Fucose-Binding Protein Variants for Stable High-Affinity Detection
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Solution Overview
Problem
Existing fucose-binding proteins like BC2LCN suffer from low binding affinity and thermal stability, and low productivity in expression systems such as Escherichia coli, limiting their industrial application and reliability.
Innovation Solution
Modifying the amino acid sequence of BC2LCN by deleting the C-terminal region and introducing specific amino acid substitutions at positions 39, 65, 72, and 81 to enhance binding affinity and thermal stability, while improving productivity in Escherichia coli expression systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If BC2LCN is used for detection of undifferentiated cells, then binding affinity to sugar chains is achieved, but binding strength is affected by external environment such as solvent and salt concentration
Solution Approach 1:
The patent applies parameter changes by substituting specific amino acid residues at positions 39, 65, 72, and 81 to modify the protein's binding characteristics. These substitutions change the chemical parameters of the binding interface, enabling the protein to maintain stable binding affinity across different environmental conditions including varying salt concentrations and solvent types.
2Reliability
If amino acid substitution is introduced to improve function, then binding affinity or stability may be improved, but productivity in Escherichia coli expression is reduced
Solution Approach 1:
The patent optimizes amino acid substitutions to achieve a balance between improved binding affinity and maintained productivity. By carefully selecting substitutions at positions 39, 65, 72, and 81, the patent enhances functional properties while minimizing negative impacts on expression yield in E. coli systems.
3Reliability
If BC2LCN is used for industrial application, then detection capability is achieved, but productivity and thermal stability are insufficient
Solution Approach 1:
The patent introduces amino acid substitutions that simultaneously improve multiple parameters including thermal stability and productivity. The substitutions at positions 39, 65, 72, and 81 are designed to enhance the protein's overall stability and expression characteristics, making it suitable for industrial-scale production while maintaining detection capability.
4Reliability
If BC2LCN is used for detection, then binding to undifferentiated cells is achieved, but thermal stability is low
Solution Approach 1:
The patent applies amino acid substitutions at positions 39, 65, 72, and 81 to enhance the thermal stability of the protein structure. These substitutions strengthen intramolecular interactions and improve the rigidity of the binding domain, enabling the protein to maintain its structural integrity and binding function at elevated temperatures, thus facilitating industrial applications.
Data Source
AI summary
The present invention aims to provide a fucose-binding protein that shows improved productivity in cases of expression in a host such as Escherichia coli, improved binding affinity to a fucose-containing sugar chain such as a sugar chain containing a structure composed of Fucα1-2Galβ1-3GlcNAc and/or Fucα1-2Galβ1-3GalNAc, and/or improved thermal stability. The above object is achieved by deleting a plurality of amino acid residues in the C-terminal side of the amino acid sequence of the fucose-binding protein BC2LCN of SEQ ID NO: 1, and, when necessary, substituting the glycine residue at position 36 in SEQ ID NO: 1 with a cysteine residue, substituting the glutamine residue at position 39 in SEQ ID NO: 1 with a leucine residue or methionine residue, substituting the glutamine residue at position 65 in SEQ ID NO: 1 with a leucine residue, substituting the cysteine residue at position 72 in SEQ ID NO: 1 with a glycine residue or alanine residue, substituting the glutamic acid residue at position 81 in SEQ ID NO: 1 with a cysteine residue, glutamine residue, histidine residue, or methionine residue, and/or substituting the glycine residue identified as the residue at position 36 in SEQ ID NO: 1 with a cysteine residue.