Intein-Mediated Cyclization of Synthetic Polymers
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Solution Overview
Problem
Current methods for producing cyclic peptides are inefficient, particularly for smaller peptides, and often require sophisticated purification procedures, limiting their production and use in clinical studies due to sterical hindrance and the inability to incorporate non-genetically encodable amino acids.
Innovation Solution
The method involves ligating first and second recombinant intein domains to a linear synthetic polymer to form a compound that autocatalytically cyclizes, allowing for the production of cyclic polymers in a cell-free system, enabling the use of non-naturally occurring amino acids and high-throughput screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If linear peptides are cyclized in vitro by reacting the N- and C-termini together, then cyclic peptides are produced, but the reaction is inefficient due to steric hindrance at the peptide ends
Solution Approach 1:
The patent introduces an intein-mediated protein splicing system as an intermediary mechanism. Instead of directly reacting the N- and C-termini of the peptide, the intein domains facilitate the cyclization process through a multi-step mechanism involving nucleophilic attack and intermediate formation, thereby overcoming the steric hindrance that prevents direct terminal reaction
Solution Approach 2:
The patent divides the cyclization process into separate functional domains: an N-terminal intein domain and a C-terminal intein domain. These segmented domains can independently interact with the peptide termini and facilitate cyclization through their respective catalytic activities, reducing the steric conflict that would occur in a direct terminal reaction
2Productivity
If cyclic peptides are produced by cyclizing linear peptides, then cyclic structures are formed, but purification from linear peptides is difficult requiring sophisticated procedures
Solution Approach 1:
The patent extracts the intein domains as separate functional entities that can be selectively removed or retained after cyclization. The intein domains serve as purification tags or handles that enable selective extraction of cyclic peptides from the reaction mixture, simplifying the purification process compared to direct cyclization methods
3Adaptability or versatility
If peptides are synthesized in vivo using inteins, then cyclic peptides are produced, but only genetically-encodable amino acids are incorporated limiting structural diversity
Solution Approach 1:
The patent creates a universal intein-mediated cyclization system that can process both naturally occurring and non-naturally occurring amino acids. The intein domains are designed to recognize and catalyze cyclization of peptide bonds regardless of the specific amino acid composition, enabling the incorporation of non-genetically encodable amino acids while maintaining cyclic peptide production capability
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 enables the efficient production of cyclic peptides with structural diversity, suitable for clinical investigations and therapeutic applications, overcoming the limitations of existing methods by facilitating the incorporation of non-genetically encodable amino acids and simplifying purification.
Implementation Method 1
D1-X(n)-D2 compounds autocatalytically cyclize the X(n) polymer to produce a cyclic polymer
Data Source
AI summary
The invention provides methods and compositions for production of a cyclic polymer in a cell free system. In general, the methods of the invention involve ligating first and second recombinant intein domains to a linear synthetic polymer to form a compound containing the structure: D1-X(n)-D2, where D1 is a first catalytic domain of an intein; D2 is a second catalytic domain of an intein; where the second catalytic domain has at its N-terminus a first reactive site for the intein; and X(n) is a polymer of a number n of monomer X, where the polymer N-terminus has a second reactive site for the intein. D1-X(n)-D2 compounds autocatalytically cyclize the X(n) polymer to produce a cyclic polymer. The invention finds use in a variety of drug discovery, clinical and therapeutic applications.