Tencon Fibronectin Scaffold Thermal Stability
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Solution Overview
Problem
Current protein scaffolds lack sufficient thermal and chemical stability, which limits their effectiveness in maintaining functional activity and stability under physiological conditions, particularly in high osmotic strength and high concentration environments.
Innovation Solution
A protein scaffold based on a consensus sequence of multiple fibronectin type III (FN3) domains from human Tenascin, with specific substitutions such as N46V, E14P, and E86I, enhancing thermal stability by up to 11°C and shifting the mid-point of GdmCl-induced denaturation from 3.4 M to greater than 5 M, allowing for improved purification, formulation, and shelf-life, and enabling the formation of binding domains selective for cellular targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If conventional protein scaffolds are used, then they can bind to target molecules, but they lack sufficient thermal and chemical stability under physiological conditions
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (N46V, E14P, E86I) into the FN3 domain sequence to enhance thermal stability by up to 11°C and increase resistance to chemical denaturation (shifting GdmCl mid-point from 3.4 M to greater than 5 M), while preserving the protein's binding function
Solution Approach 2:
The patent applies local quality by making targeted substitutions at specific positions within the FN3 domain structure rather than globally modifying the entire protein, allowing stability enhancement at critical locations while maintaining overall functional integrity
2Quantity of substance
If protein concentration is increased to improve activity, then binding affinity increases, but solubility and stability decrease due to aggregation
Solution Approach 1:
The stabilized FN3 domain exhibits improved solubility at high concentrations due to enhanced stability against aggregation, allowing the protein to maintain functional activity at higher concentrations without precipitating or forming aggregates
3Manufacturing precision
If purification is performed under stringent conditions to remove impurities, then purity increases, but protein stability and activity decrease
Solution Approach 1:
The enhanced stability of the modified FN3 domain allows it to withstand more stringent purification conditions, enabling higher purity to be achieved without compromising protein stability or functional activity
Data Source
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AI summary
A protein scaffold based on a consensus sequence of fibronectin type III (FN3) proteins, such as the tenth FN3 repeat from human fibronectin (human Tenascin), including isolated nucleic acids that encode a protein scaffold, vectors, host cells, and methods of making and using thereof. The protein scaffold molecules of the present invention exhibit enhanced thermal and chemical stability while presenting six modifiable loop domains which can be engineered to form a binding partner capable of binding to a target for applications in diagnostic and/or therapeutic compositions, methods and devices.