Dual Cistronic Expression System for Equimolar Fab Chain Production
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Solution Overview
Problem
Current methods for producing recombinant Fab fragments in E. coli often face challenges with unequal expression levels of heavy and light chains, limiting the yield of functional Fab molecules due to the use of single promoters for both chains.
Innovation Solution
A dual, independent cistron expression system in a single vector is employed, utilizing two different promoters to independently regulate the expression of heavy and light chains, ensuring equimolar expression and higher yields by allowing separate control of each chain's production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If a single promoter is used to drive expression of both heavy and light chains, then the expression system is simple, but the expression levels of the two chains are unequal, limiting the yield of functional Fab
Solution Approach 1:
The invention divides the single promoter system into two separate promoters (phoA and lambda P_L), with each promoter independently controlling the expression of one gene (heavy chain or light chain). This segmentation allows independent optimization of expression levels for each chain, resolving the contradiction between system simplicity and functional yield.
2Ease of manufacture
If a single promoter drives both chains, then the vector design is straightforward, but the expression levels are unbalanced, reducing functional Fab production
Solution Approach 1:
The vector is designed with two separate promoter-cistron units instead of one promoter driving two genes. Each unit (phoA-promoter for heavy chain, lambda P_L-promoter for light chain) can be independently constructed and optimized, maintaining ease of manufacture while enabling balanced expression and higher functional Fab production.
3Manufacturing precision
If sequential expression is used to separate light and heavy chain production, then assembly can be controlled, but the process time increases
Solution Approach 1:
The invention enables simultaneous expression of both heavy and light chains through two independently active promoters in the same cell. This continuous parallel production eliminates the time loss associated with sequential expression steps while maintaining precise control over each chain's production through promoter-specific regulation.
Data Source
AI summary
The present invention relates to the dual, independent cistron expression system in a single vector for the production of protein of interest proteins and peptides expressed as insoluble inclusion bodies formed in the bacteria E. coli. The present invention also provides the process for the expression of protein of interest using said bicistronic vector.