Fusion Polypeptide Propeptide Separation via Endopeptidase Cleavage
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Solution Overview
Problem
In recombinant expression systems, deamidase enzymes are produced as inactive proforms due to high binding affinity between the propeptide and deamidase domains, leading to reduced host cell viability and commercial production challenges, as the propeptide cannot be easily separated from the deamidase domain.
Innovation Solution
Development of fusion polypeptides with a deamidase inhibitory domain and a deamidase active domain, where the thermal unfolding temperature is between 65-82°C, allowing for separation of the propeptide and active deamidase enzyme using endopeptidases, thereby adjusting the binding affinity to facilitate recombinant expression and secretion without reducing host cell viability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the propeptide is tightly bound to the deamidase domain to reduce deamidase activity and protect host cell viability, then host cell viability is improved, but the propeptide cannot be separated from the deamidase domain by simple cleavage
Solution Approach 1:
The patent modifies the binding affinity parameter between the propeptide and deamidase domain by introducing specific mutations in the propeptide sequence. This creates an intermediate binding state that allows the fusion protein to be stable enough to protect host cells during expression, yet separable by endopeptidases after secretion, thus resolving the contradiction between maintaining viability and enabling separation.
2Ease of manufacture
If the binding affinity between propeptide and deamidase polypeptide is reduced to allow separation, then extracellular separation becomes possible, but the deamidase becomes activated and reduces host cell viability
Solution Approach 1:
The patent fine-tunes the binding affinity parameter to an optimal intermediate value through propeptide mutations. This adjusted parameter allows the fusion protein to remain stable during intracellular expression (protecting host cells) while becoming separable by endopeptidases in the extracellular environment, thus resolving the contradiction between enabling separation and maintaining host cell viability.
3Reliability
If the propeptide binding affinity is too high, then deamidase activity is suppressed and host cell viability is maintained, but commercial production becomes impossible due to inability to separate propeptide from deamidase
Solution Approach 1:
The patent optimizes the binding affinity parameter to an intermediate level that balances two requirements: sufficient stability to protect host cells during expression, and sufficient separability to enable commercial production. The mutated propeptide creates this optimal parameter state, allowing both host cell viability and commercial production feasibility.
Solution Approach 2:
The patent introduces endopeptidases as intermediary agents that facilitate the separation of propeptide from the deamidase domain. These enzymes act as mediators that can cleave the fusion protein at specific sites, enabling commercial production while maintaining host cell viability during the expression phase.
4Ease of manufacture
If the propeptide binding affinity is too low, then extracellular separation is enabled, but deamidase activity is activated prematurely and reduces host cell viability
Solution Approach 1:
The patent adjusts the binding affinity parameter to an optimal intermediate value through propeptide mutations. This modified parameter ensures the fusion protein remains stable during intracellular expression (maintaining host cell viability) while allowing endopeptidase-mediated separation in the extracellular environment (enabling manufacture).
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 approach enables the production of active deamidase enzymes by separating the propeptide from the deamidase domain, maintaining host cell viability and allowing for extracellular separation, thus overcoming the limitations of existing recombinant expression systems.
Implementation Method 1
wherein the fusion polypeptide has a thermal unfolding temperature in the range of 65-82°C
Implementation Method 2
contacting the fusion polypeptide of the invention with an endopeptidase to separate the first polypeptide from the second polypeptide
Data Source
AI summary
The present invention relates to polypeptides having deamidase 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.