Phage Display System for Disulfide-Stabilized scFv Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The instability of single chain variable fragments (scFv) leads to aggregation and biases in antibody gene product reproduction on phage surfaces, limiting their application in biotechnology and medicine, and disulfide-stabilized scFv formats have not been successfully expressed on phage surfaces due to decreased yield.
Innovation Solution
Optimized signal sequences are identified and used to facilitate the production of disulfide-stabilized single chain antibodies, enabling their expression and display on phage surfaces through the creation of a phage display system that stabilizes the sc-dsFv format, improving yield and fidelity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If disulfide-stabilized single chain antibody (sc-dsFv) format is used to stabilize the structure, then stability and fidelity of antibody gene product reproduction are improved, but expression yield on phage surface decreases severely
Solution Approach 1:
The invention changes the amino acid sequence parameters of the signal peptide by introducing 10 randomized positions (X1-X10) that can be any of the 20 naturally occurring amino acids. This creates a library of signal peptide variants that are screened to identify sequences that optimize both the stability of sc-dsFv structure and the expression yield on phage surface, resolving the contradiction between these two parameters.
Solution Approach 2:
The invention performs preliminary optimization of the signal peptide sequence before the actual expression of sc-dsFv on phage surface. By pre-screening signal peptide variants in a library format, the optimal signal sequence is identified in advance, which then enables high-yield expression of stable sc-dsFv structures without the need for subsequent optimization steps.
2Device complexity
If conventional signal sequences are used for phage-displayed sc-dsFv expression, then the expression system is simple, but expression is not viable due to severely decreased yield
Solution Approach 1:
The invention modifies the signal peptide sequence parameters by introducing 10 randomized positions (X1-X10) into the conventional signal sequence framework. This creates a diversified library of signal peptide variants that maintain the basic structure and function of conventional signals while enabling high-yield expression of sc-dsFv on phage surface, thus preserving system simplicity while dramatically improving productivity.
3Length of moving object
If single chain variable fragment (scFv) is used as scaffold, then tissue-penetrating capability is improved, but structural stability decreases leading to aggregation
Solution Approach 1:
The invention creates a composite structure by combining the small scFv scaffold with a disulfide bond network. The disulfide bonds formed between cysteine residues at specific positions create a stabilized composite structure that maintains the small size and tissue-penetrating capability of scFv while adding the structural stability needed to prevent aggregation and improve reliability.
Data Source
AI summary
Disclosed are nucleic acid libraries for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and for facilitating production of a disulfide-stabilized single chain antibody. Also disclosed are host cell libraries and phage libraries including the nucleic acid libraries. Further disclosed are methods for identifying a signal peptide that facilitates production of disulfide-stabilized single chain antibody, and methods for producing a disulfide-stabilized single chain antibody and non-fusion form thereof.