CPC C12N 9/485 (2013.01) [C12P 21/06 (2013.01)] | 21 Claims |
1. A method for identifying a modified cleavase configured to cleave a labeled terminal amino acid residue or a labeled terminal dipeptide from a polypeptide, the method comprising:
(a) constructing a library of mutated cleavase genes from a gene encoding an unmodified dipeptidyl peptidase (DPP), wherein the unmodified DPP cleaves an unlabeled terminal dipeptide from a polypeptide;
(b) contacting a plurality of auxotrophic microorganism cells with a growth medium, wherein:
each auxotrophic microorganism cell i) requires uptake of an auxotrophic amino acid in order to grow, and ii) comprises a mutated cleavase gene from the library of mutated cleavase genes, and
the growth medium is substantially free of the auxotrophic amino acid and comprises a peptide substrate comprising a labeled terminal amino acid residue and the auxotrophic amino acid residue; and
(c) identifying a modified cleavase produced from the mutated cleavase gene in an auxotrophic microorganism cell of the plurality of auxotrophic microorganism cells, wherein the modified cleavase cleaves the peptide substrate between the labeled terminal amino acid residue and the auxotrophic amino acid residue, thereby releasing the auxotrophic amino acid and allowing uptake of the released auxotrophic amino acid by the auxotrophic microorganism cell and growth of the auxotrophic microorganism cell using the growth medium.
|