Truncated Asx-Specific Protein Ligases Without Acid Activation
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Solution Overview
Problem
Existing protein ligases, such as PALs and AEPs, require acid-activation, leading to heterogeneous enzyme populations due to multiple activation sites, complicating manufacturing and quality control.
Innovation Solution
Development of a truncated polypeptide with a retained α6-helix of the cap domain, expressed and purified in a constitutively active form, eliminating the need for acid-activation and ensuring stability and activity comparable to acid-activated counterparts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If acid-activation is used to activate protein ligases, then enzymatic activity is achieved, but heterogeneous enzyme populations are produced due to multiple activation sites
Solution Approach 1:
The patent extracts and removes the cap domain from the proenzyme structure, eliminating the multiple activation sites that cause heterogeneity. By taking out the problematic cap domain containing multiple cleavage sites, the invention produces a homogeneous enzyme population while maintaining catalytic activity through the retained core domain and α6-helix.
Solution Approach 2:
The patent segments the proenzyme into distinct functional domains: the core domain containing the catalytic triad and the α6-helix, separated from the cap domain. This segmentation allows the active enzyme to be expressed independently without the heterogeneity-inducing cap domain, while preserving essential catalytic functions.
2Ease of manufacture
If truncated polypeptide is used to eliminate acid-activation requirement, then manufacturing complexity is reduced, but enzyme stability may be compromised
Solution Approach 1:
The patent changes the structural parameters of the enzyme by truncating the cap domain and retaining specifically the α6-helix region. This parameter change eliminates the need for acid-activation steps while maintaining stability through the preserved α6-helix structure that is critical for enzymatic function and structural integrity.
Solution Approach 2:
The patent performs preliminary structural modification by expressing the enzyme directly in its truncated, constitutively active form without requiring subsequent acid-activation treatment. This preliminary action of expressing the mature enzyme structure directly simplifies manufacturing while ensuring stability is built into the protein structure from expression.
3Stability of the object's composition
If full-length proenzyme is expressed, then complete enzyme structure is obtained, but additional purification and activation steps are required
Solution Approach 1:
The patent extracts only the essential functional components (core domain and α6-helix) while removing the non-essential cap domain that requires activation. This extraction approach obtains complete enzymatic functionality without the structural elements that complicate the production process.
Solution Approach 2:
Instead of expressing the full-length proenzyme and then activating it through acid treatment, the patent inverts the approach by directly expressing the truncated constitutively active form. This reversal eliminates the activation step and reduces process complexity while maintaining enzyme 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
The truncated polypeptide maintains high catalytic activity and stability, facilitating cost-effective large-scale production and industrial applications like antibody-drug conjugate manufacturing.
Implementation Method 1
polypeptides having Asx-specific protein ligase and cyclase activity... PALs catalyse peptide bond formation
Data Source
AI summary
Various embodiments relate generally to the field of enzyme technology and specifically relate to polypeptides having Asx-specific protein ligase and cyclase activity and to nucleic acids encoding those as well as methods of the manufacture of said enzymes, more particularly methods of producing stable and constitutively active protein ligases.