Recombinant Prothrombin Production via Solubility Tags
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Solution Overview
Problem
Current methods for producing recombinant prothrombin in Escherichia coli and mammalian cells face challenges such as low solubility and specific activity due to the formation of insoluble aggregates, and high manufacturing costs, which hinder large-scale industrial production.
Innovation Solution
A method involving the expression of prothrombin with specific tags (MBP, SUMO, or NusA) in lepidopteran insects or cultured cells using a vector DNA, which enhances solubility and allows for high specific activity thrombin production without the need for refolding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If prothrombin is expressed in Escherichia coli, then production cost is reduced, but the protein forms insoluble aggregates and requires complicated refolding with low efficiency
Solution Approach 1:
A solubility-enhancing tag (MBP, GST, or SUMO) is introduced as an intermediary component fused to the N-terminus of prothrombin. This tag acts as a mediator that prevents aggregation of the prothrombin protein in E. coli expression systems, enabling soluble protein production without requiring complex refolding procedures. The tag can be subsequently removed by proteolytic cleavage.
2Stability of the object's composition
If prothrombin is expressed in mammalian cells, then soluble protein is obtained, but yield is very low and manufacturing cost is high
Solution Approach 1:
The expression system parameters are changed from mammalian cells to E. coli, accompanied by introducing a solubility-enhancing tag. This parameter change enables the system to achieve both high yield (characteristic of E. coli) and good solubility (through the tag), resolving the contradiction between productivity and protein solubility.
3Stability of the object's composition
If prothrombin is produced by refolding insoluble aggregates, then soluble protein is obtained, but specific activity is very low (4-7% of native thrombin)
Solution Approach 1:
The solubility-enhancing tag is introduced in advance during the expression stage to prevent aggregate formation. This preliminary action ensures that the prothrombin protein remains soluble throughout the expression and purification process, eliminating the need for refolding and preserving the high specific activity of the final thrombin product.
Data Source
Figure 1A~1D
Figure 2~3
Figure 4A
AI summary
The present invention provides a method for producing recombinant prothrombin. The method comprises: providing a vector DNA into which a gene encoding a tag and a gene encoding prothrombin are incorporated, wherein the tag is selected from the group consisting of MBP, SUMO, and NusA; and expressing a tag fusion type prothrombin in a lepidopteran insect or cultured cells of the lepidopteran insect.