Deamidase Inhibitor Polypeptides for Viable Recombinant Expression
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Solution Overview
Problem
In recombinant expression systems, the propeptide domain of deamidase enzymes is not naturally removed, leading to the secretion of inactive deamidase proforms, which negatively affects host cell viability due to high binding affinity, and traditional mutagenesis to separate the propeptide results in partly activated intracellular deamidase.
Innovation Solution
Development of polypeptides that reversibly bind to the deamidase active site, inhibiting its activity and mitigating the negative effects on host cell viability by co-expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the propeptide domain is tightly bound to the deamidase domain in the proform, then the host cell viability is protected by preventing premature activation, but the separation of propeptide and active deamidase becomes difficult
Solution Approach 1:
The patent introduces a deamidase inhibitor as an intermediary substance that binds to the deamidase active site. This inhibitor acts as a mediator to prevent premature activation of the deamidase by the propeptide, allowing the proform to be secreted without causing harmful intracellular activation. The inhibitor enables the system to maintain the tight binding necessary for viability protection while allowing controlled separation and activation outside the cell.
2Ease of manufacture
If mutagenesis is used to reduce the binding affinity between propeptide and deamidase, then the separation becomes easier, but intracellular deamidase becomes partly activated reducing host cell viability
Solution Approach 1:
The patent applies preliminary anti-action by introducing a deamidase inhibitor that preemptively blocks the deamidase active site before the propeptide can activate it. This preliminary blocking action prevents the harmful intracellular activation that would otherwise occur when mutagenesis reduces propeptide-deamidase binding affinity. The inhibitor is expressed together with the deamidase proform to ensure protection is in place before separation occurs.
3Productivity
If the inactive deamidase proform is secreted outside the host cell, then the deamidase activity is available extracellularly, but the high binding affinity causes negative effects on host cell viability
Solution Approach 1:
The deamidase inhibitor serves as a protective intermediary that travels with the deamidase proform through secretion. This intermediary prevents the propeptide from activating the deamidase inside the cell, allowing the proform to be safely secreted. Once outside the cell, the inhibitor can be removed or inactivated, allowing the deamidase to become fully active extracellularly where it can perform its productive function without harming the host cell.
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 polypeptides effectively inhibit deamidase activity, improving recombinant expression and maintaining host cell viability by preventing the secretion of inactive deamidase proforms.
Implementation Method 1
polypeptides capable of inhibiting deamidase activity by reversibly binding to the deamidase and interacting with the deamidase active site
Data Source
AI summary
The present invention relates to polypeptides having deamidase inhibitor activity and polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.