Enhanced Expression Stability Regions for Recombinant Protein Production
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current mammalian expression systems for recombinant protein production often result in low or unsatisfactory protein expression levels, requiring time-consuming cloning and amplification steps to achieve high expression levels.
Innovation Solution
The use of enhanced expression and stability regions (EESYRs) with integrated recombinase recognition sites, such as loxp, lox511, and frt sites, to enhance protein expression by positioning genes of interest within these regions, facilitating efficient recombination and stable integration in eukaryotic cells like CHO cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional mammalian expression systems are used, then recombinant protein can be produced with appropriate post-translational modifications, but protein expression levels are low or unsatisfactory
Solution Approach 1:
The patent applies preliminary action by pre-selecting and integrating specific genomic loci (EESYRs) into the mammalian genome before protein expression. These pre-selected loci have been identified to inherently support high-level expression, thereby eliminating the need for subsequent time-consuming cloning and amplification steps to achieve high expression levels. The system prepares the genomic environment in advance to favor high expression.
Solution Approach 2:
The patent applies local quality by targeting expression to specific genomic loci (EESYRs) rather than using random integration. These specific loci possess unique local genomic characteristics that favor high-level expression. By confining expression to these specialized regions, the system achieves high productivity while maintaining the reliability of post-translational modifications that mammalian cells provide.
2Productivity
If cloning and amplification steps are performed to achieve high expression levels, then protein expression level increases, but development time increases significantly
Solution Approach 1:
The patent applies preliminary action by pre-establishing cell lines with integrated EESYR loci before the actual protein expression experiment. This preliminary setup creates a genomic environment that naturally supports high expression, thereby eliminating the need for subsequent time-consuming cloning and amplification steps. The system prepares the genomic environment in advance to favor high expression from the start.
Solution Approach 2:
The patent applies the taking out principle by extracting and isolating specific high-expression genomic loci (EESYRs) from the complex genome. By identifying and separating these functional elements, the invention creates a simplified system where high expression is achieved through targeted integration at predetermined loci, rather than requiring extensive cloning and amplification of the entire expression cassette.
3Ease of manufacture
If random integration is used, then gene insertion is simple, but expression levels are low and unstable
Solution Approach 1:
The patent applies local quality by targeting expression to specific genomic loci (EESYRs) rather than using random integration. These specific loci possess unique local genomic characteristics that favor high-level expression. By confining expression to these specialized regions, the system achieves both high productivity and stability while maintaining relative ease of manufacture through targeted integration strategies.
Solution Approach 2:
The patent applies the intermediary principle by using EESYR loci as intermediary genomic regions that mediate between the simplicity of random integration and the precision of targeted expression. These intermediary loci provide a controlled environment that enhances expression levels and stability while simplifying the overall process compared to traditional cloning and amplification methods.
Data Source
AI summary
Expression-enhancing nucleotide sequences for expression in eukaryotic systems are provided that allow for enhanced and stable expression of recombinant proteins in eukaryotic cells. Enhanced expression and stability regions (EESYRs) are provided for expression of a gene of interest in a eukaryotic cell. Chromosomal loci, sequences, and vectors are provided for enhanced and stable expression of genes in eukaryotic cells.