DNA Construct TIR and Signal Peptide Design for Certolizumab Yield
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Solution Overview
Problem
Existing DNA constructs and host cells face challenges in efficiently producing recombinant proteins, particularly Certolizumab, due to cellular stress, poor growth, low protein activity, and low yield, with existing systems lacking optimized translation initiation regions and nucleotide sequences for signal peptides.
Innovation Solution
A DNA construct comprising optimized nucleotide sequences for RhaR, RhaS transcription activators, a rhaBAD promoter, and a PelB signal peptide, integrated with a host cell having mutations in rhamnose metabolism and protease genes, to enhance recombinant protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing DNA constructs and host cells are used for recombinant protein production, then the production system is simple and well-established, but cellular stress occurs resulting in damage to cellular macromolecules and poor growth
Solution Approach 1:
The patent modifies the rhaBAD promoter sequence to create an optimized version that maintains inducibility while reducing cellular stress. Specific nucleotide changes in the promoter region alter gene expression parameters to achieve higher protein yields without compromising cellular stability. The host cell genome is also modified with specific mutations in rhaB and other genes to change metabolic parameters and reduce stress responses.
Solution Approach 2:
The patent uses a inducible expression system where the recombinant protein is produced in a controlled manner using rhamnose induction. The system allows for transient high-level expression followed by harvest, rather than requiring permanent high-level expression that would continuously stress the cells. This enables multiple production cycles with the same host cell line.
2Ease of manufacture
If existing DNA constructs are used, then the construct design is straightforward, but poor growth of host cells occurs
Solution Approach 1:
The patent introduces specific mutations in the host cell genome, including mutations in rhaB and other metabolic genes, to change growth parameters. These genetic modifications are integrated into the host cell line to improve growth characteristics while maintaining the ability to support recombinant protein expression under induction.
3Ease of operation
If existing DNA constructs are used, then the system is easy to implement, but poor activity of produced recombinant proteins occurs
Solution Approach 1:
The optimized rhaBAD promoter and host cell modifications work together to change protein expression parameters, resulting in higher levels of active recombinant protein. The promoter optimization ensures proper transcriptional control while the host cell modifications improve translational efficiency and protein folding, leading to higher specific activity of the produced proteins.
4Device complexity
If existing DNA constructs and host cells are used, then the production process is simple, but recombinant proteins are obtained in low yields
Solution Approach 1:
The patent optimizes the rhaBAD promoter with specific nucleotide changes to enhance transcriptional efficiency and protein yield. The host cell is modified with mutations in rhaB and other genes to improve metabolic flux toward recombinant protein production. These parameter changes collectively increase yield without requiring complex multi-step production processes.
Solution Approach 2:
The optimized expression system enables sustained high-level protein production during the induction phase. The modified promoter and host cell work together to maintain stable, high-level expression over an extended period, maximizing total yield without requiring repeated induction cycles or complex process interventions.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The solution enables high-yield production of Certolizumab with improved cellular stability and protein activity, facilitating efficient industrial-scale production.
Implementation Method 1
The rhaBAD operon is a positively regulated catabolic operon which transcribes above mentioned rhaB, rhaA and rhaD genes divergently from the rhaSR operon
Implementation Method 2
RhaR regulates transcription of rhaSR by binding promoter DNA spanning −32 to −82 bases relative to the rhaSR transcription start site
Implementation Method 3
The metabolism of rhamnose involves L-rhamnose being taken up into cells via the permease RhaT
Implementation Method 4
L-rhamnose being taken up into cells via the permease RhaT and then isomerized into L-rhamnulose by L-rhamnose isomerase (RhaA)
Implementation Method 5
L-rhamnulose is then phosphorylated further by rhamnulokinase (RhaB)
Implementation Method 6
finally hydrolyzed by rhamnulose-1-phosphate aldolase (RhaD) to give dihydroxyacetonephosphate and L-lactaldehyde
Data Source
AI summary
A DNA construct for expressing a recombinant protein, wherein the DNA construct comprises: —at least one of the nucleotide sequences of SEQ ID No 20 and 21, wherein a nucleotide sequence of SEQ ID 20 and 21 is a TIR sequence; and —a nucleotide sequence which encodes a signal peptide; and wherein a nucleotide sequence of SEQ ID No 20 and 21 comprises at least the first 9 nucleotides of said signal peptide encoding sequence.