Methods of producing Shiga toxin B-subunit (STxB) monomers and oligomers, and uses thereof
A method of producing a monomer of a Shiga toxin B-subunit (STxB) protein or of a variant thereof by peptide chemical synthesis, as well as to a method of producing a pentamer of the STxB protein or of the variant thereof. The methods are particularly advantageous as they overcome major issues typically observed in peptide chemical synthesis, including solubility and purity issues.
1 . A method of producing a monomer 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, 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.
2 . The method according to claim 1 , wherein step a) comprises the following sequence of substeps:
a1) removing the α amino-protecting group from the support or from the N-terminal amino acid of the synthetic peptide;
a2) optionally, washing the support;
a3) coupling the next amino acid following an amino acid sequence set forth in SEQ ID NO: 2 in a linear C- to N-terminal direction;
a4) optionally, washing the support;
a5) optionally, capping unreacted amino groups; and
a6) optionally, washing the support.
3 . The method according to claim 2 , wherein:
Substep a3) is reiterated more times and/or is carried out for a longer period of time to couple amino acid residues Cys 4, Thr 12, Thr 21, Asn 35, Leu 36, and Ile 45 with respect to SEQ ID NO: 2 numbering than to couple the other amino acid residues of the STxB protein; and
dipeptides Val 5-Thr 6, Asp 18-Thr 19, Phe 30-Thr 31, Leu 41-Ser 42, and Val 50-Thr 51 with respect to SEQ ID NO: 2 numbering are coupled in substep a3) in a pseudoproline dipeptide form.
4 . The method according to claim 2 , wherein substep a3) is reiterated more times and/or carried out for a longer period of time to couple amino acid residues Cys 4, Thr 12, Thr 21, Asn 35, Leu 36, Leu 39, Ile 45, Cys 57, and Val 65 with respect to SEQ ID NO: 2 numbering than to couple the other amino acid residues of the STxB protein or of the variant thereof.
5 . The method according to claim 2 , wherein substep a3) is reiterated more times and/or carried out for a longer period of time to couple amino acid residues Cys 4, Thr 12, Thr 21, Asn 35, Leu 36, Leu 39, Ile 45, Thr 49, Cys 57, and Val 65 with respect to SEQ ID NO: 2 numbering than to couple the other amino acid residues of the STxB protein or of the variant thereof.
6 . The method according to claim 2 , wherein substep a3) is reiterated more times and/or carried out for a longer period of time to couple amino acid residues Thr 1, Cys 4, Tyr 11, Thr 12, Thr 21, Asn 35, Leu 36, Leu 39, Ile 45, Thr 46, Thr 49, Cys 57, and Val 65 with respect to SEQ ID NO: 2 numbering than to couple the other amino acid residues of the STxB protein or of the variant thereof.
7 . The method according to claim 2 , wherein dipeptides Val 5-Thr 6, Asp 18-Thr 19, Phe 30-Thr 31, Leu 41-Ser 42, Val 50-Thr 51, and Phe 63-Ser 64 with respect to SEQ ID NO: 2 numbering are coupled in substep a3) in a pseudoproline dipeptide form.
8 . The method according to claim 1 , wherein the method further comprises one or more of the steps of:
d) precipitating the free monomer of the STxB protein or of the variant thereof obtained from step c), and optionally air-drying and/or lyophilizing the precipitated STxB protein or the variant thereof,
e) oxidizing the free monomer of the STxB protein or of the variant thereof obtained from step c) or d) under conditions suitable for the formation of an intramolecular disulfide bond between Cys 4 and Cys 57 with respect to SEQ ID NO: 2 numbering.
9 . The method according to claim 1 , wherein the method does not comprise a purification step by chromatography.
10 . A monomer of a Shiga toxin B-subunit (STxB) protein or of a variant thereof obtained by the method according to claim 1 , wherein dipeptides Val 5-Thr 6, Asp 18-Thr 19, Phe 30-Thr 31, Leu 41-Ser 42, and Val 50-Thr 51 with respite to SEQ ID NO: 2 numbering are coupled in a pseudoproline dipeptide form.