Subtilase cytotoxin b subunit mutant

Abstract

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.