Truncated Bacteriophage Adhesion Proteins for Stable Gram-Negative Detection
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Solution Overview
Problem
Existing bacteriophage-based assays for bacterial detection face challenges due to reversible binding of bacteriophage adhesion proteins caused by their hydrolytic activity, leading to decreased sensitivity and stability issues, particularly with large protein sizes and modular domain arrangements that are prone to proteolytic sensitivity.
Innovation Solution
Development of bacteriophage adhesion proteins that are truncated to lack the bacteriophage binding domain and mutated to reduce hydrolytic activity, resulting in improved expression, purification, and stability, with enhanced binding specificity and reduced aggregation, allowing for more efficient capture and detection of gram-negative bacteria.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Force
If bacteriophage adhesion proteins are used for bacterial detection, then binding affinity to bacterial receptors is improved, but reversible binding caused by hydrolytic activity decreases sensitivity and stability
Solution Approach 1:
The patent extracts and removes the hydrolytic enzymatic domain from the bacteriophage adhesion protein, retaining only the O-antigen binding domain. This creates a truncated protein that maintains high binding affinity to bacterial O-antigens while eliminating the harmful hydrolytic activity that caused reversible binding and reduced detection sensitivity. The extracted binding domain can be used as a stable reagent for bacterial detection without the destabilizing enzymatic function.
2Adaptability or versatility
If full-length bacteriophage adhesion proteins are used, then binding function is maintained, but large protein size and modular domain arrangement cause proteolytic sensitivity and stability issues
Solution Approach 1:
The patent extracts only the essential O-antigen binding domain from the full-length adhesion protein, removing the vulnerable modular domains that are susceptible to proteolytic cleavage. This truncated version maintains the critical binding function while eliminating the structural vulnerabilities that led to proteolytic sensitivity and instability in the complete protein.
Solution Approach 2:
The patent segments the bacteriophage adhesion protein into functional domains, identifying and isolating the O-antigen binding domain from the hydrolytic enzymatic domain and other modular regions. By separating the essential binding function from the problematic domains, the invention creates a stable, proteolytically resistant fragment that retains the desired binding activity.
3Productivity
If bacteriophage adhesion proteins with hydrolytic activity are used, then polysaccharide penetration during infection occurs, but reversible binding and release decreases assay sensitivity
Solution Approach 1:
The patent removes the hydrolytic enzymatic function from the adhesion protein while preserving the O-antigen binding capability. This creates a non-hydrolytic binding protein that provides stable, irreversible binding to bacterial surfaces, eliminating the reversible binding and release cycle that undermined assay sensitivity. The binding function is retained for detection purposes without the destabilizing hydrolytic activity.
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 modified bacteriophage adhesion proteins exhibit improved expression rates, higher purification grades, and longer storage stability, with enhanced binding specificity and reduced hydrolytic activity, leading to more effective bacterial detection and capture, particularly for gram-negative bacteria.
Implementation Method 1
bacteriophage adhesion proteins binding to the O-antigen of gram negative bacteria
Implementation Method 2
lacking the ability of binding to a bacteriophage and of hydrolysing lipopolysaccharides
Data Source
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AI summary
The present invention relates to bacteriophage adhesion proteins binding to the O-antigen of gram negative bacteria, lacking the ability of binding to a bacteriophage and of hydrolysing lipopolysacchaiides. The invention further relates to nucleic acid molecules comprising a sequence encoding the proteins according to the present invention. In addition, the present invention relates to a method for generating bacteriophage adhesion proteins according to the present invention. The invention further relates to the use of said proteins and methods of detection, purification and enrichment of bacteria.