System and method for identification and characterization of transglutaminase species
AssigneeRoche Sequencing Solutions, Inc.
InventorsThomas Albert, Frank Bergmann, Victor Lyamichev, Jigar Patel, Michael Schraeml, Wojtek Steffen, Thomas Streidl
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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.
Claims18
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).
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.
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 Xaa1-Xaa2-Xaa3-Xaa4-Xaa5, wherein Xaa is any amino acid, wherein at least one of Xaa3, Xaa4, and Xaa5 is glutamine, wherein one of Xaa4 and Xaa5 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 Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 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).
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 Xaa1-Xaa2-Xaa3-Xaa4-Xaa5, wherein Xaa is any amino acid, wherein the amino acid sequence includes at least one lysine, wherein one of Xaa1 and Xaa2 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 Xaa1-Xaa2-Xaa3-Xaa4-Xaa5 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).
Dependencies are inferred from the claim text.