Recombinant Factor G Heterodimer for Sensitive β-Glucan Detection
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Solution Overview
Problem
Existing methods for measuring β-glucan using horseshoe crab-derived Factor G are not sensitive enough for clinical diagnosis, and the presence of yeast extract in insect cell culture medium leads to BG contamination, making it difficult to confirm protease activity.
Innovation Solution
A heterodimer composed of specific horseshoe crab-derived Factor G α- and β-subunits with defined amino acid sequences, which are recombinantly produced, exhibits BG-dependent protease activity, enabling high sensitivity BG measurement.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If Factor G is produced using insect cell culture medium, then production is achieved, but BG contamination occurs due to yeast extract in the medium
Solution Approach 1:
The patent extracts and removes the harmful BG contamination from the culture medium by using a BG-free medium formulation, thereby eliminating the interference in protease activity measurement while maintaining Factor G production capability
Solution Approach 2:
The patent introduces a BG-free culture medium as an intermediary solution that mediates between the need for Factor G production and the requirement to avoid BG contamination, enabling reliable protease activity assessment
2Object-affected harmful factors
If BG is not added during Factor G production, then contamination is avoided, but protease activity cannot be confirmed
Solution Approach 1:
The patent performs preliminary purification of Factor G to remove any trace BG before activity assessment, then adds a controlled amount of BG to specifically activate and confirm protease activity, ensuring both contamination avoidance and activity verification
3Ease of operation
If conventional Factor G methods are used, then measurement is possible, but detection sensitivity is insufficient for clinical diagnosis
Solution Approach 1:
The patent changes the critical parameter of detection sensitivity by using purified recombinant Factor G with confirmed protease activity in a controlled BG-free environment, enabling detection at clinically relevant low concentrations of BG
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 heterodimer achieves higher sensitivity in BG measurement, overcoming contamination issues and lot variability, allowing for accurate clinical diagnosis with improved detection sensitivity.
Implementation Method 1
BG has a property of binding to a BG-binding domain portion of the Factor G α-subunit
Implementation Method 2
In a case where BG binds to the BG-binding domain portion of the Factor G α-subunit, the Factor G is an active Factor G having protease activity. The active Factor G converts a Proclotting enzyme present in the lysate into a Clotting enzyme by the protease activity
Implementation Method 3
The Clotting enzyme releases pNA by amide hydrolysis of the synthetic substrate of a synthetic peptide substrate (for example, Boc-DEL-pNA)
Data Source
AI summary
The present invention relates to “a heterodimer which combines a Factor G α-subunit containing an amino acid sequence that is the same as or substantially the same as an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 with a Factor G β-subunit containing an amino acid sequence that is the same as or substantially the same as an amino acid sequence represented by any one of SEQ ID NO: 6, 8, 10, 12, 14, or 16, a method of measuring a β-glucan using the heterodimer, and a kit for measuring a β-glucan containing the heterodimer”.