Monodisperse random coil proteins and bioconjugates thereof

A method for preparing a substantially monodisperse random coil polypeptide preparation comprising a plurality of random coil polypeptides, the method comprising the steps:

• a) expressing a random coil polypeptide precursor consisting of the random coil polypeptide and a C-terminal affinity tag using a plasmid expression vector which comprises a nucleic acid sequence which encodes the random coil polypeptide precursor, wherein the precursor comprises the random coil polypeptide linked at its C-terminus to the C-terminal affinity tag via an intein domain or a GluC cleavage site consisting of the sequence IEGR (SEQ ID NO:22), wherein the random coil polypeptide has an electrostatic charge less than +18, and wherein the random coil polypeptide comprises repeating amino acid sequences of 30-250, wherein each of repeating sequences comprises amino acids independently selected from glycine, alanine, threonine, serine, and arginine;
• b) contacting the expressed polypeptide precursor with an affinity column having affinity for the C-terminal tag;
• c) completely cleaving the affinity tag by exposing the expressed polypeptide precursor to endoproteinase GluC that cleaves within said GluC cleavage site, or exposing to dithiothreitol which induces the intein domain's self-cleavage;
• and
• d) purifying the random coil polypeptide from which the affinity tag has been completely cleaved off by column chromatography thereby preparing plurality of substantially monodisperse random coil polypeptide preparation;
• wherein the degree of the monodispersity of the plurality of random coil polypeptides after removal of the C-terminal affinity tag is much greater than the degree of the monodispersity of corresponding polypeptides which are obtained after the cleavage of the affinity tag from the precursor consisting of the random coil polypeptide and the affinity tag at its N-terminus of said polypeptide, and wherein the monodispersity is determined by free-solution conjugate electrophoresis.