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US 7,094,530 B1Granted
α-1,3-fucosyltransferase
AssigneeKyowa Hakko Kogyo Co., Ltd.
InventorsKatsutoshi Sasaki, Kazumi Miura, Nobuo Hanai, Tatsunari Nishi
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AssigneeKyowa Hakko Kogyo Co., Ltd.
InventorsKatsutoshi Sasaki, Kazumi Miura, Nobuo Hanai, Tatsunari Nishi
Estimating the term…
The invention provides a novel species of α-1,3-fucosyltransferase expressed by a gene cloned from animal cells, a cDNA coding for the α-1,3-fucosyltransferase, a method of detecting, or inhibiting the production of, the α-1,3-fucosyltransferase which involves the use of the cDNA, a recombinant vector containing the DNA as an insert, a cell harboring the recombinant vector, and a method of producing same. The α-1,3-fucosyltransferase of the invention is useful in the production of carbohydrate chains having useful physiological activity, for example sialyl Lewis x, and modifications thereof.
An isolated species of α-1,3-fucosyltransferase which comprises the amino acid sequence defined in SEQ ID NO:2.
A cDNA coding for the α-1,3-fucosyltransferase comprising the amino acid sequence of SEQ ID NO:2.
A cDNA which comprises the base sequence defined in SEQ ID NO:1.
A recombinant vector comprising, as an insert, a cDNA sequence coding for the α-1,3-fucosyltransferase comprising the amino acid sequence of SEQ ID NO:2.
A recombinant vector comprising, as an insert, a DNA sequence comprising the base sequence defined from position 123 td position 1148 of SEQ ID NO:1.
The plasmid pUC119-TH21R.
Escherichia coli JM105/pUC119-TH21 R (FERM BP4193).
An isolated nucleic acid sequence encoding α-1,3-fucosyltransferase comprising bases 123 through 1148 of SEQ ID NO:1.
An Isolated species of α-1,3-flucosyltransferase that cannot synthesize Lewis X carbohydrate chain by transferring fucose to a carbohydrate chain having a Galβl-4GlcNAc structure at a nonreducing terminus thereof comprising the amino acid sequence of SEQ ID NO:2, or modification thereof wherein one or more amino acids of residues 1 to 37 of said SEQ ID NO:2 sequence is substituted, deleted or added.
A method of introducing the sialyl Lewis x structure onto a carbohydrate chain of a glycoprotein or glycolipid comprising contacting α-1,3-fucosyltransferase having an amino acid sequence of SEQ ID NO:2 with said chain under conditions such that said introduction is effected.
A method of introducing the sialyl Lewis x structure onto a carbohydrate chain of a glycoprotein or glycolipid comprising culturing a cell harboring a recombinant vector comprising, as an insert, a DNA sequence coding for α-1,3-fucosyltransferase comprising SEQ ID NO:2, under conditions such that said DNA sequence is expressed and said α-1,3-fucosyltransferase is thereby produced, and such that said α-1,3-fucosyltransferase effects the introduction of said Lewis x structure into a carbohydrate chain of a glycoprotein or glycolipid present in said cell.
A method of detecting the presence of a sample DNA sequence encoding α-1,3-fucosyltransferase comprising SEQ ID NO:2 in a sample, comprising contacting the sample with a DNA coding for the α-1,3-fucosyltransferase comprising the amino acid sequence of SEQ ID NO:2 or with a DNA which comprises the base sequence defined by positions 123 to 1148 of SEQ ID NO:1, under conditions such that hybridization of said encoding DNA sequence or said DNA which comprises the base sequence defined by positions 123 to 1148 of SEQ ID NO:1 and said sample DNA sequence can occur whereby a complex is formed and detecting the presence of said complex.
Dependencies are inferred from the claim text.
From the US filing history. Foreign family members aren’t listed.
Glycosyltransferases: hexosyltransferases
Microorganisms or enzymes
Term ended 22 Aug 2023 (estimated)
USPTO file history isn’t available for this patent, so only indexed dates are shown.
The present invention relates to a novel species of α-1,3-fucosyltransferase, a cDNA coding for the fucosyltransferase, a recombinant vector containing the cDNA as an insert and a cell harboring the recombinant vector as well as method of producing same. The invention further relates to a method of producing carbohydrate chains using the fucosyltransferase and to a method of producing carbohydrate chains through production of the fucosyltransferase in transformed cells. Still further, the invention relates to a method of detecting the fucosyltransferase and a method of inhibiting the production of the fucosyltransferase, both using DNA coding for the α-1,3-fucosyltransferase of the invention. The α-1,3-fucosyltransferase of the invention is useful, in particular, in the production of carbohydrate chains having a useful physiological activity, for example sialyl Lewis x, and modifications thereof.
While proteins produced in prokaryotes, for example Escherichia coli, have no carbohydrate chain, proteins and lipids produced in eukaryotes, such as yeast, fungi, plant cells and animal cells, have a carbohydrate chain bound thereto in many instances.
Carbohydrate chains bound to proteins in animal cells include N-glycoside bond type carbohydrate chains (also called N-glycans) bound to an asparagine (Asn) residue in the protein and O-glycoside bond type carbohydrate chains (also called O-glycans) bound to a serine (Ser) or threonine (Thr) residue. It has recently been revealed that a certain kind of lipid containing a carbohydrate chain is covalently bound to a number of proteins and that those proteins are attached to the cell membrane through the lipid. This carbohydrate chain-containing lipid is called glycosyl phosphatidylinositol anchor.
FIG. 1 shows a construction scheme for the plasmid pAGEL106.
FIG. 2 shows a construction scheme for the plasmid pASLB3-3-1.
FIG. 3 shows a construction scheme for the plasmid pASLB3-3.
The symbols used in the figures respectively have the following meanings: dhfr: Dihydrofolate reductase gene hG-CSF: Human granulocyte colony stimulating factor gene bp: Base pairs kb: Kilobase pairs G418/Km: Transposon 5 (Tn 5)-derived G418/kanamycin resistance gene hyg: Hygromycin resistance gene Ap: pBR322-derived ampicillin resistance gene Tc: pBR322-derived tetracycline resistance gene P1: pBR322-derived P1 promoter Ptk: Herpes simplex virus (HSV) thymidine kinase (tk) gene promoter Sp. βG: Rabbit β globin gene splicing signal A. βG: Rabbit β globin gene poly(A) addition signal A.SE: Simian virus 40 (SV40) early gene poly(A) addition signal Atk: Herpes simplex virus (HSV) thymidine kinase (tk) gene poly(A) addition signal Pse: Simian virus 40 (SV40) early gene promoter Pmo: Moloney murine leukemia virus long terminal repeat (LTR) promoter HTLV-1: Human T cell leukemia virus type-1 (HTLV-1) gene EBNA-1: Epstein-Barr virus EBNA-1 gene oriP: Epstein-Barr virus replication origin ori: pUC119 replication origin Lac'Z: Part of Escherichia coli β galactosidase gene IG: M13 phage DNA intergenic region G-CSF der.: Human granulocyte colony stimulating factor derivative gene S: Gene portion coding for human granulocyte colony stimulating factor signal peptide A or ProA: Gene portion coding for binding region of Staphylococcus aureus protein A to IgG TH21: α-1,3-Fucosyltransferase gene obtained from THP-1 cells (full-length gene or active region gene)
Certain aspects of the invention are described in greater detail in the non-limiting Examples that follow.
1. Construction of Direct Expression Cloning Vectors pAMoERC3Sc and pAMoPRC3Sc