Plant Aspartic Protease Secretion via PSI Domain Deletion
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Solution Overview
Problem
Current methods for producing recombinant plant aspartic proteases are hindered by the need for large culture volumes and multiple steps, making it difficult to obtain significant amounts of these enzymes, particularly in non-plant expression systems, due to the role of the plant-specific insert (PSI) in vacuolar sorting and secretion.
Innovation Solution
Inactivating the normal vacuolar sorting function of the PSI domain through recombinant DNA manipulation to enhance secretion of plant aspartic proteases in non-plant eukaryotic cells, such as yeast, while retaining protease activity, by deleting or replacing the PSI domain with a linker, allowing for higher expression and secretion levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If the PSI domain is intact in plant aspartic proteases, then the enzyme is properly sorted to vacuoles, but secretion into the extracellular medium is reduced
Solution Approach 1:
The PSI domain is completely removed from the plant aspartic protease sequence. This extraction of the vacuolar sorting signal prevents the enzyme from being targeted to vacuoles, thereby redirecting it to the secretory pathway for extracellular accumulation.
Solution Approach 2:
The sorting destination parameter is changed by removing the PSI domain. This fundamental change in the protein's trafficking parameters redirects the enzyme from vacuolar localization to extracellular secretion, resolving the contradiction between vacuolar sorting and secretion.
2Quantity of substance
If large culture volumes are used to produce recombinant plant aspartic proteases, then sufficient enzyme quantity is obtained, but production complexity and cost increase
Solution Approach 1:
The secretion parameter is optimized by removing the PSI domain, which fundamentally changes the protein's trafficking behavior. This enables high-level extracellular secretion in standard culture conditions, eliminating the need for large culture volumes and complex production processes.
Solution Approach 2:
The enzyme is produced in heterologous expression systems (yeast, mammalian cells) rather than plant cells. This copying of the production system allows for better control and higher yields without the complexity of plant tissue culture.
3Manufacturing precision
If multiple production steps are used, then purification quality is maintained, but processing time and complexity increase
Solution Approach 1:
The PSI domain is extracted to enable direct extracellular secretion of the active enzyme. This eliminates the need for complex intracellular extraction and purification steps, as the enzyme is already in the extracellular medium in its active form.
Solution Approach 2:
The enzyme is secreted directly into the extracellular medium in its active form through PSI deletion. This preliminary action of secretion with inherent purification reduces the need for extensive downstream processing steps.
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
This approach enables the production of high volumes of active plant aspartic proteases that are convenient to isolate and purify, facilitating applications like cheese production by ensuring efficient secretion and maintaining enzymatic activity.
Implementation Method 1
each containing an aspartate residue which are within hydrogen-bonding distance of each other and act together to activate a water molecule which results in cleavage of the substrate peptide bond (via nucleophilic attack)
Implementation Method 2
activate a water molecule which results in cleavage of the substrate peptide bond (via nucleophilic attack)
Implementation Method 3
In the mature two-chain form both polypeptide chains are held together by hydrophobic interactions and hydrogen bonds
Implementation Method 4
In the mature two-chain form both polypeptide chains are held together by hydrophobic interactions and hydrogen bonds
Data Source
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AI summary
The invention relates to aspartic proteases, and particularly to aspartic proteases for plants. Disclosed are modified plant aspartic proteases, and methods for their manufacture, and uses thereof. Particularly contemplated are the uses of aspartic proteases in clotting milk.