Engineered ClyO Lysin for Broad-Spectrum Staphylococcus Killing
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Solution Overview
Problem
Current lysins are not effectively solubly expressed or have high activity, making them unsuitable for broad-spectrum killing of Staphylococcus aureus, especially methicillin-resistant strains, and are not adaptable to protein-rich environments or wide pH ranges, limiting their application in treating and detecting staphylococcal infections.
Innovation Solution
Development of a novel staphylococcal lysin, ClyO, with high activity and broad-spectrum killing capabilities, expressed and purified using the E. coli expression system, capable of functioning across a wide pH range and in protein-rich environments, and used for both therapeutic and diagnostic purposes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If normal egg white lysozyme is used, then it is inexpensive and readily available, but it has no obvious lytic effect on staphylococcal cell wall due to the tough cell wall structure
Solution Approach 1:
The patent modifies the amino acid sequence of traditional lysozyme to create engineered variants (such as LysK, Ply187, and their chimeras) with altered catalytic properties. These parameter changes in the protein structure enable effective hydrolysis of staphylococcal peptidoglycan while maintaining solubility and activity across broader pH ranges (pH 5-9), directly resolving the contradiction between availability and lytic effectiveness.
Solution Approach 2:
The patent creates chimeric lysins by combining the N-terminal catalytic domain of phage lysins (which provide staphylococcal cell wall binding capability) with the C-terminal domain of LysK (which provides catalytic activity). This composite structure integrates the advantages of both parent proteins, achieving high lytic activity against MRSA while maintaining solubility and stability in protein-rich environments like milk.
2Reliability
If lysostaphin is used, then it has good lytic activity against staphylococcal cell wall, but it is too expensive to be widely applied
Solution Approach 1:
The patent employs genetically engineered lysins that can be produced recombinantly in E. coli expression systems, replacing the need for expensive lysostaphin extraction from Staphylococcus lugdunensis. These engineered proteins (LysK, Ply187, and their chimeras) achieve comparable or superior lytic activity at a fraction of the cost, making them suitable for widespread clinical and food safety applications.
Solution Approach 2:
The patent substitutes the natural extraction process of lysostaphin (which requires complex purification from bacterial sources) with recombinant protein expression systems. This mechanical/biological substitution enables scalable, cost-effective production of lysins with controlled amino acid sequences, directly addressing the cost barrier while maintaining high lytic activity.
3Reliability
If existing natural lysins and chimeric lysins are used, then they can kill Staphylococcus aureus in vivo and in vitro, but they are difficult to be solubly expressed or have low activity and cannot adapt to protein-rich environment
Solution Approach 1:
The patent systematically modifies the amino acid sequences of lysins to improve their biophysical properties. Specific mutations are introduced to enhance solubility, broaden pH activity ranges (extending to pH 5-9), and improve stability in protein-rich environments. These parameter changes maintain or enhance lytic activity while resolving the expressibility and adaptability issues of natural lysins.
Solution Approach 2:
The patent focuses modifications on specific domains of the lysin structure. The N-terminal cell wall binding domain is optimized for staphylococcal specificity, while the C-terminal catalytic domain is engineered for enhanced activity and stability. This localized optimization allows each domain to contribute its specialized function, achieving both high killing effect and improved environmental adaptability.
4Reliability
If existing lysins are used, then they have specific killing activity, but the pH range to keep them active is narrow, generally in pH 5-8
Solution Approach 1:
The patent engineers lysins with altered pH stability profiles through amino acid sequence modification. Specific residues in the catalytic domain are mutated to maintain protonation states and catalytic efficiency across broader pH ranges (pH 5-9). This parameter change enables the lysins to remain active in diverse physiological and environmental conditions while preserving their specificity for staphylococcal cell walls.
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
ClyO effectively kills various Staphylococcus aureus strains, including MRSA, with low cytotoxicity and broad-spectrum activity, suitable for fermentation production, and facilitates rapid detection and identification through ATP or DNA release methods, demonstrating potential as an anti-infective medicine and diagnostic tool.
Implementation Method 1
Phage lysin is a kind of cell wall hydrolase expressed in late stage after host bacteria being infected with dsDNA phage
Implementation Method 2
the C-terminal cell wall binding domain (CBD) that determines cell binding sites
Data Source
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AI summary
The present invention discloses a lysin that is capable of killing Staphylococcus and the use thereof, belonging to the field of biological agents. The present invention discloses the amino acid sequence and the encoding gene sequence of the lysin. This lysin keeps active in a wide range of pH. It has lytic activity against Staphylococcus in pH 4-11. The recombinant protease constructed by the encoding gene can be solubly expressed in E.coli strain BL21 (DE3). The lysin can be used to effectively kill multiple species Staphylococcus in vitro, including methicillin sensitive Staphylococcus aureus (MSSA) and methicillin resistant Staphylococcus aureus (MRSA) isolated in clinics. This lysin can be used as an antibiotic for the treatment of staphylococcal infections in vivo. This lysin is also able to rapidly lyse staphylococcal cell wall; as a result, intracellular substances such as ATP and DNA are released. Those released substances can be used to detect the type of Staphylococcus.