Synthesis of oligosaccharides, glycolipids, and glycoproteins using bacterial glycosyltransferases
View Patent ↗This invention provides nucleic acid and amino acid sequences of fucosyltransferases from Helicobactor pylori . The invention also provides methods to use the fucosyltransferases to synthesize oligosaccharides, glycoproteins, and glycolipids.
1. A method for producing a fucosylated glycoprotein, the method comprising: contacting a recombinant fucosyltransferase protein with a mixture comprising a donor substrate comprising a GDP-fucose residue, and an acceptor substrate on a glycoprotein, wherein the acceptor substrate comprises an N-acetylglucosamine residue, under conditions where the fucosyltransferase catalyzes the transfer of the fucose residue from the donor substrate to the acceptor substrate on the glycoprotein, thereby producing a fucosylated glycoprotein, wherein the recombinant fucosyltransferase protein comprises a polypeptide having greater than 95% identity to the amino acid sequence of SEQ ID NO: 4.
2. The method of claim 1 , wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 4.
3. The method of claim 1 , wherein the polypeptide further comprises an amino acid tag.
4. The method of claim 1 , wherein the method further comprises a step of purifying the fucosylated glycoprotein.
5. The method of claim 1 , wherein an acceptor substrate on the glycoprotein comprises Galβ,3/4GlcNAc-OR, NeuAca2,3Galβ,3/4GlcNAc-OR, wherein R is an amino acid, a saccharide, an oligosaccharide, or a radical group having at least one carbon atom.
6. A method of making a fucosylated oligosaccharide, the method comprising: contacting the recombinant fucosyltransferase with a mixture comprising a donor substrate comprising a GDP-fucose residue, and an acceptor substrate comprising a sugar or oligosaccharide, wherein the acceptor substrate comprises an N-acetylglucosamine residue, under conditions where the fusion protein catalyzes the transfer of a fucose residue from the donor substrate to the acceptor substrate, thereby producing a fucosylated oligosaccharide, wherein the recombinant fucosyltransferase protein comprises a polypeptide having greater than 95% identity to the amino acid sequence of SEQ ID NO:4.
7. The method of claim 6 , wherein the method further comprises a step of purifying the fucosylated oligosaccharide.
8. The method of claim 6 , wherein the fucosyltransferase comprises an amino acid tag.
9. The method of claim 6 , wherein the acceptor substrate is Lacto-N-neo-Tetraose (LNnT).
10. The method of claim 9 , wherein the fucosylated oligosaccharide is Lacto-N-Fucopentaose III (LNFP III).
11. The method of claim 6 , wherein the mixture further comprises lactose, a β-1,3-N-acetylglucosaminyltransferase, and a β1,4-galactosyltransferase.
12. The method of claim 11 , wherein the β-1,3-N-acetyiglucosaminyltransferase is a bacterial enzyme.
13. The method of claim 12 , wherein the β-1,3-N-acetyiglucosaminyltransferase is from Neisseria gonococcus.
14. The method of claim 11 , wherein the β-1,4-galactosyltransferase is a bacterial enzyme.
15. The method of claim 14 , wherein the β-1,4-galactosyltransferase is from Neisseria gonococcus.
16. The method of claim 11 , wherein the fucosylated oligosaccharide is Lacto-N-Fucopentaose III (LNFP III).
17. A method for producing a fucosylated glycolipid, the method comprising: contacting the recombinant fucosyltransferase protein of with a mixture comprising a donor substrate comprising a GDP-fucose residue, and an acceptor substrate on a glycolipid, wherein the acceptor substrate comprises an N-acetylglucosamine residue, under conditions where the fucosyltransferase catalyzes the transfer of the fucose residue from the donor substrate to the acceptor substrate on the glycolipid, thereby producing a fucosylated glycolipid, wherein the recombinant fucosyltransferase protein comprises a polypeptide having greater than 95% identity to the amino acid sequence of SEQ ID NO:4.