Engineered T4 DNA Ligase Variants for Faster DNA Fragment Joining
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing T4 DNA ligases have limitations in ligation efficiency, requiring multiple production runs and longer reaction times, which affects the efficiency and cost-effectiveness of molecular biology protocols.
Innovation Solution
Engineering T4 DNA ligase mutants with specific amino acid substitutions, such as E89K, E271K, D340R, D371Q, D371R, E419K, E438K, E440R, E440W, D452R, and K470E, to enhance ligation activity, allowing for higher efficiency at lower concentrations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If wild-type T4 DNA ligase is used, then the enzyme can perform ligation function, but the ligation efficiency is limited requiring multiple production runs and longer reaction times
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the T4 DNA ligase sequence (positions 89, 271, 340, 371, 419, 438, 440, 452, and 470) to create mutant variants with enhanced catalytic activity. These point mutations alter the enzyme's biochemical parameters to achieve faster ligation rates and improved efficiency compared to wild-type ligase
2Productivity
If wild-type T4 DNA ligase is used, then the enzyme can catalyze bond joining, but higher enzyme concentrations and longer incubation times are required
Solution Approach 1:
The patent modifies the enzyme's intrinsic catalytic parameters through amino acid substitutions, enabling the mutant ligases to achieve higher turnover numbers and catalytic efficiency. This allows the reaction to proceed effectively at lower enzyme concentrations, reducing the quantity of substance required while maintaining or improving productivity
3Quantity of substance
If multiple production runs are conducted to achieve sufficient product, then the final product quantity increases, but the manufacturing time and cost increase
Solution Approach 1:
By changing the kinetic parameters of the ligase enzyme through targeted mutations, each production run using mutant ligase achieves higher yield in shorter time. This consolidates the production process, allowing sufficient final product quantity to be obtained in fewer runs, thereby improving overall production efficiency and reducing manufacturing costs
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 engineered mutants exhibit increased ligation activity, reducing the need for enzyme quantity and reaction time, thereby improving the efficiency and cost-effectiveness of molecular biology applications.
Implementation Method 1
Ligases are commonly used in molecular biology for forming phosphodiester bonds between duplex nucleic acid fragments at the intersection of juxtaposed 5′ phosphate and 3′ hydroxyl termini. T4 DNA Ligase is a versatile enzyme that catalyzes the bond joining duplex DNA or RNA
Data Source
AI summary
The invention includes a mutant T4 DNA ligase or a biologically active fragment thereof, which has greater activity than wild type T4 DNA ligase. The mutant T4 DNA ligase, or the biologically active fragment, has one or more substitutions differing from the wild type, as described more fully in the Summary.