Yeast Expression Vector for Recombinant Plasminogen Production
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Solution Overview
Problem
Current methods for producing large quantities of recombinant plasminogen and its derivatives, such as miniplasminogen and microplasminogen, are inefficient, making it difficult to treat ischemic and thrombotic disorders effectively.
Innovation Solution
The use of Pichia pastoris yeast for high-yield production of recombinant mammalian plasminogen and its derivatives, involving a yeast expression vector with a mammalian nucleotide sequence linked to a promoter, allowing for stable integration and secretion of active plasminogen forms, which are then purified and stabilized for clinical use.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If conventional methods are used for producing recombinant plasminogen, then production cost and time are reduced, but production yield and purity are insufficient for clinical application
Solution Approach 1:
The patent uses a yeast secretion signal peptide as an intermediary to facilitate the secretion of recombinant plasminogen from yeast cells into the culture medium. This mediator enables efficient recovery of the protein without complex cell disruption steps, resolving the contradiction between high yield and ease of manufacture by simplifying the purification process while maintaining high productivity
Solution Approach 2:
The patent optimizes multiple parameters including yeast strain selection, promoter strength, culture conditions, and secretion signal peptide sequence to maximize plasminogen production yield. By systematically changing these parameters, the invention achieves clinical-grade purity and yield while maintaining feasible manufacturing complexity
2Quantity of substance
If plasminogen is produced in high quantities, then therapeutic effectiveness is improved, but purification difficulty and cost increase
Solution Approach 1:
The secretion signal peptide acts as an intermediary that directs plasminogen to the extracellular space, where it can be easily separated from cellular components. This mediator enables simple purification by filtration and concentration, resolving the contradiction between high quantity production and purification ease
Solution Approach 2:
The patent separates the production system into intracellular synthesis (where plasminogen is made) and extracellular secretion (where it is collected). This segmentation allows high-yield production while simplifying purification, as the protein is already separated from most contaminants by being secreted into the medium
3Reliability
If recombinant plasminogen derivatives are produced for clinical use, then therapeutic efficacy is improved, but production efficiency remains insufficient
Solution Approach 1:
The yeast secretion signal peptide serves as a mediator that enables efficient recovery of recombinant plasminogen derivatives from the expression system. This intermediary function resolves the contradiction between producing clinically effective quantities and maintaining high production efficiency by simplifying the isolation process
Solution Approach 2:
The yeast expression system automatically performs folding, post-translational modifications, and secretion of the recombinant plasminogen derivatives. This self-service capability ensures clinical-grade quality while maintaining high production efficiency, as the system handles complex processing without additional intervention
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 method enables the production of high-purity, clinically applicable recombinant plasminogen derivatives, effectively treating ischemic stroke, acute myocardial infarction, and extracorporeal arteriovenous circulation thrombosis, with improved therapeutic efficacy and safety.
Implementation Method 1
a yeast expression vector comprising a mammalian nucleotide sequence operably linked to a promoter, wherein the said mammalian nucleotide sequence codes for the catalytic domain of plasminogen and further optionally codes for one of more kringle domains of plasminogen
Implementation Method 2
allowing for stable integration and secretion of active plasminogen forms
Data Source
AI summary
Vectors for the expression in yeast of mammalian plasminogen derivatives such as microplasminogen and miniplasminogen are presented. Methods for expression of these proteins in a methylotrophic yeast expression system are disclosed as well as the activation and stabilisation of the recombinant proteins. The proteins of this invention are used In the treatment of focal cerebral ischemic infarction and other thrombotic diseases.