Host Cell Protease Inactivation for Protein Production Stability
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Solution Overview
Problem
Microorganisms used for protein production, such as fungi, inherently produce proteolytic enzymes that degrade proteins of interest, leading to reduced yield and quality, especially for sensitive or unstable proteins, making it challenging to achieve long-term stability and shelf life.
Innovation Solution
Inactivation of the pea1 gene, which regulates endogenous protease expression, to suppress protease production in host cells, thereby enhancing protein yield and stability by using a polynucleotide encoding a protein with at least 90% sequence identity to amino acids 402-533 of SEQ ID NO: 13, and incorporating this into a vector for genetic modification of host cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If microorganisms are used as host cells for protein production, then protein expression capability is improved, but endogenous proteases degrade the protein of interest reducing yield and quality
Solution Approach 1:
The invention extracts and removes the harmful element (endogenous proteases) from the host cell system by deleting or inactivating protease genes, while preserving the beneficial protein expression capability of the microorganism
Solution Approach 2:
The invention introduces protease inhibitor proteins as intermediary substances that mediate between the host cell and the protein of interest, preventing protease degradation without eliminating the host cell's natural proteolytic functions entirely
2Stability of the object's composition
If multiple endogenous protease genes are individually deleted to reduce proteolytic activity, then protein stability is improved, but the complexity of strain development increases significantly
Solution Approach 1:
The invention merges multiple protease gene deletion targets into a coordinated strain development approach, using systematic methods to simultaneously address multiple protease genes rather than individually deleting each one
Solution Approach 2:
The invention changes the parameter of proteolytic activity by deleting protease genes, thereby altering the biochemical environment to favor protein stability while maintaining host cell functionality
3Duration of action of stationary object
If protease inhibitors are added to protein compositions to inhibit endogenous protease activity, then protein shelf life is extended, but the complexity of composition formulation increases
Solution Approach 1:
The invention enables the host cell to self-regulate protease production by incorporating protease gene deletions directly into the strain, eliminating the need for external protease inhibitors and simplifying the overall composition formulation
Data Source
AI summary
The present description is related to the field of protein production. It introduces novel host cells with low protease activity, a novel protease regulator, its use in expression systems and protein production, and a method of producing host cells for protein production.