Bifidobacterium Expression Cassette for Antibody Secretion
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Solution Overview
Problem
Current methods for producing heterologous polypeptides in bacteria of the genus Bifidobacterium lack efficient secretory signal peptides, resulting in suboptimal secretion of these proteins outside the bacterial cell, which limits their therapeutic potential, particularly for anti-cancer applications.
Innovation Solution
Development of an expression cassette comprising a promoter DNA, a DNA encoding a secretory signal peptide, a DNA encoding a linker peptide, and a terminator DNA, specifically optimized for Bifidobacterium, which includes secretory signal peptides like SP7 and linker peptides to enhance the secretion efficiency of heterologous polypeptides, such as anti-PD-1 and anti-CTLA-4 antibodies, outside the bacterial cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional secretory signal peptides are used in Bifidobacterium expression systems, then the expression cassette can be constructed, but the secretion efficiency of heterologous polypeptides outside the bacterial cell is suboptimal
Solution Approach 1:
The patent applies parameter changes by optimizing the amino acid sequence of the secretory signal peptide (specifically SP7 from Bifidobacterium longum) to enhance secretion efficiency. The signal peptide was modified through site-directed mutagenesis to improve its cleavage efficiency and secretion performance, directly addressing the suboptimal secretion problem while maintaining expression cassette functionality
Solution Approach 2:
The patent introduces a linker peptide as an intermediary component between the secretory signal peptide and the heterologous polypeptide. This linker peptide facilitates proper protein folding and secretion while maintaining the therapeutic function of the expressed protein, thereby improving both secretion efficiency and therapeutic potential
2Productivity
If signal peptides from membrane proteins and secretory proteins are used, then expression can occur, but secretion efficiency outside the bacterial cell is insufficient
Solution Approach 1:
The patent segments the expression cassette into distinct functional modules: promoter region, secretory signal peptide (SP7), linker peptide, heterologous polypeptide coding sequence, and terminator region. This modular segmentation allows for independent optimization of each component, particularly the signal peptide and linker, to achieve high secretion efficiency while maintaining ease of construction
Data Source
AI summary
An object is to provide a signal peptide that can secrete a heterologous polypeptide with high efficiency outside the bacterial cell in a bacterium of the genus Bifidobacterium, an expression cassette that can secrete a heterologous polypeptide with high efficiency outside the bacterial cell, a heterologous polypeptide expression vector, a bacterium of the genus Bifidobacterium capable of secreting a heterologous polypeptide. Means for attaining the object is a bacterium of the genus Bifidobacterium transformed by a vector having an expression cassette sequentially comprising a promoter DNA functioning in a bacterium of the genus Bifidobacterium, a DNA encoding the secretory signal peptide, a DNA encoding a scFv antibody having an antitumor activity, and a terminator DNA functioning in the bacterium of the genus Bifidobacterium; and capable of secreting the scFv antibody with high efficiency outside the bacterial cell.