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 protein which comprises the amino acid sequence set forth in SEQ ID NO: 1, or a variant thereof, wherein:
the variant comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 1; and,
the variant 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, or the variant thereof is capable of binding α2-3-linked N-glycolylneuraminic acid and α2-6-linked N-glycolylneuraminic acid.
2 . The isolated protein of claim 1 , which comprises a deletion of one or more of the third and fourth amino acid residues of the amino acid sequence TTSTE (SEQ ID NO: 3).
3 - 29 . (canceled)
30 . The isolated protein of claim 1 , wherein the variant comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 1.
31 . The isolated protein of claim 1 , wherein the variant comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 1.
32 . The isolated protein of claim 1 , which comprises a deletion of the third and fourth amino acid residues of the amino acid sequence TTSTE (SEQ ID NO: 3).
33 . An isolated molecular complex comprising the isolated protein of claim 1 and a glycan comprising α2-3-linked N-glycolylneuraminic acid and/or a α2-6-linked N-glycolylneuraminic acid.
34 . The isolated molecular complex of claim 33 , 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 a feline blood cell.
35 . A composition comprising the isolated protein of claim 1 .
36 . 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 protein of claim 1 , or
(b) a composition comprising the isolated protein of claim 1 , with a sample to thereby form a detectable complex comprising said isolated protein and α2-3-linked N-glycolylneuraminic acid and/or α2-6-linked N-glycolylneuraminic acid.
37 . The method of claim 36 , wherein the α2-3-linked N-glycolylneuraminic acid and/or the α2-6-linked N-glycolylneuraminic acid is from a tumour cell or a feline blood cell.
38 . 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 protein of claim 1 ; or
(b) a composition comprising the isolated protein of claim 1 , with a sample to thereby form a complex comprising said isolated protein and the glycan comprising the α2-3-linked N-glycolylneuraminic acid and/or α2-6-linked N-glycolylneuraminic acid; and isolating the complex.
39 . An isolated nucleic acid encoding the isolated protein of claim 1 .
40 . A genetic construct comprising the isolated nucleic acid of claim 39 .
41 . A host cell comprising the genetic construct of claim 40 .
42 . 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 protein of claim 1 ; or
(b) a composition comprising the isolated protein of claim 1 ,
with a sample comprising a cell to thereby form a complex comprising said isolated protein and the glycan comprising the α2-3-linked N-glycolylneuraminic acid and/or α2-6-linked N-glycolylneuraminic acid; and isolating the cell.