US 11,788,080 B2
Modified cleavases, uses thereof and related kits
Kevin Desai, San Diego, CA (US); Kevin L. Gunderson, San Diego, CA (US); Robert C. James, San Diego, CA (US); Lei Shi, San Diego, CA (US); and Stephen Verespy, III, San Diego, CA (US)
Assigned to Encodia, Inc., San Diego, CA (US)
Filed by Encodia, Inc., San Diego, CA (US)
Filed on Jun. 27, 2022, as Appl. No. 17/850,516.
Application 17/850,516 is a continuation of application No. 17/213,169, filed on Mar. 25, 2021, granted, now 11,427,814.
Application 17/213,169 is a continuation in part of application No. PCT/US2020/024521, filed on Mar. 24, 2020.
Claims priority of provisional application 63/085,977, filed on Sep. 30, 2020.
Claims priority of provisional application 62/931,737, filed on Nov. 6, 2019.
Claims priority of provisional application 62/824,157, filed on Mar. 26, 2019.
Claims priority of provisional application 62/823,927, filed on Mar. 26, 2019.
Prior Publication US 2023/0021091 A1, Jan. 19, 2023
This patent is subject to a terminal disclaimer.
Int. Cl. C12N 9/48 (2006.01); C12P 21/06 (2006.01)
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.