EBCatcher Peptide Mutant for High-Efficiency Ester Bond Formation
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Solution Overview
Problem
The low covalent bond formation efficiency of molecular peptide pairs like Catcher and Tag in acidic environments limits their application in protein separation and other fields, as they require specific pH adjustments and additional components like glycerol and CaCl2 for ester bond formation.
Innovation Solution
A molecular peptide mutant, EBCatcher, is designed with three specific mutations (F31Y, F94I, and Q97E) to enhance covalent bond formation efficiency with EBTag, allowing for improved stability and binding efficiency without altering the spatial structure, and a method for purifying this mutant using recombinant plasmids and Ni-NTA resin is introduced.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the original Catcher and Tag molecular peptide pair is used in acidic environment, then ester bond formation can occur with glycerol and CaCl2 addition, but the covalent bond formation efficiency is low
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the Catcher protein through three specific mutations (F31Y, F94I, Q97E). These mutations change the chemical parameters of the protein, enabling high-efficiency ester bond formation without requiring glycerol and CaCl2 additives, thus improving covalent bond formation efficiency while reducing system complexity
Solution Approach 2:
The mutated Catcher protein (EBCatcher) achieves self-service capability by inherently possessing the ability to form stable ester bonds with EBTag in acidic environment without external assistance from glycerol or CaCl2. The protein mutations enable the system to self-catalyze the bond formation process, eliminating the need for additional chemical components
2Reliability
If pH is adjusted to 8.0 for ester bond formation, then covalent bond can form, but the ester bond can be hydrolyzed after glycerol and Ca2+ are dialyzed out
Solution Approach 1:
The patent changes the operational parameters by enabling ester bond formation to occur efficiently at acidic pH (pH 6.0) rather than requiring pH 8.0 adjustment. The EBCatcher mutant forms stable covalent bonds that remain intact after dialysis, eliminating the need for pH adjustment and subsequent rebinding steps, thus improving ease of operation while maintaining bond stability
Solution Approach 2:
The protein mutations in EBCatcher provide beforehand cushioning by pre-configuring the active site structure to stabilize the ester bond against hydrolysis. The mutations create a protective microenvironment that prevents bond cleavage during dialysis, ensuring the covalent link remains intact without requiring additional stabilizing agents or complex operational protocols
3Productivity
If three mutations (F31Y, F94I, Q97E) are introduced to Catcher, then covalent bond formation efficiency is significantly improved, but protein structure modification is required
Solution Approach 1:
The patent applies parameter changes through site-directed mutagenesis of three specific amino acid positions in the Catcher protein. These targeted sequence modifications (F31Y, F94I, Q97E) enhance the catalytic efficiency and binding affinity, resulting in significantly improved bond formation efficiency while maintaining compatibility with standard bacterial expression systems
Solution Approach 2:
The patent uses copying by creating a mutated version of the Catcher protein through genetic engineering. The mutant sequence is copied into the bacterial expression system, allowing production of the modified protein using standard recombinant protein techniques. This approach maintains ease of manufacture by utilizing established molecular biology workflows despite the sequence modifications
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 EBCatcher mutant significantly increases covalent bond formation efficiency with EBTag, enabling more effective protein separation and purification processes, as demonstrated by increased ligation efficiencies and stability in aqueous solutions.
Implementation Method 1
Thr11 of the Catcher and Gln14 of the Tag can form a covalent bond (ester bond)
Implementation Method 2
carrying out purification dialysis to obtain purified protein
Data Source
AI summary
The invention relates to a molecular peptide mutant with a high ester bond formation efficiency, the amino acid sequence of which is as shown in SEQ ID NO: 1. The invention introduces mutations at three sites on the basis of not affecting the spatial structure of Catcher, and through three mutations, obtains the Catcher mutant EBCatcher, which significantly improves the stability of the Catcher in water solution and can improve the binding efficiency to EBTag.