Engineered PSD Protein for Phosphatidylserine Removal on Extracellular Vesicles
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Solution Overview
Problem
Current methods for obtaining PS(-)-EVs are inefficient, with low yield and high cost, and existing PSD protein purification methods result in low protein yield and contamination due to low solubility and slow reaction rates.
Innovation Solution
An engineered PSD protein with specific functional domains and mutations is developed, which is secreted by engineered cells to catalyze the reduction of phosphatidylserine on EVs, thereby reducing macrophage phagocytosis and prolonging circulation time.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If Tim4-coupled magnetic beads are used to capture PS(+)-EVs and reverse screen to obtain PS(-)-EVs, then PS(-)-EVs can be obtained with reduced macrophage recognition, but the yield is very low because PS(-)-EVs account for only about 10% of natural extracellular vesicles
Solution Approach 1:
The invention enables EVs to self-modify their surface properties by incorporating PSD enzyme activity directly into the EV preparation process. The EVs undergo spontaneous PS depletion through the catalytic action of PSD during co-incubation, eliminating the need for complex Tim4-based capture and reverse screening procedures while achieving both high yield and reduced macrophage recognition.
Solution Approach 2:
The invention changes the chemical parameter of EV surface composition by catalytically removing phosphatidylserine through PSD enzyme action. This parameter change (PS content reduction) directly achieves reduced macrophage recognition while preserving the bulk EV population, thereby maintaining high yield unlike the reverse screening method.
2Reliability
If PSD is purified through prokaryotic expression and added for in vitro co-incubation to catalyze PS reduction, then PS(-)-EVs can be obtained, but the protein yield is low due to low solubility of PSD protein
Solution Approach 1:
Instead of purifying and adding PSD protein separately, the invention uses crude cell lysate containing endogenous PSD enzyme. The cellular system itself provides the catalytic function, eliminating the need for protein purification steps and associated solubility problems, while maintaining full catalytic activity for PS reduction.
Solution Approach 2:
The invention uses crude cell lysate as an intermediary medium that contains the necessary PSD enzyme activity. This intermediary approach avoids the need to isolate and purify the enzyme, providing sufficient catalytic activity for PS reduction while simplifying the overall process and improving protein availability.
3Reliability
If in vitro PSD catalysis is performed with purified PSD protein, then PS(-)-EVs can be obtained, but the reaction is slow and long incubation time can affect extracellular vesicles, requiring re-purification that increases process complexity and results in yield loss
Solution Approach 1:
The cellular system provides self-contained catalytic activity through endogenous PSD enzyme in crude lysate, eliminating the need for separate protein purification and addition steps. This self-service approach reduces process complexity while maintaining catalytic efficiency, avoiding the need for re-purification steps that cause yield loss.
Solution Approach 2:
The PSD enzyme is pre-prepared in crude cell lysate form, which already contains the necessary catalytic activity. This preliminary preparation eliminates the need for subsequent purification steps and reduces the complexity of the overall process, allowing direct use in the co-incubation step without affecting EV integrity or requiring re-purification.
4Reliability
If PSD protein is purified through prokaryotic expression, then the enzyme can be added for catalysis, but post-expression protein incubation is prone to introducing other contamination
Solution Approach 1:
Crude cell lysate serves as an intermediary that provides the necessary PSD enzyme activity without requiring purification. This intermediary approach accepts the presence of other cellular components as acceptable, focusing on obtaining sufficient catalytic activity rather than high protein purity, thereby avoiding contamination issues associated with purification procedures.
Solution Approach 2:
The invention uses crude cell lysate as a disposable reagent that does not require high purification. The acceptable level of impurity in crude lysate is tolerated because the catalytic function is sufficient for the application, and the cost and complexity of achieving high purity would outweigh the benefits. This approach prioritizes functional adequacy over manufacturing precision.
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 engineered PSD protein significantly reduces the surface PS content of EVs, leading to reduced macrophage phagocytosis and increased circulation time, while also improving protein yield and solubility, and simplifying the production process.
Implementation Method 1
The engineered PSD protein catalyzes the phosphatidylserine on the surface of the extracellular vesicles
Implementation Method 2
phosphatidylserine decarboxylase (PSD protein) comprising the following three functional domains
Data Source
AI summary
The application discloses an engineered phosphatidylserine decarboxylase (PSD protein) and use thereof, a fusion protein comprising the engineered PSD protein and use thereof, a nucleic acid molecule for encoding the engineered PSD protein or the fusion protein, an expression vector comprising the nucleic acid molecule, an engineered cell comprising the nucleic acid molecule or the expression vector, a method for preparing an engineered extracellular vesicle, an engineered extracellular vesicle and use thereof. In this application, an engineered phosphatidylserine decarboxylase is used for catalyzing phosphatidylserine on the surface of an extracellular vesicle, so that phagocytosis of macrophages is reduced, in-vivo clearance is reduced, and the circulation time of the extracellular vesicle is prolonged.