Recombinant Factor C for Salt-Resistant Endotoxin Detection
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Solution Overview
Problem
Existing methods for detecting endotoxin, such as the limulus test, rely on extracts from horseshoe crabs, which are unsustainable and prone to reaction inhibition by salts and ions commonly found in pharmaceutical products.
Innovation Solution
Production of recombinant horseshoe crab Factor C using human or Chinese hamster cells, which maintains activity even in the presence of salts and ions, allowing for a reconstituted cascade reaction system that overcomes the limitations of traditional methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional limulus test using horseshoe crab lysate is used for endotoxin detection, then endotoxin detection capability is provided, but the method is unsustainable and prone to reaction inhibition by salts and ions
Solution Approach 1:
The patent extracts and isolates Factor C from horseshoe crab lysate through purification processes, separating it from other components that cause reaction inhibition. This extracted Factor C can then be used in a controlled manner without the harmful effects of salts and ions present in the original lysate.
Solution Approach 2:
The patent modifies the properties of Factor C through recombinant DNA technology and protein engineering, changing parameters such as amino acid sequence, molecular structure, or post-translational modification to reduce sensitivity to salts and ions while maintaining endotoxin detection activity.
2Reliability
If horseshoe crab lysate is used for endotoxin detection, then detection function is achieved, but biological resources are depleted and supply is limited
Solution Approach 1:
The patent creates a recombinant Factor C using human or Chinese hamster ovary cells that copies the functional properties of the original horseshoe crab Factor C. This recombinant version can be produced indefinitely through cell culture without depleting biological resources, effectively copying the detection function while eliminating the sustainability problem.
Solution Approach 2:
The patent replaces the biological extraction system (horseshoe crab lysate) with a recombinant protein production system using mammalian cell culture. This substitution transitions from harvesting natural biological materials to synthesizing proteins through genetic engineering, enabling unlimited supply without depleting wild populations.
3Reliability
If recombinant Factor C is produced using human or Chinese hamster cells, then sustainability and salt/ion resistance are improved, but production complexity increases
Solution Approach 1:
The patent employs Chinese hamster ovary cells as a universal production system that can express various proteins through standardized recombinant DNA techniques. This universal cell line system allows for the production of Factor C with salt and ion resistance properties while using established, relatively simple biotechnology protocols.
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 recombinant Factor C enables effective endotoxin detection in the presence of salts and ions, providing a sustainable and reliable alternative to traditional methods.
Implementation Method 1
Factor C of the present invention has been found to have low susceptibility to inhibition of activity by an ion. Specifically, Factor C of the present invention has been found to have 10% or higher residual activity in the presence of 21 mM sodium citrate, 25% or higher residual activity in the presence of 52 mM sodium hydrogencarbonate, 25% or higher residual activity in the presence of 214 mM sodium chloride, 15% or higher residual activity in the presence of 16 mM magnesium sulfate, and 35% or higher residual activity in the presence of 2.5 mM calcium chloride.
Implementation Method 2
Factor C, which is a serine protease zymogen present in the lysate, is activated to thereby form activated Factor C. The thus-formed activated Factor C activates Factor B present in the lysate, to thereby form activated Factor B.
Implementation Method 3
The Clotting enzyme hydrolyzes a specific site of a coagulogen molecule present in the lysate, thereby coagulin gel is formed, and thus the lysate is coagulated.
Implementation Method 4
there is known a method for measuring endotoxin by use of a hematocyte extract of a horseshoe crab (i.e. an amebocyte lysate of a horseshoe crab, hereinafter also referred to simply as a lysate) which employs a cascade reaction of a variety of proteins present in the lysate
Data Source
AI summary
A horseshoe crab Factor C protein having activity of Factor C, wherein the horseshoe crab is selected from Tachypleus tridentatus, Limulus polyphemus, and Carcinoscorpius rotundicauda, and wherein the horseshoe crab Factor C protein is produced through being recombinantly expressed from a Chinese Hamster Ovary (CHO) DG44 cell or HEK cell.