Transamidase Ligation Efficiency via Modified Recognition Sequence
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Solution Overview
Problem
Existing methods of transamidase ligation face efficiency issues due to competition from peptide fragments released during the reaction, which hinder the formation of amide bonds between acyl donor and nucleophilic acyl acceptor compounds.
Innovation Solution
The use of a modified transamidase recognition sequence with a moiety of poor nucleophilicity, such as an ester group, to enhance the efficiency of the ligation reaction by reducing competition from by-products.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If a standard transamidase recognition sequence (e.g., LPXTG) is used as the acyl donor, then the ligation reaction can proceed, but the released peptide fragment (containing N-terminal glycine) competes with the protein nucleophile for the acyl-enzyme intermediate, reducing ligation efficiency
Solution Approach 1:
The patent modifies the C-terminal residue of the transamidase recognition sequence from an amino acid (in standard sequences like LPXTG) to a non-amino acid moiety with poor nucleophilicity (such as an ester group). This parameter change prevents the released fragment from competing for the acyl-enzyme intermediate, thereby improving ligation efficiency without affecting the enzyme's ability to process the acyl donor substrate.
2Productivity
If the C-terminal residue of the transamidase recognition sequence is changed to enhance ligation efficiency, then competition from by-products is reduced, but the recognition sequence must still be recognized by the transamidase enzyme
Solution Approach 1:
The patent applies local quality by modifying only the C-terminal residue of the transamidase recognition sequence while keeping the rest of the recognition motif (e.g., LPXT) intact. This localized modification ensures that the transamidase enzyme still recognizes and processes the acyl donor substrate effectively, while the modified C-terminal residue prevents harmful competition from released fragments.
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 allows for more efficient ligation of acyl donor and nucleophilic acyl acceptor compounds, improving the yield and effectiveness of the transamidase-mediated reaction.
Implementation Method 1
The invention provides novel reagents and methods for ligating an acyl donor compound with a nucleophilic acyl acceptor compound to form an amide bond using a transamidase
Data Source
AI summary
The present invention relates to methods for ligation. The invention provides novel reagents and methods for ligating an acyl donor compound with an acyl acceptor compound. Provided acyl donor compounds comprise a transamidase recognition sequence that allows ligation with a nucleophilic acyl acceptor in the presence of transamidase. The invention further provides kits comprising acyl donor compounds and optionally comprising other reagents for ligation.