Methods of producing shiga toxin b-subunit (stxb) monomers and oligomers, and uses thereof

A method of producing a pentamer of a Shiga toxin B-subunit (STxB) protein or a variant thereof by peptide chemical synthesis, comprising the steps of:

• a) stepwisely coupling amino acid residues onto a support following an amino acid sequence of the STxB protein or the variant thereof, wherein said amino acid residues comprise α amino-protecting groups and optionally side chain-protecting groups, thereby obtaining a synthetic peptide;
• b) deprotecting the synthetic peptide obtained from step a) by removing:
• b1) the final α amino-protecting group, and
• b2) optionally, the side-chain protecting-groups,
• thereby obtaining a deprotected synthetic peptide;
• and
• c) cleaving the deprotected synthetic peptide obtained from step b) from the support,
• thereby obtaining a free monomer of the STxB protein or the variant thereof,
• d) refolding the free monomer of the STxB protein or of the variant thereof by dialyzing said protein or the variant thereof against at least one dialysate, thereby obtaining a free pentamer of the STxB protein or of the variant thereof,
• wherein said amino acid sequence of the STxB protein is SEQ ID NO:
• 2, said variant thereof consists of one of the peptides with amino acid sequence selected from SEQ IDs NO:
• 1 and 3 to 21,
• wherein the monomer of the STxB protein or the variant thereof is produced by peptide chemical synthesis of at least two fragments of said STxB protein or variant thereof, wherein said at least two fragments are ligated to obtain the monomer of the STxB protein or the variant thereof, and wherein said at least two fragments consist of SEQ ID NO:
• 22 and SEQ ID NO:
• 23.