Recombinant Factor C Protein Production in Leishmania tarentolae
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Solution Overview
Problem
Recombinant production of active Factor C protein from horseshoe crabs has been challenging due to the failure of previous methods in achieving biologic activity required for endotoxin detection, with most attempts using prokaryotic, simple eukaryotic, and higher eukaryotic host cells resulting in inactive or insoluble forms.
Innovation Solution
A novel method using a parasitic protozoan host cell, specifically Leishmania tarentolae, to express and produce recombinant Factor C protein, which is secreted in a soluble and active form, overcoming the limitations of previous host systems by providing correct folding and cleavage of the pre-pro-enzyme into an active protease.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If prokaryotic, simple eukaryotic, or higher eukaryotic host cells are used for recombinant Factor C production, then production is achieved, but the protein is inactive or insoluble
Solution Approach 1:
The patent changes the host cell parameter from conventional prokaryotic/eukaryotic systems to a parasitic protozoan system (Leishmania tarentolae), which provides the appropriate cellular environment for correct protein folding and cleavage of the pre-pro-enzyme into active protease form, resolving the contradiction between production capability and protein activity/solubility
Solution Approach 2:
The parasitic protozoan host cell acts as an intermediary system that mediates the expression and processing of Factor C, providing the necessary cellular machinery for correct folding and proteolytic cleavage that conventional host cells lack, thereby enabling production of active, soluble protein
2Productivity
If conventional host cells are used for Factor C expression, then protein production is achieved, but the pre-pro-enzyme is not correctly folded or cleaved into active protease
Solution Approach 1:
The patent changes the host cell parameter to a parasitic protozoan system that possesses the appropriate cellular machinery for correct protein folding and proteolytic processing, thereby achieving both high productivity and manufacturing precision in producing active Factor C protease
Solution Approach 2:
The Factor C gene is expressed as a pre-pro-enzyme that requires sequential processing (folding then cleavage) to become active. The parasitic protozoan host provides the segmented processing pathway necessary to convert the initial transcript into the final active protease form with correct structural organization
3Quantity of substance
If recombinant Factor C is produced in conventional systems, then protein is obtained, but it is insoluble and inactive for endotoxin detection
Solution Approach 1:
The patent changes the host cell parameter to parasitic protozoa, which produce Factor C in a soluble, active form that retains endotoxin detection capability, thereby achieving both sufficient quantity and reliable functional activity for its intended application
Data Source
AI summary
The present invention provides a novel method for the recombinant production of Factor C protein from horseshoe crab using a parasitic protozoan expressing the Factor C protein. In particular, the present invention provides a parasitic protozoan host cell harbouring a polynucleotide encoding horseshoe crab Factor C protein, and a method for producing Factor C protein comprising culturing said parasitic protozoan host cell under conditions such that the cells express the horseshoe crab Factor C protein. Furthermore, the present invention provides recombinant Factor C protein produced by the novel method and its use in the detection and/or removal of endotoxin.