α1,2 fucosyltransferase
AssigneeThe Governors of the University of Alberta
InventorsDiane Taylor, Ge Wang, Monica Palcic
Life of this patent
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Abstract
A bacterial α1,2-fucosyltransferase gene and deduced amino acid sequence is provided. The gene is useful for preparing α1,2-fucosyltransferase polypeptide, and active fragment thereof, which can be used in the production of oligosaccharides such as Lewis X, Lewis Y, Lewis B and H type 1, which are structurally similar to certain tumor-associated carbohydrate antigens found in mammals. These product glycoconjugates also have research and diagnostic utility in the development of assays to detect mammalian tumors.
Claims24
A method for producing an alpha-1,2-fucosylated oligosaccharide blood type antigen, the method comprising contacting a Helicobacter alpha-1,2-fucosyltransferase polypeptide or bioactive fragment thereof with a type 1 or type 2 oligosaccharide substrate for sufficient time and under conditions such that an alpha-1,2-fucosylated oligosaccharide blood type antigen is produced, wherein the alpha-1,2-fucosyltransferase polypeptide is encoded by a polynucleotide identical to a polynucleotide that is amplified using Helicobacter genomic DNA as a template by PCR using a first primer comprising 5′-GAACACTCACACGCGTCTT-3′ (SEQ ID NO:3) and a second primer comprising 5′-TAGAATTAGACGCTCGCTAT-3′ (SEQ ID NO:4), wherein the bioactive fragment has alpha-1,2-fucosyltransferase activity.
A method for producing an alpha-1,2-fucosylated oligosaccharide blood type antigen, the method comprising contacting an alpha 1,2-fucosyltransferase that has a sequence as set forth in SEQ ID NO:2 with a type 1 or type 2 oligosaccharide substrate for sufficient time and under conditions such that an alpha-1,2-fucosylated oligosaccharide blood type antigen is produced.
A system for producing an alpha-1,2-fucosylated oligosaccharide blood type antigen, the system comprising:
- (a) a host cell transfected or transformed with a polynucleotide encoding an alpha 1,2-fucosyltransferase polypeptide or bioactive fragment thereof, wherein the alpha-1,2-fucosyltransferase polypeptide is encoded by a polynucleotide identical to a polynucleotide that is amplified using Helicobacter genomic DNA as a template by PCR using a first primer comprising 5′-GAACACTCACACGCGTCTT-3′ (SEQ ID NO:3) and a second primer comprising 5′-TAGAATTAGACGCTCGCTAT-3′ (SEQ ID NO:4);
- wherein the bioactive fragment has alpha-1,2-fucosyltransferase activity,
- (b) expressing a polypeptide from the polynucleotide;
- (c) contacting the host cell with a type 1 or type 2 oligosaccharide substrate under conditions and for sufficient period of time such that the substrate is acted upon by the alpha 1,2-fucosyltranferase or bioactive fragment;
- and
- (d) recovering the alpha-1,2-fucosylated oligosaccharide blood type antigen.
A method for producing an H type blood antigen, the method comprising contacting an alpha-1,2-fucosyltransferase polypeptide or bioactive fragment thereof with a type 1 or type 2 oligosaccharide substrate for sufficient time and under conditions such that an H type blood antigen is produced, wherein the alpha-1,2-fucosyltransferase polypeptide is encoded by a polynucleotide identical to a polynucleotide that is amplified using Helicobacter genomic DNA as a template by PCR using a first primer comprising 5′-GAACACTCACACGCGTCTT-3′ (SEQ ID NO:3) and a second primer comprising 5′-TAGAATTAGACGCTCGCTAT-3′ (SEQ ID NO:4), wherein the bioactive fragment has alpha-1,2-fucosyltransferase activity.
A method for producing an H type blood type antigen, the method comprising contacting an alpha 1,2-fucosyltransferase that has a sequence as set forth in SEQ ID NO:2 with a type 1 or type 2 oligosaccharide substrate for sufficient time and under conditions such that an H type blood type antigen is produced.
A system for producing H type blood antigen, the system comprising:
- (a) a host cell transfected or transformed with a polynucleotide encoding an alpha 1,2-fucosyltransferase or bioactive fragment thereof, wherein the alpha-1,2-fucosyltransferase polypeptide is encoded by a polynucleotide identical to a polynucleotide that is amplified using Helicobacter genomic DNA as a template by PCR using a first primer comprising 5′-GAACACTCACACGCGTCTT -3′ (SEQ ID NO:3) and a second primer comprising 5′-TAGAATTAGACGCTCGCTAT-3′ (SEQ ID NO:4);
- wherein the bioactive fragment has alpha-1,2-fucosyltransferase activity,
- (b) expressing the polynucleotide;
- (c) contacting the host cell with a type 1 or type 2 oligosaccharide substrate under conditions and for sufficient period of time such that the substrate is acted upon by the alpha 1,2-fucosyltransferase or bioactive fragment;
- and
- (d) recovering the H type blood antigen.
Dependencies are inferred from the claim text.