Recombinant Polypeptide Ester Bond Ligation
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Solution Overview
Problem
Current protein ligation technologies rely on non-permanent interactions, limiting the complexity and flexibility of protein complexes, and there is a need for new methods that enable the creation of complex protein structures using reversible covalent bonds.
Innovation Solution
Development of recombinant polypeptides with immunoglobulin-like domains split into truncated proteins and peptide tags capable of forming spontaneously-formed, reversibly hydrolysable ester bonds, allowing for the assembly of complex protein structures through peptide tag and binding partner pairs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If non-covalent interactions (ionic bonds, hydrogen bonds, hydrophobic bonds, van der Waals forces) are used for protein ligation, then the protein complexes can be formed, but the stability and permanence of the protein structures are limited
Solution Approach 1:
The patent changes the chemical nature of the bonding parameter from non-covalent to covalent, specifically introducing isopeptide bonds between lysine and asparagine/aspartate residues. This parameter change enables both stable covalent attachment and reversible formation under physiological conditions, resolving the contradiction between stability and flexibility.
Solution Approach 2:
The patent introduces dynamic reversibility to the covalent isopeptide bond system. The bonds can form spontaneously under physiological conditions and be reversibly cleaved by specific proteases, allowing the protein complexes to be assembled and disassembled dynamically. This enables flexible reconfiguration of protein structures while maintaining stability during assembly.
2Reliability
If isopeptide bonds are used for protein ligation, then spontaneous formation and protease resistance are achieved, but the irreversibility limits the complexity and flexibility of protein complexes
Solution Approach 1:
The patent modifies the reversibility parameter of the isopeptide bond system by introducing specific protease cleavage sites. This allows the bonds to remain stable and protease-resistant under physiological conditions while being reversibly cleavable by specific proteases, enabling dynamic reconfiguration of protein complexes for increased complexity and flexibility.
3Adaptability or versatility
If only two known isopeptide systems are available, then the current protein ligation technologies are limited, but new protein ligation technologies are needed to overcome these limitations
Solution Approach 1:
The patent describes a universal isopeptide bond formation mechanism that can be applied across multiple protein systems. The method uses a general approach of introducing lysine and asparagine/aspartate residues at specific positions, which can be universally applied to create various protein complexes, fusion proteins, and multimeric structures, thereby increasing technological diversity without proportionally increasing 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
Enables the controlled assembly of complex protein structures with increased stability and flexibility, allowing for the formation of multimeric protein complexes with specific binding properties and reversible self-polymerization.
Implementation Method 1
capable of binding to each other by forming a spontaneously-formed ester bond
Implementation Method 2
reversibly hydrolysable ester bonds
Data Source
AI summary
The invention relates to protein ligation technologies, purified or recombinant peptides, methods for making peptides and proteins with covalent bonds including reversible covalent bonds such as reversible intermolecular covalent bonds, and uses thereof. In particular, this invention relates to intermolecular ester bonds, particularly reversible ester bonds between the hydroxyl and amide groups of amino acid side chains present in recombinant chimeric peptides and proteins and the use of such peptides and proteins in protein engineering, for example in the preparation of multimeric protein complexes, including functionalised multimeric protein complexes.