Peptide Framework for Stable Single Domain Antibody Library
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Solution Overview
Problem
Conventional peptide library methods face challenges in stability, limited library size, and inability to predict and design peptides with desired structural characteristics, particularly for single domain antibodies like VHH, due to issues with mRNA degradation and cellular expression requirements.
Innovation Solution
A peptide framework consisting of three complementarity determining regions and four framework regions with specific amino acid sequences, allowing for the design and synthesis of peptides with desired structural characteristics, enabling efficient screening of single domain antibodies and construction of a peptide library.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If mRNA display method is used to obtain diverse candidate peptides, then library size is improved, but stability deteriorates due to mRNA degradation by RNase
Solution Approach 1:
The patent uses a cell-free translation system to synthesize peptides based on DNA templates without requiring living cells or mRNA intermediates. This copying approach allows the peptide sequence to be determined by DNA while avoiding mRNA degradation issues, enabling both large library diversity and stable peptide production.
Solution Approach 2:
The invention replaces the biological mRNA-based display system with a cell-free biochemical system. By substituting the cellular machinery with in vitro translation components (ribosomes, tRNAs, amino acids), the system achieves both large-scale peptide synthesis and stability without RNase degradation.
2Reliability
If phage display method is used to ensure stability of display, then stability is improved, but library size deteriorates due to complex cellular expression requirements
Solution Approach 1:
The patent extracts the essential translation function from living cells, using only the necessary cellular components (ribosomes, tRNAs, amino acids, enzymes) in a cell-free system. This extraction eliminates the complexity of cellular expression while maintaining peptide synthesis capability, enabling larger library sizes with stable production.
Solution Approach 2:
The invention introduces a cell-free translation mixture as an intermediary between DNA templates and peptide products. This intermediary system performs peptide synthesis without requiring complete cellular machinery, simplifying the process while enabling large-scale stable peptide production.
3Ease of manufacture
If conventional peptide library screening is performed, then ease of manufacture is improved, but ability to predict and design peptides with desired structural characteristics deteriorates
Solution Approach 1:
The patent performs preliminary design of the peptide framework sequence before synthesis, incorporating desired structural characteristics into the framework. By pre-designing the framework with specific properties (beta-sheet structure, stability features) and then using cell-free translation to synthesize the library, the method combines ease of manufacture with precise structural control.
Data Source
AI summary
Provided are a peptide consisting of three complementarity determining regions and four framework regions, in which a framework 1 comprises an amino acid sequence represented by SEQ ID NO: 1, a framework 2 comprises an amino acid sequence represented by SEQ ID NO: 2, a framework 3 comprises an amino acid sequence represented by SEQ ID NO: 7, a framework 4 comprises an amino acid sequence represented by SEQ ID NO: 32, a complementarity determining region 1 is an amino acid sequence consisting of 10 amino acids, a complementarity determining region 2 is an amino acid sequence consisting of 16 or 17 amino acids, and a complementarity determining region 3 is an amino acid sequence consisting of 6, 12 or 15 amino acids; and a peptide library including the peptide.