Multi-Copy Gene Expression System for Recombinant Protein Yield
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Solution Overview
Problem
Recombinant protein expression often faces challenges of low quantity production and genetic instability due to recombination of identical expression cassettes, leading to decreased protein quality and stability over time.
Innovation Solution
Introducing multiple expression cassettes with different nucleotide sequences that code for the same mature recombinant protein, utilizing degenerate genetic code and varying vector elements like promoters, terminators, and signal sequences to reduce recombination and overload on protein synthesis machinery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If multiple copies of the same expression cassette are introduced into a cell to increase protein yield, then protein quantity increases, but genetic stability decreases due to recombination between identical sequences
Solution Approach 1:
The patent divides the expression cassette into multiple separate cassettes (at least two), each containing the complete set of elements (promoter, coding sequence, terminator, signal sequence) required for protein expression. By segmenting the system into multiple independent but functionally equivalent units, the patent achieves both high protein yield through multiple copies while reducing recombination risk through sequence differentiation.
Solution Approach 2:
The patent applies local quality by making each expression cassette locally different in its nucleotide sequence while maintaining the same functional output. Specific regions such as the promoter sequence, coding sequence, or terminator sequence are varied between cassettes using degenerate codons or alternative sequence designs, ensuring that each cassette has unique local characteristics that prevent recombination while preserving the identical amino acid sequence of the expressed protein.
2Quantity of substance
If multiple copies of the same expression cassette are introduced into a cell to increase protein yield, then protein quantity increases, but protein quality decreases due to recombination events
Solution Approach 1:
The patent segments the expression system into multiple independent cassettes with differentiated sequences, preventing recombination events that would otherwise lead to truncated, mutated, or duplicated protein variants. This segmentation strategy maintains manufacturing precision by ensuring each cassette produces the correct full-length protein sequence.
Solution Approach 2:
By introducing local sequence variations in non-critical regions (such as synonymous codon changes in the coding sequence or variations in promoter/terminator regions), the patent prevents recombination without affecting the amino acid sequence of the expressed protein, thereby maintaining high manufacturing precision and protein quality.
3Quantity of substance
If a high copy number of expression cassette is introduced into a cell to increase protein yield, then protein quantity increases, but the molecular machinery becomes overburdened and expression rate decreases
Solution Approach 1:
The patent divides the high-copy expression system into multiple separate expression cassettes distributed throughout the cell. This segmentation allows the cellular machinery to handle multiple lower-intensity expression tasks simultaneously rather than being overwhelmed by a single high-copy cassette, thereby maintaining efficient transcription and translation rates while achieving high overall protein yield.
Data Source
AI summary
The present invention belongs to the field of biotechnology, specifically to the field of recombinant protein expression. The present invention focuses on two problems commonly encountered during recombinant protein expression, low quantity of protein expression and genetic instability of cell lines used for recombinant protein expression. The basic principle of the present invention is to introduce several expression cassettes into a cell which expression cassettes all code for the same mature recombinant protein of interest, but which expression cassettes have different nucleotide sequences. Expression cassette means a polynucleotide sequence which comprises at least a promoter sequence, a start codon, a polynucleotide sequence coding for a protein which is intended to be recombinant expressed (POI), a stop codon and a terminator.