(2) Preparation of FUT6 substrate
LNnT sugar chain (manufactured by Glyko: Galβ1-4GlcNAcβ1-3Galβ1-4Glc-OH) 2.0 mg was purified by fluorescence labeling and gel filtration with 2 aminopyridine according to a conventional method (LNnT-PA (Galβ1 -4GlcNAcβ1-3Galβ1-4Glc-PA), 616 nmole was obtained.
[0090]
(3) FUT6 activity measurement
0.5 μM MES buffer pH 8.0 4 μl, 200 mM MgCl 2 1 μl, 1 mM LNnT-PA (obtained in (2) above) 1 μl, 5 mM GDP-Fuc (manufactured by Wako Pure Chemical Industries) 1 μl, above After reacting 20 μl of the reaction solution consisting of 5 μl of the MBP-hFUT6 fusion protein solution obtained in (1) at 37 ° C. for 15 minutes, the reaction was stopped by boiling for 1 minute. 80 μl of MilliQ water was added to the reaction solution, mixed, and then 20 μl of the supernatant obtained by centrifugation was analyzed by HPLC to measure the amount of reaction product GM1a-PA. As a result, Escherichia coli JM109 transformed with pMF6-C101 showed high FUT6 activity (FIG. 2). E. coli JM109 transformed with pMF6-P101 also showed FUT6 activity although it was weak (FIG. 2). When FUT6 activity is defined as the amount of enzyme that transfers 1 μmole of fucose per minute to LNnT-PA according to the above reaction conditions, the productivity of FUT6 of E. coli JM109 transformed with pMF6-C101 is 170 mU / L- Calculated as Broth.
[0091]
Example 3 MBP − hFUT6 Examination of various properties of fusion proteins
Various properties of the MBP-hFUT6 fusion protein derived from E. coli JM109 transformed with pMF6-C101 obtained in Example 2 were examined.
[0092]
(1) Examination of optimum reaction pH
Under the same conditions, FUT6 activity was measured at pH 4.0 to pH 8.0. As a result, FUT6 activity was detected at pH 5.5 to 8.0, and the optimum reaction pH was around pH 8 to 8.5. It was confirmed (FIG. 3).
[0093]
(2) Examination of the effects of manganese
As a result of investigating the influence on the activity of manganese under the same conditions, it was found that in the absence of manganese, the activity was reduced to about 1/7 in the presence of 20 mM manganese (FIG. 4). From these results, it was considered that manganese is necessary for the activity of FUT6.
[0094]
[Table 1]

[0095]
【The invention's effect】
In the method of the present invention, a fusion protein having α1,3-fucosyltransferase VI (FUT6) activity can be easily and efficiently produced in large quantities as a soluble protein. In addition, α1,3-fucosyltransferase VI (FUT6) itself can be easily obtained from the fusion protein.
[0096]
The α1,3-fucosyltransferase VI (FUT6) obtained by the present invention is not only useful as a reagent for research on sugar chain engineering, but also by utilizing the obtained α1,3-fucosyltransferase VI (FUT6). , Various Lewis structure sugar chains useful for research use or pharmaceutical use, or synthesis of sugar chains such as various chemical substances including Lewis structure sugar chain analogs, fucose acid-containing sugar chains, fucose acid-containing sugar chains, etc. . Furthermore, it can be used for sugar chain modification of various useful proteins and various sugar chain-containing chemical substances. In addition, α1,3-fucosyltransferase VI (FUT6) antibody useful for research use or pharmaceutical use can be easily obtained.
[0097]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows a reaction catalyzed by FUT6. Specifically, the sugar nucleotide GDP-fucose (GDP-Fuc) is used as a fucose donor and the sugar acceptor R is used.₁-Galβ1-4GlcNAc-R₂Transfer fucose to α1-3 bond to R₁-Galβ1-4 (Fucα1-3) GlcNAc-R₂It is the figure which showed the reaction which produces | generates.
FIG. 2 shows the results of FUT6 activity measurement of MBP-FUT6 fusion protein expressed in E. coli.
FIG. 3 is a graph showing the effect of pH of a reaction solution on FUT6 activity of MBP-FUT6 expressed in E. coli.
FIG. 4 shows the effect of manganese concentration on FUT6 activity of MBP-FUT6 expressed in E. coli.

Claims (21)

A recombinant fusion protein of a sugar-binding protein and α1,3-fucosyltransferase VI. The recombinant fusion protein according to any one of claims 1 to 4, wherein α1,3-fucosyltransferase VI is derived from human. The recombinant fusion protein according to claim 1 or 2, wherein α1,3-fucosyltransferase VI is the protein described in (a) or (b).
(A) a protein comprising the amino acid sequence set forth in SEQ ID NO: 2 (b) (a) comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and α1,3-fucosyltransferase VI activity Protein with The protein according to claim 3, wherein α1,3-fucosyltransferase VI comprises at least the amino acid sequence represented by amino acid numbers 41 to 359 in the amino acid sequence represented by SEQ ID NO: 2. The protein according to any one of claims 1 to 4, wherein α1,3-fucosyltransferase VI comprises an amino acid sequence in which amino acids corresponding to all or part of the transmembrane region of the protein are deleted. The recombinant fusion protein according to any one of claims 1 to 5, comprising a protease recognition sequence between the sugar-binding protein and α1,3-fucosyltransferase VI. The recombinant fusion protein according to any one of claims 1 to 8, wherein the sugar-binding protein is a maltose-binding protein. The recombinant fusion protein according to claim 6, wherein the protease is a protease selected from the group consisting of blood coagulation factor Xa, enterokinase, and genenase I. DNA encoding the recombinant fusion protein according to any one of claims 1 to 8. An expression vector comprising the DNA according to claim 9. A transformant transformed with the expression vector according to claim 10. The method for producing a recombinant fusion protein according to any one of claims 1 to 8, comprising the following steps (1) to (3).
(1) Expression in which a DNA encoding a sugar-binding protein and a DNA encoding α1,3-fucosyltransferase VI are linked so as to be expressed as a fusion protein of both proteins under the control of a promoter capable of functioning in E. coli Using a vector, E. coli is transformed,
(2) culturing the obtained transformant to produce a fusion protein of a sugar-binding protein and α1,3-fucosyltransferase VI;
(3) Separating the fusion protein from the obtained culture The method according to claim 12, wherein α1,3-fucosyltransferase VI is derived from a human. The method according to claim 13, wherein the DNA encoding α1,3-fucosyltransferase VI comprises at least a base sequence encoding an amino acid sequence represented by amino acid numbers 41 to 359 in the amino acid sequence represented by SEQ ID NO: 2. The DNA encoding α1,3-fucosyltransferase VI comprises a base sequence encoding an amino acid sequence from which amino acids corresponding to all or part of the transmembrane site of the protein have been deleted. the method of. Claims wherein the DNA encoding α1,3-fucosyltransferase VI comprises a base sequence obtained by deleting the base sequence encoding amino acids corresponding to all or part of the transmembrane site of the protein from the base sequence shown in SEQ ID NO: 1. Item 16. The method according to any one of Items 12 to 15. The method according to any one of claims 12 to 16, wherein the sugar binding protein is a maltose binding protein. 18. The DNA encoding a maltose binding protein is derived from a vector selected from the group consisting of pMAL p2, pMAL p2X, pMAL p2E, pMAL p2G, pMAL c2, pMAL c2X, pMAL c2E, pMAL c2G. the method of. Production of α1,3-fucosyltransferase VI, comprising the step of removing the sugar-binding protein portion from the fusion protein obtained by the method according to any one of claims 12 to 18 and collecting α1,3-fucosyltransferase VI. Method. The DNA encoding a sugar binding protein contains a base sequence encoding a protease recognition sequence on the C-terminal side of the protein, and the sugar binding protein portion is removed from the fusion protein by the action of the protease. The method described. 21. The method according to claim 20, wherein the protease is a protease selected from the group consisting of blood coagulation factor Xa, enterokinase, and genenase I.

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