Chimeric Receptor Binding Proteins Resistant to Proteolytic Digestion
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Solution Overview
Problem
Protein-based DNA delivery vectors, such as packaged phagemids, face stability issues due to proteolytic digestion in the gastrointestinal tract, particularly by enzymes like pancreatin, which affects their functionality and stability.
Innovation Solution
Designing a chimeric lambda-based STF protein with a specific linker region containing point mutations in phenylalanine and lysine residues, or incorporating a helix-forming sequence from the V10 tail fiber, to enhance resistance to proteolytic digestion by pancreatin, thereby increasing stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If a protein-based DNA delivery vector is designed for oral administration, then it can reach target bacterial cells in the gastrointestinal tract, but it is degraded by proteolytic enzymes such as pancreatin, reducing its stability and functionality
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the linker region in the chimeric STF protein. Specific parameters such as proteolytic cleavage resistance are improved through targeted mutations of amino acid residues (e.g., replacing trypsin-sensitive lysine and arginine residues with protease-resistant alternatives), thereby changing the chemical properties of the protein to withstand gastrointestinal conditions while maintaining oral administration capability
Solution Approach 2:
The patent creates a composite structure by fusing two different STF proteins from different bacteriophages into a chimeric protein. This composite STF combines domains from parent phages to achieve both functional activity (binding to bacterial receptors) and enhanced stability against proteolytic enzymes, resolving the contradiction between ease of operation and reliability
2Ease of manufacture
If the linker region of a chimeric STF protein contains standard amino acid sequences, then the protein can be easily constructed, but it becomes susceptible to proteolytic cleavage by enzymes like trypsin and chymotrypsin
Solution Approach 1:
The patent applies local quality by making targeted modifications only to specific residues within the linker region rather than redesigning the entire protein. The N-terminal and C-terminal domains remain unchanged, while only the linker region undergoes localized amino acid substitutions to confer protease resistance, maintaining ease of construction through modular design while protecting against harmful proteolytic factors
Solution Approach 2:
The patent converts the harmful susceptibility to proteolytic cleavage into a benefit by strategically introducing protease-resistant amino acid sequences. The linker region is redesigned to contain sequences that are naturally resistant to common gastrointestinal proteases, thereby transforming what would be a vulnerability into a protective feature that enhances overall protein stability without compromising constructability
Data Source
AI summary
The present disclosure provides a chimeric receptor binding protein (RBP) resistant to proteolytic digestion wherein said RBP comprises a portion of a receptor binding protein derived from a bacteriophage fused through a designed linker region consisting of 1 to 70 amino acids, to a portion of a receptor binding protein derived from a different bacteriophage, wherein said linker region is designed to be resistant to proteolytic digestion.