Sortase-Mediated Protein Ligation for Chimeric Antibody Assembly
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Solution Overview
Problem
Conventional protein ligation technologies, such as native chemical ligation and recombinant technologies, face difficulties in efficiently preparing synthetic peptide esters and expressing correctly folded fusion proteins with inteins, limiting the application of protein ligation methods.
Innovation Solution
The use of sortase-mediated transacylation reactions to install reactive chemical groups, specifically click chemistry handles, on proteins, allowing for the conjugation of proteins at their C-terminus or N-terminus, enabling the formation of chimeric proteins and protein dimers through covalent bonds.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If native chemical ligation is used to join proteins, then protein-protein fusions can be generated, but efficient preparation of synthetic peptide esters is technically difficult
Solution Approach 1:
The patent introduces sortase as an enzymatic intermediary that mediates protein ligation through a recognition motif (LPXTG) rather than requiring direct chemical reaction between protein termini. The sortase enzyme catalyzes the transpeptidation reaction between the C-terminal motif of one protein and the N-terminal glycine of another, eliminating the need for complex synthetic peptide ester preparation while maintaining ligation efficiency
Solution Approach 2:
The patent replaces the mechanical/chemical approach of native chemical ligation (requiring synthetic peptide esters and specific reaction conditions) with a biological enzymatic system (sortase-mediated ligation). This substitution simplifies the manufacturing process by using a readily available enzyme and peptide motif system instead of technically difficult synthetic chemistry
2Productivity
If intein-based protein ligation is used, then proteins can be joined, but the target protein must be expressed as a correctly folded fusion with the intein which is often challenging
Solution Approach 1:
The patent segments the ligation system into two independent components: a small C-terminal recognition motif (LPXTG, 5 amino acids) that can be easily added to any target protein without requiring full intein fusion expression, and the sortase enzyme that performs the ligation. This segmentation eliminates the need to express challenging intein fusion proteins while maintaining ligation capability
Solution Approach 2:
The patent extracts only the essential C-terminal recognition motif (LPXTG) from the intein system, separating it from the complex intein protein structure. This extracted motif can be easily incorporated into target proteins without the burden of expressing large intein fusion proteins, while still enabling efficient sortase-mediated ligation
3Adaptability or versatility
If conventional sortase modifications are used, then proteins can be derivatized with modifications, but the modification is limited to C-terminal conjugation
Solution Approach 1:
The patent creates a universal sortase modification system where the same sortase enzyme and recognition motif can be used for multiple purposes: C-terminal conjugation (traditional), N-terminal conjugation (by adding motif to N-terminus), and site-specific labeling anywhere in the protein sequence (by inserting motif at desired location). This multi-functional approach greatly enhances adaptability without increasing system complexity
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
Facilitates the generation of chimeric proteins and protein dimers that cannot be achieved by conventional means, offering flexibility in protein engineering by allowing post-translational modification and conjugation of proteins under physiological conditions.
Implementation Method 1
these artificial sortase substrates undergo a transacylation reaction resulting in the exchange of residues C-terminal to the threonine residue with the synthetic oligoglycine peptide, resulting in the protein C-terminus being ligated to the N-terminus of the synthetic peptide
Data Source
AI summary
In some aspects, polypeptides comprising single domain antibodies and methods of identifying single domain antibodies are provided. In some embodiments polypeptides comprising a single domain antibody and a sortase recognition sequence, are provided. In some aspects, products and methods of use in modulating the immune system, e.g., modulating an immune response, are provided.