Split Intein Fragments for Rapid Protein Splicing Kinetics
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Solution Overview
Problem
Current split inteins used in protein splicing and purification are limited by slow kinetics and context-dependent efficiency, which restricts their practical utility in various applications.
Innovation Solution
Development of split intein N- and C-fragments and their variants, which can form covalent or non-covalent interactions, allowing for efficient protein purification and modification by binding to supports and interacting with nucleophiles to form conjugates.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional split inteins are used for protein splicing, then the splicing process can occur, but the kinetics are slow and efficiency is limited
Solution Approach 1:
The intein is divided into N-terminal and C-terminal fragments that can function independently. The N-fragment contains the nucleophile and the C-fragment contains the electrophile, allowing them to interact with substrate proteins in a stepwise manner that accelerates the splicing reaction compared to intact inteins
Solution Approach 2:
The split intein fragments act as intermediaries in the splicing process. The N-fragment first forms a thioester intermediate with the substrate protein, which then reacts with the C-fragment to complete the splicing reaction, providing a more efficient pathway than conventional inteins
2Productivity
If conventional split inteins are used, then protein splicing can occur, but the efficiency is context-dependent on flanking extein sequences
Solution Approach 1:
The split intein design places specific functional residues at defined positions within the N- and C-fragments. The N-fragment contains a nucleophilic cysteine at a specific position, and the C-fragment contains an electrophilic serine or threonine, creating localized reactive sites that reduce dependence on the surrounding extein sequence context
Solution Approach 2:
The patent modifies the chemical parameters of the intein fragments by introducing specific amino acid substitutions that enhance nucleophilicity and electrophilicity. These parameter changes make the splicing reaction more robust across different sequence contexts by optimizing the chemical reactivity of the fragments themselves rather than relying on favorable extein sequences
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 split intein fragments enable rapid and efficient protein splicing and purification, improving the kinetics and tolerance of sequence variations, thus enhancing the utility of split inteins in protein chemistry applications.
Implementation Method 1
The complex of the fusion protein and C-fragment or variant thereof can be via a covalent interaction between the fusion protein and C-fragment or variant
Implementation Method 2
via a non-covalent interaction (e.g., ionic, H-bonding, and/or van der Waals interaction)
Implementation Method 3
contacting the intein intermediate with a nucleophile to form a conjugate of the protein and the nucleophile
Data Source
AI summary
Disclosed herein are split inteins, fused proteins of split inteins, and methods of using split inteins to efficiently purify and modify proteins of interest.