Photobacterium sp. alpha-2-6-sialyltransferase variants
View Patent ↗The present invention provides Photobacterium sp. JH-ISH-224 α2-6-sialyltransferase Psp26ST variants and expression cassettes, vectors, and host cells for expressing the Psp26ST variants. Methods of synthesizing sialylated products are also described.
1. An isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.
2. The polypeptide of claim 1 , comprising the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6.
3. The polypeptide of claim 1 , comprising the amino acid sequence set forth in SEQ ID NO: 2.
4. The polypeptide of claim 1 , further comprising one or more heterologous amino acid sequences located at the N-terminus and/or the C-terminus of the polypeptide.
5. An isolated nucleic acid comprising a polynucleotide sequence encoding the polypeptide of claim 1 .
6. The nucleic acid of claim 5 , wherein the polynucleotide sequence comprises the polynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.
7. The nucleic acid of claim 5 , wherein the polynucleotide sequence comprises the polynucleotide sequence set forth in SEQ ID NO: 1.
8. An expression cassette comprising the nucleic acid of claim 5 and a promoter, wherein the nucleic acid is operably linked to the promoter.
9. A vector comprising the nucleic acid of claim 5 .
10. An isolated host cell comprising the nucleic acid of claim 5 .
11. A method of synthesizing a sialylated product, the method comprising;
forming a reaction mixture comprising:
an acceptor glycoside,
a sialic acid donor, and
the polypeptide of claim 1 , and
incubating the reaction mixture under conditions sufficient to form the sialylated product.
12. The method of claim 11 , wherein the polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2.
13. The method of claim 11 , wherein the sialic acid donor is a cytidine-5′-monophosphate sialic acid.
14. The method of claim 11 , wherein the sialylated product comprises a sialylated α-linked GalNAc moiety or a sialylated β-linked Gal moiety.
15. The method of claim 14 , wherein the sialylated α-linked GalNAc moiety is a Neu5Acα2-6GalNAc moiety.
16. The method of claim 11 , wherein the sialylated product is a Siaα2-6GalNAcα1-O-Ser/Thr (sialyl Tn) antigen.
17. The method of claim 16 , wherein the acceptor glycoside has a structure according to the formula:
or a salt thereof, wherein:
Ac is acetyl
R a is selected from the group consisting of H and CH 3 ,
R b is selected from the group consisting of H, an amino acid residue, an oligopeptide, and a polypeptide, and
R c is selected from the group consisting of OH, an amino acid residue, an oligopeptide, and a polypeptide.
18. The method of claim 16 , wherein the sialyl Tn antigen has a structure according to the formula:
or a salt thereof, wherein:
Ac is acetyl
R a is selected from the group consisting of H and CH 3 ,
R b is selected from the group consisting of H, an amino acid residue, an oligopeptide, and a polypeptide, and
R c is selected from the group consisting of OH, an amino acid residue, an oligopeptide, and a polypeptide.
19. The method of claim 17 , wherein R b and Re are independently a polypeptide.
20. The method of claim 19 , wherein R b and Re are optionally and independently glycosylated.