Functionally modified polypeptides and radiobiosynthesis
Provided herein are compositions and methods for generating polypeptides using non-natural amino acids (nnAAs) and genetic machinery, wherein the modified polypeptides, such as therapeutic polypeptides, bind to albumin, such as serum albumin. Methods of substituting a non-natural amino acid in a first polypeptide to obtain a modified polypeptide, the nnAA in some instances comprising an albumin targeting group, are disclosed, as are methods for making populations of such modified polypeptides. A therapeutic polypeptide, interleukin-1 receptor antagonist (IL-1RA) is exemplified using the disclosed methods.
1. A method of substituting a natural amino acid in a first polypeptide with a non-natural amino acid to obtain a modified polypeptide, wherein the non-natural amino acid comprises a moiety selected from the group consisting of: an albumin-targeting group and a radiolabelled albumin-targeting group, comprising:
(a) selecting at least one amino acid residue in the first polypeptide to be substituted with the non-natural amino acid;
(b) selecting a polynucleotide encoding the first polypeptide;
(c) modifying the polynucleotide such that the amino acid residue to be substituted is encoded by a nonsense codon;
(d) expressing the modified polynucleotide in a cell, wherein the cell is in the presence of the non-natural amino acid and expresses a suppressor tRNA and its cognate tRNA synthetase wherein the suppressor tRNA recognizes the nonsense codon of step (c), and the cell incorporates the non-natural amino acid into the modified polypeptide at the nonsense codon of step (c) during translation,
wherein the albumin-targeting group or radiolabelled albumin-targeting group is selected from the group consisting of:
2. The method of claim 1 , wherein the non-natural amino acid is Nε-(4-(4-iodophenyl)butanoyl)lysine, or a pharmaceutically acceptable salt thereof.
3. The method of claim 1 , wherein the non-natural amino acid is Nε-(4-(4-iodophenyl)butanethioyl) lysine, or a pharmaceutically acceptable salt thereof.
4. The method of claim 1 , further comprising step (e), purifying the modified polypeptide.
5. The method of claim 1 , wherein the radiolabel is 123 I, 124 I, 125 I, or 131 I.
6. The method of claim 1 , wherein the non-naturally occurring amino acid is selected from the group consisting of: