Subtilase cytotoxin B subunit mutant
A mutant subtilase cytotoxin B subunit protein is provided which can bind glycans having α2-3-linked N-glycolylneuraminic acid and glycans having α2-6-linked N-glycolylneuraminic acid. The mutant SubB protein has deletions of one or more of the amino acid sequence TTSTE and has a previously undescribed ability to bind glycans having α2-6-linked N-glycolylneuraminic acid, while not losing the ability to bind glycans having α2-3-linked N-glycolylneuraminic acid.
1. An isolated subtilase cytotoxin B subunit (SubB) protein comprising an amino acid sequence of SEQ ID NO:2, or a fragment, a variant, or a derivative thereof, which comprises a deletion of one or more of the amino acid residues of the amino acid sequence TTSTE (SEQ ID NO: 3) and/or a substitution of one or more of the third, fourth, and fifth amino acid residues of the amino acid sequence TTSTE (SEQ ID NO: 3), and wherein the isolated protein, the fragment, the variant, or the derivative thereof is capable of binding α2-3-linked glycolylneuraminic acid and α2-6-linked N-glycolylneuraminic acid.
2. The isolated SubB protein of claim 1 , which comprises a substitution or deletion of at least one of the third or fourth amino acid residues of TTSTE (SEQ ID NO:3).
3. The isolated SubB protein, fragment of claim 2 , which further comprises a deletion of the fifth amino acid residue of TTSTE (SEQ ID NO:3).
4. The isolated SubB protein of claim 3 , which comprises a deletion of the fourth and fifth amino acid residues of TTSTE (SEQ ID NO: 3).
5. The isolated SubB protein of claim 1 , which comprises a deletion of at least one of the third or fourth amino acid residues of TTSTE (SEQ ID NO:3).
6. The isolated SubB protein of claim 1 , wherein the isolated SubB protein comprises the amino acid sequence of SEQ ID NO: 1.
7. A composition comprising, the isolated SubB protein of claim 1 .
8. An isolated molecular complex comprising the isolated SubB protein of claim 1 and a glycan comprising, α2-3-linked N-glycolylneuraminic acid and/or a α2-6-linked N-glycolylneuraminic acid.
9. The isolated molecular complex of claim 8 , Wherein the glycan comprising the α2-3-linked N-glycolylneuraminic acid and/or the α2-6-linked N-glycolylneuraminic acid is from a tumour cell or by a feline blood cell.
10. An isolated nucleic acid encoding the isolated SubB protein of claim 1 .
11. A genetic construct comprising the isolated nucleic acid of claim 10 .
12. A host cell comprising the genetic construct of claim 11 .
13. A method of detecting α2-3-linked N-glycolylneuraminic acid and/or α2-6-linked N-glycolylneuraminic acid, said method including the step of combining:
(a) the isolated SubB protein of claim 1 , or
(b) a composition comprising the isolated SubB protein of claim 1 , with a sample to thereby form a detectable complex comprising said isolated SubB protein and α2-3-linked N-glycolylneuraminic acid and/or α2-6-linked N-glycolylneuraminic acid.
14. The method of claim 13 , wherein the α2-3-linked N-glycolylneuraminic acid and/or the α2-6-linked N-glycolylneuraminic acid is from a tumour cell or by a feline blood cell.
15. A method of isolating a glycan comprising α2-3-linked N-glycolylneuraminic acid and/or an α2-6-linked N-glycolylneuraminic acid, the method including the steps of: combining:
(a) the isolated SubB protein of claim 1 ; or
(b) a composition comprising the isolated SubB protein of claim 1 , with a sample to thereby form a complex comprising said isolated SubB protein and the glycan comprising the α2-3-linked N-glycolylneuraminic acid and/or α2-6-linked N-glycolylneuraminic acid; and isolating the complex.
16. A method of isolating a cell expressing a glycan comprising α2-3-linked N-glycolylneuraminic acid and/or an α2-6-linked N-glycolylneuraminic acid, said method including the steps of combining:
(a) the isolated SubB protein of claim 1 ; or
(b) a composition comprising the isolated SubB protein of claim 1 ,
with a sample comprising a cell to thereby form a complex comprising said isolated SubB protein and the glycan comprising the α2-3-linked N-glycolylneuraminic acid and/or α2-6-linked N-glycolylneuraminic acid; and isolating the cell.