Monodisperse random coil proteins and bioconjugates thereof
The present disclosure provides substantially monodisperse random coil polypeptides, vectors encoding the polypeptides, conjugates containing the polypeptides, methods for their preparation, and their uses in nucleic acid separations, DNA sequencing, and other applications requiring high monodispersity.
1. 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.
2. The method of claim 1 , wherein the polypeptide is expressed in E. coli.
3. A substantially monodisperse random coil polypeptide made by the method of claim 1 .
4. The method of claim 1 , wherein the polypeptide has at least 10 of the repeating sequences wherein each repeating sequence has at least five amino acids.
5. The method of claim 1 , wherein the polypeptide has 30 to 70 of the repeating sequences wherein each repeating sequence has at least seven amino acids.
6. The method of claim 1 , wherein each of the repeating sequences is selected from GAGTGSA (SEQ ID NO: 1) and GAGTGRA (SEQ ID NO: 2).
7. The method of claim 1 , wherein the polypeptide comprises at its N-terminus a T7 tag comprising the amino acid sequence MASMTGGQQMG (SEQ ID NO: 3).
8. The method of claim 1 , wherein when the C-terminal tag is not an intein, the polypeptide is greater than 250 amino acids in length.
9. The method of claim 1 , wherein the polypeptide further comprises a second non-identical repeating amino sequence.
10. The method of claim 1 , wherein the polypeptide comprises, on average, evenly spaced arginines.
11. The method of claim 1 , wherein the arginines are spaced every 18 amino acids, on average.
12. The method of claim 1 , wherein the affinity tag is a polyhistidine tag.
13. The method of claim 1 , wherein the random coil polypeptide has 30 to 70 repeats of the repeating amino acid sequence, wherein the amino acid sequence is GAGTGSA (SEQ ID NO: 1) and GAGTGRA (SEQ ID NO: 2).
14. The method of claim 1 , wherein the random coil polypeptide is less than 250 amino acids in length and each of the repeating amino acid sequences is 7 amino acids in length.
15. The method of claim 1 , wherein the plurality of random coil polypeptides in the preparation are at least 95% identical to each other.
16. The method of claim 1 , wherein the plurality of random coil polypeptides in the preparation are at least 99% identical to each other.