Protease Fragmentation for Rapid Protein Structural Analysis
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Solution Overview
Problem
Current protein analysis methods require lengthy pretreatment processes, limiting throughput and efficiency in determining protein higher-order structures and complex formations.
Innovation Solution
Employing a protease with multiple cleavage sites, such as thermolysin, for rapid chemical modification and fragmentation of proteins at elevated temperatures, allowing for quicker analysis of protein structures and complex formations without the need for denaturation or reduction steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional protein analysis methods are used, then detailed structural information can be obtained, but the pretreatment time is excessively long
Solution Approach 1:
The patent changes the parameter of protease selection from conventional specific proteases to broad-specificity proteases (such as proteinase K, pronase, or thermolysin) that can cleave at multiple sites. This parameter change enables simultaneous achievement of complete fragmentation and reduced pretreatment time, resolving the contradiction between structural information quality and pretreatment time
Solution Approach 2:
The patent applies preliminary chemical modification treatment before proteolysis to expose hidden cleavage sites and enhance protease accessibility. This preliminary action ensures that even proteins with complex higher-order structures can be efficiently fragmented by broad-specificity proteases, maintaining measurement precision while reducing overall pretreatment time
2Productivity
If proteins are rapidly fragmented, then throughput increases, but fragmentation completeness may be compromised
Solution Approach 1:
The patent employs broad-specificity proteases that can recognize and cleave at multiple different amino acid sequences (e.g., proteinase K cleaving at Lysine and Arginine residues). This multi-functionality ensures comprehensive fragmentation across diverse protein structures while maintaining rapid processing speed, thus achieving both high throughput and complete fragmentation
Solution Approach 2:
The patent uses proteases with high catalytic efficiency and broad substrate specificity that can continuously cleave peptide bonds at multiple sites throughout the protein molecule. This continuous action ensures that even complex proteins are completely fragmented into analyzable peptides within a short time frame, maintaining both productivity and fragmentation completeness
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
This approach significantly reduces pretreatment time, enabling rapid determination of protein stability and identification of interacting substances, including potential pharmaceuticals, while minimizing false positives in screening processes.
Implementation Method 1
fragmenting a protein to be analyzed
Implementation Method 2
by using a protease having many types of cleavage sites
Implementation Method 3
it is preferable to use a heat-resistant enzyme as the protease, and in that case, pretreatment can be performed at a high temperature
Data Source
AI summary
Provided is a pretreatment method for structural analysis of a protein, including chemical modifying and fragmenting a protein to be analyzed, in which in the fragmenting, the protein is treated at a temperature of 60 to 99° C. in the presence of a heat-resistant protease that has four or more cleavage sites and is soluble or immobilized on a carrier.