Alpha1,3-fucosyltransferase
AssigneeGovernors of the University of Alberta, a Canada corporation
InventorsDiane E. Taylor, Zhongming Ge
Life of this patent
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Abstract
A bacterial α1,3-fucosyltransferase gene and deduced amino acid sequence is provided. The gene is useful for preparing α1,3-fucosyltransferase polypeptide, and active fragment thereof, which can be used in the production of oligosaccharides such as Lewis X, Lewis Y, and siayl Lewis X, which are structurally similar to certain tumor-associated carbohy-drate antigens found in mammals. These product glycoconjugates also have research and diagnostic utility in the development of assays to detect mammalian tumors. In addition the polypeptide of the invention can be used to develop diagnostic and research assays to determine the presence of H. pylon in human specimens.
Claims54
A substantially purified transmembrane segment-free α1,3-fucosyltransferase polypeptide.
A substantially purified transmembrane segment-free α1,3-fucosyltransferase comprising a polypeptide having at least one repeat of the sequence comprising X1X2LRX3X4Y, wherein X1 is D or N;
- X2 is D or N;
- X3 is I, V or A;
- X4 is N or D.
A polynucleotide selected from the group consisting of:
- a) SEQ ID NO:
- 4;
- b) SEQ ID NO:
- 4, wherein T is U;
- c) nucleic acid sequences complementary to a) or b);
- and
- d) fragments of a), b), or c) that are at least 15 nucleotide bases in length and that hybridize to DNA which encodes any one of the polypeptide set forth in SEQ ID NO:
- 1, SEQ ID NO:2and SEQ ID NO:3.
A method for detecting transmembrane segment-free α1,3-fucosyltransferase polynucleotide in a sample, comprising:
- a) contacting a sample suspected of containing α1,3-fucosyltransferase polynucleotide with a nucleic acid probe that hybridizes to α1,3-fucosyltransferase polynucleotide;
- b) detecting hybridization of the probe with α1,3-fucosyltransferase polynucleotide, wherein the detection of hybridization is indicative of α1,3-fucosyltransferase polynucleotide in the sample.
A method for detecting transmembrane segment-free α1,3-fucosyltransferase polynucleotide in a sample, comprising amplifying the α1,3-fucosyltransferase polynucleotide.
A recombinant method for producing transmembrane segment-free α1,3-fucosyltransferase polypeptide, comprising:
- a) growing a recombinant host cell containing a polynucleotide encoding the α1,3-fucosyltransferase polypeptide under conditions which allow expression and secretion of α1,3-fucosyltransferase polypeptide;
- and
- b) isolating the polypeptide.
A method of producing a transmembrane segment-free α1,3-fucosyltransferase fusion protein comprising:
- a) growing a host cell containing a polynucleotide encoding α1,3-fucosyltransferase polypeptide operably linked to a polynucleotide encoding a polypeptide or peptide of interest under conditions which allow expression and secretion of the fusion protein;
- and
- b) isolating the fusion protein.
A gene expression system for producing transmembrane segment-free α1,3-fucosyltransferase comprising a host cell modified with a polynucleotide encoding α1,3-fucosyltransferase polypeptide or an enzymatically active portion thereof.
A method for producing transmembrane segment-free α1,3-fucosyltransferase polypeptide, comprising the steps of:
- (a) culturing a gene expression system comprising a host cell modified with a polynucleotide encoding the α1,3-fucosyltransferase polypeptide or an enzymatically active portion thereof;
- and
- (b) harvesting the α1,3-fucosyltransferase.
A method for producing fucosylated oligosaccharides, the method comprising contacting a transmembrane segment-free α1,3-fucosyltransferase polypeptide with a substrate for a suitable time and under suitable conditions to produce the oligosaccharides.
A method for producing fucosylated oligosaccharides, the method comprising the steps of:
- (a) culturing a gene expression system comprising a host cell modified with a polynucleotide encoding a transmembrane segment-free a 1,3 -fucosyltransferase polypeptide or an enzymatically active portion thereof;
- and
- (b) contacting the host cell with a substrate, under conditions and for sufficient time to produce the oligosaccharides.
Dependencies are inferred from the claim text.