Optimized DNA Sequence Prevents Truncated By-Product in scFv-Fusion Protein Expression
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Solution Overview
Problem
Current methods for producing the scFv(FRP5)-ETA antibody-toxin fusion protein result in a truncated by-product that cannot be completely eliminated using classical protein purification techniques, leading to impurities that may cause adverse reactions and increase production costs.
Innovation Solution
Modifying the DNA sequence encoding the scFv(FRP5) domain by exchanging a specific codon to prevent internal start of protein translation, thereby preventing the generation of the truncated by-product during bacterial expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If current DNA sequence is used for bacterial expression, then production yield is achieved, but truncated by-product is generated causing impurity
Solution Approach 1:
The patent applies preliminary action by modifying the DNA sequence in advance to include a mutated start codon (ATG changed to ACG) at position 92 of the scFv(FRP5) sequence. This preventive modification ensures that during bacterial translation, the ribosome cannot initiate at this internal position, thereby preventing the formation of truncated by-products before they can be generated. The solution addresses the impurity problem at its root cause rather than attempting to remove the by-product after formation.
2Manufacturing precision
If classical purification techniques are used, then some by-product removal is achieved, but complete elimination is not possible increasing costs
Solution Approach 1:
The patent implements preliminary action by pre-modifying the DNA sequence with a mutated start codon before expression. This prevents the formation of truncated by-products at the source, eliminating the need for costly and complex purification steps. The approach transforms the problem from one requiring extensive post-production purification to one where high purity is achieved directly during production, significantly reducing manufacturing costs.
3Productivity
If internal start codon is present, then translation initiation occurs at position 92, but truncated protein fragment is produced as by-product
Solution Approach 1:
The patent applies local quality by making a specific localized change to the DNA sequence at position 92, where the start codon ATG is mutated to ACG. This localized modification affects only the translation initiation at this specific position without altering the rest of the sequence or affecting overall expression efficiency. The solution demonstrates local quality by targeting the precise problematic site while maintaining the integrity and functionality of the entire protein sequence.
Solution Approach 2:
The patent uses preliminary action by pre-modifying the start codon at position 92 before bacterial expression. This preventive measure ensures that the truncated by-product is never formed during translation, maintaining product homogeneity from the outset. The approach addresses the manufacturing precision issue by eliminating the alternative translation initiation pathway before it can generate impurities.
Data Source
Figure 1A~1B
Figure 2A~2C
AI summary
Object matter of the invention is an optimized DNA sequence encoding the scFv(FRP5) antibody fragment. This novel sequence prevents the generation of the undesired by-product in the context of an scFv(FRP5)-ETA fusion protein, and possibly also other bacterially expressed scFv(FRP5)-containing fusion proteins. The DNA sequence of the scFv(FRP5) domain of scFv(FRP5)-ETA was modified by exchanging a distinct codon, thereby preventing an otherwise possible internal start of protein translation.