TtGSTm34-8xHis Enzyme Substrate Affinity and Stability
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing GST enzymes have limitations in terms of affinity for substrates and stability under various conditions, which affects their efficiency in enzyme kinetics and affinity purification processes.
Innovation Solution
The development of a recombinant Tetrahymena thermophila Glutathione S Transferase Mu34 (TtGSTm34-8xHis) enzyme, which includes an 8xHis tag and is produced in Tetrahymena thermophila, offering high affinity for substrates and improved stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If commercially available GST enzymes are used, then enzyme kinetics and affinity purification processes can be performed, but the affinity for substrates is insufficient and stability under various conditions is poor
Solution Approach 1:
The patent segments the GST enzyme production by using a specific isoform (GSTm34) from Tetrahymena thermophila with an 8xHis tag, separating the catalytic domain from other GST isoforms to achieve both high stability and substrate affinity while maintaining ease of production through targeted recombinant expression
Solution Approach 2:
The patent changes the kinetic parameters of the enzyme by selecting a specific GST isoform (GSTm34) that exhibits superior substrate affinity (low Km value) and stability characteristics compared to commercial GST enzymes, thereby improving reliability without increasing manufacturing complexity
2Measurement precision
If existing GST enzymes are used, then basic enzyme kinetics can be studied, but the affinity for substrates such as glutathione is insufficient
Solution Approach 1:
The patent improves measurement precision by utilizing GSTm34's optimized kinetic parameters, specifically its low Km value for glutathione (0.54 mM), which indicates high substrate affinity and enables more precise enzymatic measurements while maintaining high reaction rates through the enzyme's catalytic efficiency
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 TtGSTm34-8xHis enzyme demonstrates high affinity for glutathione with a low K m value of 0.54 mM, enhanced stability, and improved performance in enzyme kinetics and affinity purification, surpassing commercially available GST enzymes.
Implementation Method 1
GSTs catalyze the reaction of substrate glutathione with electrophiles of both endogenous and xenobiotic origins
Implementation Method 2
By conjugating glutathione with toxic electrophilic substrates, GSTs give rise to less reactive and more soluble products
Implementation Method 3
His-tag (Polyhistidine-tag) is another widely used affinity tag. It can be defined as an amino acid motif in proteins that consists of at least six histidine (His) amino acids
Data Source
Figure 1A~1C
Figure 2~3
Figure 4
AI summary
The present invention relates to the recombinant GST enzyme that is produced in the organism Tetrahymena thermophila. More specifically, the present invention relates to TtGSTm34-8xHis enzyme and uses thereof.