Compositions comprising homogeneously glycosylated erythropoietin
The present invention provides isolated homogeneous polyfunctionalized proteins (e.g., erythropoietin), isolated glycopeptides, and a method for preparing polyfunctionalized peptides and/or proteins via cysteine-free native chemical ligation. In certain embodiments, the invention provides an isolated homogeneous polyfunctionalized protein having the structure (I). In certain other embodiments, the invention provides an isolated glycopeptide having Formula (II). In certain other embodiments, the inventive method is a method for preparing a polyfunctionalized peptide comprising a peptidic backbone made up of four or more amino acids, wherein two or more non-adjacent amino acids are independently substituted with a moiety having the structure (III) -LH. wherein A and L1 are as defined herein.
1. A composition of a homogenously glycosylated erythropoietin or fragment thereof, comprising:
a polypeptide whose amino acid sequence includes a sequence that:
a) is identical to that of SEQ ID NO:1, or
b) contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, substitutions, additions or combinations thereof relative to such SEQ ID NO: 1, and stimulates erythrocyte production; or
c) is a fragment of a) or b), wherein the fragment has an amino acid sequence corresponding to amino acid residues 1-28, 29-77, 78-113, or 114-166 of SEQ ID NO: 1;
the polypeptide having at least one amino acid residue site glycosylated;
wherein each glycosylated polypeptide in the composition has the same glycosylation pattern in that:
it is glycosylated on at least one amino acid residue site;
it is glycosylated at the same at least one site;
it is glycosylated at a site selected from the group consisting of Asn 24 , Asn 38 , Asn 83 , Ser 126 in SEQ ID NO: 1, and combinations thereof; and
for a given glycosylation site, it has the same glycan.
2. The composition of claim 1 , wherein the polypeptide's amino acid sequence is SEQ ID NO: 1, or is SEQ ID NO: 1 having 1-10 amino acid substitutions, additions, and/or deletions.
3. The composition of claim 1 , wherein the fragment has an amino acid sequence as follows: SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 6.
4. The composition of claim 1 , wherein the structure of the homogenously glycosylated erythropoietin is of formula:
5. The composition of claim 1 , wherein the structure of the fragment of erythropoietin is as follows:
6. A method of preparing homogeneously glycosylated erythropoietin, the method comprising steps of:
ligating to one another a set of fragments of a polypeptide whose amino acid sequence includes a sequence that:
a) is identical to that of SEQ ID NO: 1, or
b) contains 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid deletions, substitutions, additions or combinations thereof relative to such SEQ ID NO: 1, and stimulates erythrocyte production:
the polypeptide having at least one amino acid residue site glycosylated;
wherein each glycosylated polypeptide in the composition has the same glycosylation pattern in that:
it is glycosylated on at least one amino acid residue site;
it is glycosylated at the same at least one site;
it is glycosylated at a site selected from the group consisting of Asn 24 , Asn 38 , Asn 83 , Ser 126 in SEQ ID NO: 1, and combinations thereof; and
for a given glycosylation site, it has the same glycan;
which set of fragments includes fragments whose amino acid sequence corresponds to amino acid residues 1-28, 29-77, 78-113, and 114-166 of SEQ ID NO: 1, so that a homogenously glycosylated erythropoietin polypeptide is generated.
7. The composition of claim 1 , wherein at least one glycosylated residue is glycosylated with an O-linked glycans selected from the group consisting of:
or wherein at least one glycosylated residue is glycosylated with an N-linked glycans selected from the group consisting of:
8. The composition of claim 4 ,
wherein Asn 24 , Asn 38 , and Asn 83 are glycosylated with a glycan of formula:
and Ser 126 is glycosylated with a glycan of formula:
9. The composition of claim 1 , wherein Ser 126 is glycosylated with glycophorin, and wherein at least one other glycosylated residue is glycosylated with chitobiose.