Split Intein Affinity Resin for Rapid Tagless Protein Purification
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Solution Overview
Problem
Current protein purification methods using affinity tags face challenges such as inefficient cleavage kinetics, premature cleavage, and ligand leakage during resin reuse, which affect the yield and purity of target proteins, especially in large-scale processes and repeated usage of chromatography resins.
Innovation Solution
Development of an affinity chromatography resin with immobilized N-terminal split intein fragments, which allows for higher ligand densities and multiple reuses even after harsh cleaning procedures, using specific sequences and methods for expression, solubilization, refolding, and covalent attachment to agarose beads, enabling efficient and stable protein purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If intein-based affinity tag systems are used for purification, then tag removal can be achieved without chemical agents or proteases, but cleavage kinetics are slow requiring 16 hours or more of incubation
Solution Approach 1:
The intein is divided into two separate fragments: an N-terminal fragment immobilized on the resin and a C-terminal fragment fused to the target protein. This segmentation enables the cleavage reaction to occur on the resin surface, dramatically improving cleavage kinetics from 16+ hours to minutes or hours, while still achieving complete tag removal without chemical agents or proteases
2Productivity
If affinity tag systems are used for protein purification, then rapid purification can be achieved, but additional steps are required to remove the affinity tag
Solution Approach 1:
The system merges affinity purification and tag removal into a single integrated process. The N-terminal intein fragment on the resin provides affinity binding, while the same resin facilitates self-cleavage of the C-terminal intein fragment from the target protein. This eliminates the need for separate purification and tag removal steps, reducing overall process complexity
3Duration of action of stationary object
If resin is reused after harsh cleaning procedures, then resin durability is improved, but ligand leakage occurs affecting purification quality
Solution Approach 1:
The N-terminal intein fragment is covalently immobilized on the resin surface before use, creating a stable anchor that prevents ligand leakage during harsh cleaning procedures. This preliminary covalent attachment ensures the ligand remains firmly bound to the resin even after repeated exposure to strong cleaning agents, maintaining binding activity and purification quality across multiple reuse cycles
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 resin supports efficient and predictable purification of tag-less target proteins with improved yield and resin durability, reducing the need for additional steps and chemical agents, and maintaining binding activity and self-cleavage functionality for repeated use.
Implementation Method 1
immobilizing said protein as ligands on a chromatography resin
Implementation Method 2
solubilizing said inclusion bodies and releasing expressed protein
Implementation Method 3
refolding said protein
Implementation Method 4
purification of tag-less target proteins
Data Source
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AI summary
The present invention relates to the field of chromatography and more specifically to producing protein affinity chromatography resins comprising affinity ligands based on a N-terminal fragment of a split intein, such as DnaE from Nostoc punctiforme, as well as methods for using the same. The N-terminal fragments are produced in inclusion bodies in bacterial cells.