System and method for identification and characterization of transglutaminase species

Patent metadata

Field	Value
Publication number	US-10006915-B2
Application number	US-201514973472-A
Country	US
Kind code	B2
Filing date	Dec 17, 2015
Priority date	Dec 19, 2014
Publication date	Jun 26, 2018
Grant date	Jun 26, 2018

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.

First claim

What is claimed is:

1. A structure, a substrate tag attached to the structure, and a microbial transglutaminase bound to the substrate tag, the substrate tag comprising an acyl-donor tag having at least 80% sequence identity to the peptide sequence YRYRQ (SEQ ID NO:1), wherein the acyl-donor tag is a substrate for the microbial transglutaminase, wherein the structure is selected from a recombinant protein, a detectable label, and a chemically synthesized structure, and wherein the microbial transglutaminase has at least 80% sequence identity to the Kutzneria albida microbial transglutaminase (SEQ ID NO:6).

2. The structure of claim 1 , wherein the detectable label is selected from a biotin moiety, a fluorescent dye, a ruthenium label, a radiolabel, and a chemiluminescent label.

3. The structure of claim 1 , the acyl-donor tag having the peptide sequence APRYRQRAA (SEQ ID NO:24).

4. The structure of claim 1 , wherein the recombinant microbial transglutaminase is the Kutzneria albida microbial transglutaminase (SEQ ID NO:6).

5. The structure of claim 1 , wherein the chemically synthesized structure comprises at least one of a protein, a peptide, an oligonucleotide, a carboxybenzyl group and a polyethylene glycol (PEG) polymer.

6. 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.

7. The kit of claim 6 , further comprising a first substrate including an acyl-donor tag having at least 80% sequence identity to the peptide sequence YRYRQ (SEQ ID NO:1).

8. The kit of claim 7 , the acyl-donor tag having the peptide sequence APRYRQRAA (SEQ ID NO:24).

9. The kit of claim 7 , 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).

10. The kit of claim 9 , wherein at least one of the first substrate and the second substrate includes a detectable label.

11. The kit of claim 10 , wherein the detectable label is selected from a biotin moiety, a fluorescent dye, a ruthenium label, a radiolabel, and a chemiluminescent label.

12. The kit of claim 6 , wherein the ammonium was present at a concentration of at least about 10 μM.

13. The kit of claim 6 , 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.

14. An acyl-donor substrate for a microbial transglutaminase, the acyl-donor substrate comprising a structure having an acyl-donor tag attached thereto, and the microbial transglutaminase bound to the acyl-donor tag, the acyl-donor tag comprising an amino acid sequence having the formula Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 , wherein Xaa is any amino acid, wherein at least one of Xaa 3 , Xaa 4 , and Xaa 5 is glutamine, wherein one of Xaa 4 and Xaa 5 is arginine, wherein the amino acid sequence includes at least one arginine sequentially adjacent to a glutamine, and wherein the total number of amino acids in the amino acid sequence selected from arginine, glutamine, phenylalanine, tryptophan, and tyrosine is at least four, wherein the structure is selected from a recombinant protein, a detectable label, and a chemically synthesized structure, wherein the amino acid sequence having the formula Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 is a substrate for the microbial transglutaminase, and wherein the microbial transglutaminase has at least 80% sequence identity to the Kutzneria albida microbial transglutaminase (SEQ ID NO:6).

15. The acyl-donor substrate of claim 14 , wherein Xaa 5 and at least one of Xaa 1 , Xaa 2 , and Xaa 3 is arginine.

16. An amine-donor substrate for a microbial transglutaminase, the amine-donor substrate comprising a structure having an amine-donor tag attached thereto, and the microbial transglutaminase bound to the amine-donor tag, the amine-donor tag comprising an amino acid sequence having the formula Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 , wherein Xaa is any amino acid, wherein the amino acid sequence includes at least one lysine, wherein one of Xaa 1 and Xaa 2 is selected from tyrosine and arginine, and wherein the total number of amino acids in the amino acid sequence selected from arginine, serine, tyrosine, and lysine is at least three, wherein the structure is selected from a recombinant protein, a detectable label, and a chemically synthesized structure, wherein the amino acid sequence having the formula Xaa 1 -Xaa 2 -Xaa 3 -Xaa 4 -Xaa 5 is a substrate for the microbial transglutaminase, and wherein the microbial transglutaminase has at least 80% sequence identity to the Kutzneria albida microbial transglutaminase (SEQ ID NO:6).

17. The amine-donor substrate of claim 16 , wherein one of Xaa 4 and Xaa 5 is lysine.

18. The amine-donor substrate of claim 16 , wherein the amino acid sequence includes no more than two of the amino acid lysine.

Assignees

• Roche Sequencing Solutions Inc

Inventors

• Albert Thomas
• Bergmann Frank
• Lyamichev Victor
• Patel Jigar
• Schraeml Michael
• Steffen Wojtek
• Streidl Thomas

Classifications

• C07K1/1075 Primary

  by covalent attachment of amino acids or peptide residues · CPC title

• G01N33/573 Primary

  for enzymes or isoenzymes · CPC title

• G01N2333/91085

  Transglutaminases; Factor XIIIq (2.3.2.13) · CPC title

• C07K7/06 Primary

  having 5 to 11 amino acids · CPC title

• C12N9/1044

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