FKBP Transglutaminase Substrate for Controlled Protein Labeling
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Conventional methods for labeling antigens and antibodies in immunological diagnostic systems face challenges such as difficulty in controlling stoichiometry, heterogeneity of immunoconjugates, and potential interference with immunoreactivity due to non-specific conjugation of markers, especially with chemical methods.
Innovation Solution
A recombinant transglutaminase (TG) substrate is developed, comprising an FKBP domain with a Q-tag sequence that allows for site-specific and controlled labeling of proteins, using the TG function of Kutzneria albida, ensuring precise and stoichiometric binding of labels without interfering with the protein's function.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If chemical methods are used for labeling antigens and antibodies, then labeling can be achieved, but stoichiometry control is difficult and heterogeneity of immunoconjugates occurs
Solution Approach 1:
The patent introduces a transglutaminase enzyme as an intermediary to mediate the labeling reaction. The enzyme specifically catalyzes the formation of isopeptide bonds between glutamine residues on the antigen/antibody and amine-containing labels, providing precise stoichiometric control and homogeneous immunoconjugates, thereby resolving the contradiction between labeling efficiency and precision control
Solution Approach 2:
The patent changes the reaction parameters from non-specific chemical conjugation to enzyme-catalyzed specific conjugation. By controlling enzyme concentration, reaction time, and substrate availability, precise stoichiometric control is achieved, transforming the labeling process from heterogeneous to homogeneous and controllable
2Reliability
If chemical labeling methods are used, then labeling can be performed, but non-specific conjugation interferes with immunoreactivity
Solution Approach 1:
The transglutaminase enzyme acts as a selective intermediary that recognizes specific glutamine residues as substrates. This enzymatic specificity ensures that only designated sites on the antigen or antibody are labeled, preventing non-specific conjugation that would interfere with immunoreactivity, thus simultaneously achieving high labeling precision and maintained immunoreactivity
Solution Approach 2:
The patent implements local quality control by directing labeling to specific glutamine residues within the antigen or antibody structure. The transglutaminase enzyme recognizes and labels only specific local sites, ensuring that critical immunoreactive regions remain unlabeled and functional, thereby maintaining immunoreactivity while achieving precise labeling
3Reliability
If the FKBP domain is used as a scaffold for protein engineering, then solubility and folding are improved, but the structure must be modified to enable transglutaminase labeling
Solution Approach 1:
The patent extracts the essential chaperone function from the complete FKBP domain while removing or modifying regions that would interfere with transglutaminase labeling. By taking out the core folding-promoting capability and separating it from the portions requiring modification, the solution achieves both solubility improvement and labeling compatibility with minimal overall structural change
Solution Approach 2:
The patent applies local quality modification by making targeted changes only to specific regions of the FKBP domain that need to accommodate transglutaminase labeling, while preserving the core chaperone domains that provide solubility and folding assistance. This localized approach maintains the beneficial solubility properties while enabling the new labeling functionality
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The solution enables highly controlled and specific labeling of proteins, maintaining their immunoreactivity and allowing for multiple labeling, which is essential for precise quantification in immunological assays.
Implementation Method 1
The enzyme catalyzes the formation of an isopeptide bond between an acyl-group, e.g. a glutamine (Q) side chain and an alkyl-amine, e.g. a lysine (K) side chain
Implementation Method 2
the enzymatic reaction results in the formation of an isopeptide bond
Data Source
Figure 1A~1D
Figure 2A~2B
Figure 3A~3B
AI summary
The present invention relates to a recombinant transglutaminase (TG) substrate comprising an amino acid sequence of the FKBP domain of an FKBP polypeptide, wherein the "insert-in-flap" (IF) domain thereof is, at least in part, replaced by an amino acid sequence ("Q-tag") of 5 to 20 amino acids comprising a sequence having at least 80% sequence identity to the YRYRQ portion of the peptide sequence X -YRYRQ-X2 (SEQ ID NO. 1), and wherein said TG substrate is a substrate for the TG function of the Kutzneria albida TG. The present invention furthermore relatesuses of said substrate.