System and method for identification and characterization of transglutaminase species

Patent metadata

Field	Value
Publication number	US-11280791-B2
Application number	US-202016991679-A
Country	US
Kind code	B2
Filing date	Aug 12, 2020
Priority date	Dec 19, 2014
Publication date	Mar 22, 2022
Grant date	Mar 22, 2022

Abstract

In one aspect, the present disclosure provides a system and method for the identification and characterization of a transglutaminase. Further, the present disclosure provides transglutaminase enzymes for forming isopeptide bonds, methods of forming isopeptide bonds in the presence of transglutaminases, and substrate tags for use with transglutaminases. In another aspect, the present disclosure provides glutamine-containing substrates (or Q-tag substrates) that are more resistant to proteases/clipping and therefore, more stable, than other Q-tag substrates, and their uses in substrate tags for cross-linking to an amine-donor tag via an isopeptide bond mediated by a microbial transglutaminase.

First claim

What is claimed is:

1. A kit for forming an isopeptide bond in the presence of a microbial transglutaminase, the kit comprising a recombinant protein construct including an isolated microbial transglutaminase having at least 80% sequence identity to the Kutzneria albida microbial transglutaminase (SEQ ID NO:6), wherein the isolated microbial transglutaminase was expressed and isolated in the presence of ammonium, and a first substrate including an acyl-donor tag having at least 80% sequence identity to the peptide sequence RYRQR (SEQ ID NO:14).

2. The kit of claim 1 , wherein the acyl-donor tag has the peptide sequence of APRYRQRAA (SEQ ID NO:24).

3. The kit of claim 1 , wherein the acyl-donor tag has the peptide sequence of RVRQR (SEQ ID NO:113).

4. The kit of claim 1 , further comprising a second substrate including an amine-donor tag having at least 80% sequence identity to the peptide sequence RYESK (SEQ ID NO:2).

5. The kit of claim 4 , wherein at least one of the first substrate and the second substrate includes a detectable label.

6. The kit of claim 5 , wherein the detectable label is selected from a biotin moiety, a fluorescent dye, a ruthenium label, a radiolabel, and a chemiluminescent label.

7. The kit of claim 1 , wherein the ammonium was present at a concentration of at least 10 μM.

8. The kit of claim 1 , wherein the isolated microbial transglutaminase was prepared from a modular expression construct selected from one of SEQ ID NO:8 and SEQ ID NO:11.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Albert Thomas
• Bergmann Frank
• Lyamichev Victor
• Mohamed Mohamed Yosry Hassan
• Oelschlaegel Tobias
• Patel Jigar
• Schraeml Michael
• Steffen Wojtek
• Streidl Thomas

Classifications

• G01N2333/91085

  Transglutaminases; Factor XIIIq (2.3.2.13) · CPC title

• C07K16/00

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C12N9/1044 Primary

  Protein-glutamine gamma-glutamyltransferase (2.3.2.13), i.e. transglutaminase or factor XIII · CPC title

• C07K7/06

  having 5 to 11 amino acids · CPC title

• C12Y203/01013

  Glycine N-acyltransferase (2.3.1.13) · CPC title