SpyDock Polypeptide for Reversible SpyTag Affinity Purification
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Solution Overview
Problem
Existing affinity tags, such as His-tag and C-tag, pose challenges by inhibiting protein solubility, structure, and function, and require additional purification steps that reduce yield and introduce immunogenicity, while isopeptide bond-forming peptide tag systems necessitate separate purification tags.
Innovation Solution
A modified SpyCatcher polypeptide (SpyDock) with specific mutations allows SpyTag to function as both a purification and conjugation tag, enabling high-affinity, reversible non-covalent binding for efficient purification without additional tags, and can be immobilized on a solid substrate for reuse.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If traditional affinity tags (His-tag, C-tag) are used for purification, then purification can be achieved, but the tags inhibit protein solubility, structure, and function, and require additional purification steps that reduce yield and introduce immunogenicity
Solution Approach 1:
The invention divides the purification tag system into two separate functional components: a small peptide tag (SpyTag) for affinity recognition and a larger binding partner (SpyCatcher) that provides the purification function. This segmentation allows the peptide tag to be minimal and non-interfering, while the binding partner handles the purification task, thereby resolving the contradiction between purification capability and protein functionality.
Solution Approach 2:
The invention extracts the essential purification function from traditional large affinity tags and relocates it to a separate binding partner protein (SpyCatcher). The original peptide tag (SpyTag) is retained only for its recognition capability, removing the harmful effects of large tags on protein solubility and function while maintaining purification efficiency.
2Reliability
If isopeptide bond-forming peptide tag systems (SpyTag/SpyCatcher) are used for conjugation, then stable covalent binding is achieved, but separate purification tags are still necessary
Solution Approach 1:
The invention makes the peptide tag (SpyTag) universal by enabling it to serve dual functions: affinity purification through reversible non-covalent binding to SpyDock, and stable conjugation through isopeptide bond formation with SpyCatcher. This multi-functionality eliminates the need for separate purification and conjugation tags, resolving the contradiction between conjugation reliability and system complexity.
Solution Approach 2:
The invention merges the purification and conjugation functions into a single peptide tag system. By designing SpyTag to bind reversibly to SpyDock for purification and irreversibly to SpyCatcher for conjugation, the system combines what were previously separate functions into one unified approach, thereby reducing the number of components needed.
3Ease of manufacture
If affinity tags are used for purification, then purification is achieved, but the tags produce unhelpful immune response in vivo
Solution Approach 1:
The invention uses a minimal peptide tag (SpyTag) that can be easily removed or degraded after serving its purification purpose. The small size of the peptide tag makes it less immunogenic compared to large protein tags, and it can be cleaved off after purification, eliminating the source of immunogenicity while maintaining purification efficiency.
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 SpyDock system achieves high-purity purification of SpyTag-fusions, maintaining stability under various conditions and allowing multiple uses without significant loss of activity, thus avoiding the need for additional purification steps and immunogenicity.
Implementation Method 1
capable of forming a stable and reversible non-covalent complex (i.e. a polypeptide: ligand complex) that can be dissociated under appropriate conditions
Implementation Method 2
Proteins that are capable of spontaneous isopeptide bond formation have been used to develop peptide tag/binding partner pairs which covalently bind to each other and provide irreversible interactions
Data Source
AI summary
The present invention relates to an affinity purification system that utilises a polypeptide comprising:(i) an amino acid sequence as set forth in SEQ ID NO: 1, wherein X at position 79 is selected from alanine, glycine, serine, asparagine, or threonine;(ii) a portion of (i) comprising an amino acid sequence as set forth in SEQ ID NO: 2, wherein X at position 56 is selected from alanine, glycine, serine, asparagine or threonine;(iii) an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 1, wherein the polypeptide comprises alanine, glycine, serine, asparagine or threonine at a position equivalent to position 79 of SEQ ID NO: 1; or(iv) a portion of (iii) comprising an amino acid sequence with at least 80% sequence identity to a sequence as set forth in SEQ ID NO: 2, wherein the polypeptide comprises alanine, glycine, serine, asparagine or threonine at a position equivalent to position 56 of SEQ ID NO: 2,wherein the polypeptide binds selectively and reversibly to a peptide comprising an amino acid sequence as set forth in SEQ ID NO: 3, 4 or 5.