Controllable prfA Gene Expression for Recombinant Protein Production
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Solution Overview
Problem
The low yield of recombinant proteins with site-directed insertion of unnatural amino acids in Escherichia coli due to premature translation termination by the peptide chain releasing factor RF1, leading to significant production of truncated proteins and hindered growth rates, making industrial fermentation challenging.
Innovation Solution
Modifying the expression of the prfA gene in Escherichia coli to be controllable, either suppressed or inducible, by using plasmids with specific promoters and replication origins, and introducing a plasmid encoding an unnatural amino acid tRNA/tRNA synthetase to enable efficient production of recombinant proteins with site-directed insertion of unnatural amino acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If the prfA gene is directly knocked out to inhibit RF1 competition, then the yield of recombinant protein with unnatural amino acids is improved, but the growth rate of the strain is significantly reduced
Solution Approach 1:
The patent applies dynamics by making the prfA gene expression controllable rather than static knockout. The prfA gene is placed under an inducible promoter (e.g., T7 promoter) that allows dynamic control of RF1 expression levels. During growth phase, RF1 expression is suppressed to maintain fast growth; during protein production phase, RF1 expression is induced to enable efficient incorporation of unnatural amino acids. This dynamic control resolves the contradiction between growth rate and protein yield.
Solution Approach 2:
The patent implements periodic action through two-stage induction: first inducing RF1 expression during growth phase to maintain cellular health, then inducing recombinant protein expression in the second stage when unnatural amino acid incorporation is needed. This periodic switching of gene expression states allows the system to optimize for growth initially, then for protein production, resolving the trade-off between growth rate and productivity.
2Reliability
If RF1 is present in Escherichia coli to recognize amber codon as termination codon, then the natural translation termination function is maintained, but the yield of recombinant protein with site-directed insertion of unnatural amino acids is low and truncated proteins are generated
Solution Approach 1:
The patent uses dynamic control of RF1 expression to resolve this contradiction. By placing the prfA gene under an inducible promoter, the system can dynamically adjust RF1 levels: keeping RF1 suppressed during the phase when unnatural amino acid incorporation is needed, thereby preventing premature termination at amber codons, while maintaining the ability to restore RF1 function when natural termination is required. This dynamic regulation allows selective modulation of translation fidelity based on process stage.
3Stability of the object's composition
If the structure of special recombinant proteins hinders the insertion of unnatural amino acids at certain sites, then the protein structure integrity is maintained, but it is difficult to obtain an intact recombinant protein containing unnatural amino acids
Solution Approach 1:
The patent applies parameter changes by systematically optimizing multiple parameters: the inducible promoter type (T7, lacUV5, araBAD), the induction timing, the concentration of unnatural amino acids, and the growth conditions. By adjusting these parameters, the system can accommodate different protein structures and their specific requirements for unnatural amino acid incorporation, resolving the issue where fixed conditions fail for structurally diverse recombinant proteins.
Data Source
AI summary
Provided in the present invention are a method for constructing a strain for producing a recombinant protein containing unnatural amino acids, and the strain obtained therefrom. The method comprises modifying the expression of a prfA gene contained in the strain to be controllable. The strain constructed by the method of the present invention can efficiently produce in intact protein containing unnatural amino acids, greatly reduce the production of a truncated protein and can also maintain high-speed growth.