Expression Vector with Dual Selection Markers for Mammalian Protein Production
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Solution Overview
Problem
Current expression vectors for recombinant protein production in mammalian cells exhibit variability in protein expression levels and require cumbersome selection processes, leading to increased production costs.
Innovation Solution
An expression vector design incorporating a gene expression regulatory site, a protein-coding gene, an internal ribosome entry site, a glutamine synthetase gene, and a dihydrofolate reductase gene, optimized with specific regulatory elements and mutation strategies to enhance protein expression and selection efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional expression vectors with single selection markers are used, then selection process is simple, but protein expression levels vary and production efficiency is low
Solution Approach 1:
The patent combines multiple selection marker genes (DHFR gene and GS gene) into a single expression vector along with the target protein gene. This merging of multiple functional elements into one vector enables simultaneous selection for both markers, ensuring only cells with high-level expression of all three genes survive, thereby improving protein production efficiency while managing vector complexity through integrated design.
Solution Approach 2:
The expression vector is designed to perform multiple functions: it contains the target protein gene for expression, the DHFR gene as a first selection marker for methotrexate resistance, and the GS gene as a second selection marker for sulfanilomide resistance. This multi-functionality allows a single vector to handle both protein production and dual-marker selection, improving productivity without requiring separate vectors for each function.
2Reliability
If multiple selection markers are incorporated in the expression vector, then selection accuracy for high-level expressers is improved, but vector design and selection process complexity increases
Solution Approach 1:
Multiple selection markers (DHFR and GS genes) are merged into a single expression vector alongside the target protein gene. This integration ensures that cells must express all three genes at high levels to survive dual selection pressure, improving selection accuracy for high-level expressers while managing design complexity through unified vector construction.
Solution Approach 2:
The internal ribosome entry site (IRES) acts as an intermediary element between the CMV promoter and the target protein gene, enabling cap-independent translation initiation. This intermediary mechanism ensures high-level expression of the target protein in conjunction with the selection markers, improving reliability of expression while simplifying the overall design by using a well-characterized viral element.
Data Source
AI summary
Disclosed are a novel expression vector for efficient expression of recombinant proteins in mammalian cells, a mammalian cell transformed with the vector, and a method for production of the mammalian cell. The expression vector is an expression vector for expression of a mammalian protein and includes a gene expression regulatory site, and a gene encoding the protein downstream thereof, and an internal ribosome entry site further downstream thereof, and a gene encoding a glutamine synthetase further downstream thereof, and a dihydrofolate reductase gene downstream of either the same gene expression regulatory site or another gene expression regulatory site in addition to the former.