Split Intein Affinity Chromatography for Tag-Less Protein Recovery
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Solution Overview
Problem
Existing split intein systems for protein purification are limited by amino acid requirements at the splice junction, slow cleavage kinetics, solubility issues, and lack of scalability, particularly for tag-less protein recovery.
Innovation Solution
Development of N-intein protein variants with mutations at positions 24 and/or 25, enhancing solubility and enabling efficient large-scale affinity purification using a split intein system with broad amino acid tolerance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If the prototypical split intein DNAE from Nostoc punctiforme is used for protein purification, then rapid kinetic properties are achieved, but amino acid dependency at the +2 position severely limits general applicability
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues in the N-intein sequence (positions 24 and/or 25) to alter the chemical properties of the splice junction. This enables the intein to tolerate various amino acids at the +2 position of the C-extein, transforming the system from highly specific to broadly applicable while preserving rapid cleavage kinetics.
Solution Approach 2:
The engineered N-intein variants achieve universality by being able to process multiple different amino acid sequences at the splice junction. The modified intein can now function with any amino acid at the +2 position, making it a universal tool for protein purification rather than being limited to specific substrate sequences.
2Reliability
If split intein systems are used for affinity purification, then tag-less protein recovery is enabled, but solubility issues and lack of scalability limit large-scale application
Solution Approach 1:
The patent improves solubility and scalability by changing the amino acid parameters at positions 24 and/or 25 of the N-intein. These parameter changes enhance the solubility characteristics of the intein fusion protein, enabling efficient large-scale expression and purification while maintaining the tag-less protein recovery capability.
3Quantity of substance
If existing split intein systems are used, then affinity purification is achieved, but amino acid requirements at the splice junction prevent recovery of proteins without extraneous amino acids
Solution Approach 1:
The patent resolves this contradiction by changing the amino acid parameters at positions 24 and/or 25 of the N-intein, which broadens the tolerance for amino acids at the splice junction. This enables the system to efficiently purify proteins without requiring extraneous amino acids, achieving both high purification efficiency and versatility.
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 N-intein variants achieve high solubility and efficient cleavage of proteins from the intein tag, facilitating large-scale, tag-less protein recovery with improved yield and reduced environmental impact.
Implementation Method 1
Inteins are protein elements expressed as in-frame insertions that interrupt enzyme sequences and catalyze their own excision and ligation of two flanking polypeptides
Implementation Method 2
affinity chromatography using a split intein system comprising a C-intein tag and N-intein ligand
Data Source
AI summary
The present invention relates to protein purification, primarily in the chromatographic field. More closely, the invention relates to affinity chromatography using a split intein system comprising a C-intein tag and N-intein ligand, wherein the N-intein ligand provides increased solubility suitable for large scale purification of any recombinant target protein.