Genetic engineering approach to develop next-generation reagents for endotoxin quantification

Area of Science:

• Biotechnology
• Biochemistry
• Analytical Chemistry

Background:

• The bacterial endotoxin test (BET) traditionally relies on amebocyte lysate (LAL) derived from horseshoe crabs.
• Horseshoe crab populations are facing pressure, necessitating sustainable alternatives for LAL reagents.

Purpose of the Study:

• To develop sustainable, recombinant raw materials for lysate reagents.
• To engineer recombinant cascade reagents (RCRs) that mimic the natural LAL reaction cascade.
• To evaluate the performance of RCRs as an alternative to traditional LAL reagents.

Main Methods:

• Genetic engineering was used to prepare three recombinant protease zymogens: factor C (from mammalian cells), factor B, and proclotting enzyme (from insect cells).
• Recombinant cascade reagents (RCRs) were constructed to replicate the amebocyte lysate reaction cascade.
• The protease activity, signal amplification efficiency, and endotoxin sensitivity of RCRs were compared to natural LAL reagents.

Main Results:

• RCRs demonstrated efficient signal amplification, with protease activity significantly higher than recombinant factor C alone.
• RCRs containing mammalian cell-derived factor C showed reduced interference from injectable drug components compared to insect cell-derived factor C.
• RCRs with mammalian cell-derived recombinant factor C exhibited a steeper standard curve slope, indicating greater endotoxin sensitivity than natural LAL reagents.

Conclusions:

• Recombinant reagents can be produced without relying on natural resources like horseshoe crabs.
• Mammalian cell-derived recombinant factor C-based RCRs offer improved sensitivity and reduced interference for endotoxin testing.
• This study supports the future production of sustainable, recombinant reagents for bacterial endotoxin detection.