Yeast Microvesicles for Hemorrhage Treatment
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Solution Overview
Problem
Current methods for producing biologically active tissue factor (TF) protein are inefficient, with low yields and high costs due to the challenges of expressing and purifying TF in bacterial systems, and existing recombinant TF production in eukaryotic systems results in lower yields and functional issues.
Innovation Solution
Development of tissue factor-bearing yeast-derived microvesicles comprising a yeast membrane and a TF protein or fragment with pro-coagulant activity, produced through recombinant yeast fermentation, homogenization, and separation processes to generate high-yield, biologically active TF for therapeutic use.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If TF protein is produced in bacterial systems, then production cost is reduced, but biological activity and yield are low
Solution Approach 1:
The patent changes the host system parameter from bacterial to yeast, and modifies the TF protein structure by adding a transmembrane domain and signal sequence, transforming it from a soluble protein to a membrane-associated protein. This parameter change enables high-yield production of biologically active TF in yeast systems.
Solution Approach 2:
The patent creates a recombinant copy of the TF gene with added sequences (signal sequence and transmembrane domain) that directs the protein to the yeast cell membrane. This copied and modified gene is then expressed in yeast, producing membrane-associated TF that mimics natural TF structure and function.
2Reliability
If TF protein is produced in eukaryotic systems, then biological activity is improved, but yield and production efficiency decrease
Solution Approach 1:
The yeast cell membrane serves as both the expression platform and the functional environment for TF. The TF protein is inserted into the yeast membrane where it naturally localizes, and the membrane provides the necessary phospholipid environment for TF biological activity without requiring additional purification or reconstitution steps.
Solution Approach 2:
The patent creates a composite structure where the TF protein is integrated into the yeast cell membrane, combining the eukaryotic expression system with the natural membrane environment. This composite membrane-protein complex maintains high biological activity while enabling scalable production in yeast.
3Productivity
If soluble TF is expressed in bacteria, then expression yield is high, but the protein lacks biological activity
Solution Approach 1:
The patent transitions TF from a soluble three-dimensional structure to a membrane-associated structure by adding a transmembrane domain. This dimensional change anchors the protein to the membrane, providing the necessary structural context for biological activity while maintaining high expression levels in the yeast system.
Solution Approach 2:
The patent applies different structural qualities to different parts of the TF protein: the extracellular domain maintains the native soluble structure for ligand binding, while the added transmembrane domain provides membrane anchoring. This local differentiation enables both high expression and biological activity.
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 process enables the production of high-yield, biologically active TF microvesicles suitable for therapeutic applications, effectively addressing the limitations of existing TF production methods by providing a scalable and efficient source of TF for treating hemorrhages and promoting angiogenesis and cell migration.
Implementation Method 1
subjecting a culture of recombinant yeast cells which express TF protein or a fragment thereof having pro-coagulant activity to fermentation
Implementation Method 2
subjecting said fermentation product from step b) to homogenization, to render a fermentation homogenate
Implementation Method 3
subjecting said fermentation homogenate from step c) to separation, to render a pellet and a clarified yeast extract (CYE) containing said TF-bearing yeast derived microvesicle
Data Source
Figure 1A~2
Figure 3
Figure 4A~5
AI summary
Tissue factor-bearing yeast derived microvesicles comprising a yeast membrane and a tissue factor protein, or a fragment thereof, or a tissue factor protein or a fragment thereof fused to another peptide as a fusion protein having pro-coagulant activity are disclosed. Said products can be used as pro-coagulant agents in the treatment of hemorrhages in a subject.