Protein and peptide purification methods

Patent metadata

Field	Value
Publication number	US-11655271-B2
Application number	US-201916972923-A
Country	US
Kind code	B2
Filing date	Jun 7, 2019
Priority date	Jun 8, 2018
Publication date	May 23, 2023
Grant date	May 23, 2023

Abstract

Provided herein are methods, reagents, and kits for isolating polypeptides, such as a proteome. Also provided herein is a modified trypsin polypeptide that is resistant to autolysis, and that can be selectively-separated from a biological sample once digestion is complete.

First claim

We claim:

1. A coupling compound having the structure:

2. A method of purifying a polypeptide, comprising:
mixing a sample having a basic pH, comprising a polypeptide or a protease-digested polypeptide with an amount of a coupling compound as claimed in claim 1 ;
coupling polypeptides in the sample to a second member of the bio-orthogonal coupling pair linked to a substrate;
optionally washing polypeptides bound to the substrate to remove any unbound materials from the substrate-bound polypeptides; and
eluting the polypeptides from the substrate in an elution solution having an acidic pH.

3. The method of claim 2 , wherein the substrate is a bead, a solid surface or a porous matrix.

4. The method of claim 2 , wherein:
the first member of the bio-orthogonal coupling pair is the tetrazinyl group, and the second member of the bio-orthogonal coupling pair is a strained cycloalkene.

5. The method of claim 2 , wherein the polypeptides of the sample are digested with a modified trypsin polypeptide, comprising:
a trypsin polypeptide;
biotin moieties attached to the trypsin polypeptide; and
groups comprising a member of a bio-orthogonal coupling pair attached to the trypsin polypeptide,
wherein the ratio of groups comprising a member of a bio-orthogonal coupling pair to biotin moieties on the trypsin polypeptide ranges from 1:4 to 1:6.

6. The method of claim 4 , wherein the strained cycloalkene is trans-cyclooctene.

Assignees

• Univ Carnegie Mellon

Inventors

• Minden Jonathan
• Lucas Amber

Classifications

• C07D403/12

  linked by a chain containing hetero atoms as chain links · CPC title

• C07K1/14 Primary

  Extraction; Separation; Purification · CPC title

• C07K19/00

  Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes} · CPC title

• C07K1/10

  using coupling agents {(C07K1/006 takes precedence)} · CPC title

• C12N9/6427 Primary

  Chymotrypsins (3.4.21.1; 3.4.21.2); Trypsin (3.4.21.4) · CPC title