Two-Chain Protein Expression with Chromosomal Chaperone Integration
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Solution Overview
Problem
Existing methods for producing recombinant two-chain proteins in prokaryotic host cells face challenges in achieving efficient folding, assembly, and purification, particularly for antibodies and antibody fragments, due to the formation of inclusion bodies and the need for separate expression of chaperone proteins from plasmids, which increases time and cost.
Innovation Solution
Integrating translational units encoding chaperone proteins, such as peptidyl-prolyl isomerases and protein disulfide oxidoreductases, into the host cell chromosome using non-native promoters, allowing for efficient folding and assembly of two-chain polypeptides without the need for separate plasmid-based chaperone expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If chaperone proteins are expressed from plasmids to improve folding and assembly of two-chain proteins, then protein folding efficiency is improved, but device complexity and manufacturing cost increase due to separate plasmid construction and optimization
Solution Approach 1:
The patent merges the chaperone protein expression system with the target protein expression system by placing both chaperone and target protein coding sequences on the same expression vector. This eliminates the need for separate plasmid construction and integration, reducing device complexity while maintaining the folding efficiency benefits of chaperone co-expression.
Solution Approach 2:
The patent creates a universal expression vector system that can simultaneously express both chaperone proteins and various target two-chain proteins. The vector is designed with multiple multiple cloning sites and regulatory elements that allow flexible insertion of different target sequences while maintaining consistent chaperone co-expression, reducing the need for vector re-engineering for different products.
2Manufacturing precision
If chaperone proteins are expressed from plasmids to improve folding and assembly, then protein assembly quality is improved, but time and cost for plasmid construction and optimization increase
Solution Approach 1:
The patent incorporates chaperone protein coding sequences pre-configured on the expression vector before target protein insertion. This preliminary arrangement of the chaperone expression framework eliminates the need for time-consuming plasmid construction and optimization steps for each new target protein, as the chaperone co-expression system is already in place and functional.
3Productivity
If plasmid-based chaperone expression is used to increase antibody titer, then productivity is improved, but additional purification steps are required to remove chaperone proteins
Solution Approach 1:
The patent applies different tagging strategies to different protein components within the same expression system. The target protein can be tagged with specific affinity tags while the chaperone protein is either left untagged or tagged differently, enabling selective purification of the target protein through affinity chromatography without requiring additional steps to remove chaperone contaminants.
4Adaptability or versatility
If separate plasmids are used for chaperone and target protein expression, then expression levels can be independently optimized, but ease of manufacture decreases due to multiple vector components
Solution Approach 1:
The patent incorporates multiple regulatable promoters within the single vector system, allowing independent control of chaperone and target protein expression levels. The vector may contain inducible promoters with different induction conditions (e.g., different inducers or inducible at different times), enabling dynamic optimization of expression levels for both proteins without requiring separate plasmids or complex vector construction.
Data Source
AI summary
Provided herein are methods and host cells for producing a polypeptide containing two chains, such as an antibody, half-antibody, antibody fragment, or one-armed antibody. The methods and host cells allow for two-chain polypeptide production using expression of polynucleotides encoding the polypeptide chains from extra-chromosomal polynucleotide(s), and expression of one or more chaperone protein(s) (e.g., peptidyl-prolyl isomerases and/or protein disulfide oxidoreductases) from the host cell chromosome using non-native combination(s) of promoters and translational units encoding a chaperone protein.