Baculovirus expression system-based expression of horseshoe crab factor C and its activity.

Lan LAN; Huanlei LIU; Hao NAN; Sijun HE; Wangcheng SONG; Yunlong WANG; Xinjuan FAN; Xiangbo WAN; Xiaodong XU

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Baculovirus expression system-based expression of horseshoe crab factor C and its activity.

Author: Lan LAN ¹ ; Huanlei LIU ² ; Hao NAN ¹ ; Sijun HE ¹ ; Wangcheng SONG ¹ ; Yunlong WANG ² ; Xinjuan FAN ² ; Xiangbo WAN ² ; Xiaodong XU ¹
Author Information

1. College of Life Sciences, Northwest A & F University, Yangling 712100, Shaanxi, China.
2. Tianjian Laboratory of Advanced Biomedical Sciences, Zhengzhou University, Zhengzhou 450001, Henan, China.
Publication Type:Journal Article
Keywords: Tachypleus tridentatus; baculovirus expression system; endotoxin detection; recombinant factor C
MeSH: Animals; Recombinant Proteins/genetics*; Horseshoe Crabs/chemistry*; Baculoviridae/metabolism*; Endotoxins/analysis*; Protein C/biosynthesis*; Genetic Vectors/genetics*; Arthropod Proteins/genetics*; Enzyme Precursors; Serine Endopeptidases
From: Chinese Journal of Biotechnology 2025;41(4):1428-1439
CountryChina
Language:Chinese
Abstract: Endotoxins are common exogenous pyrogens. Excessive endotoxins in medical devices and injections can lead to serious consequences such as sepsis, septic shock, and even death. Therefore, endotoxin detection plays a crucial role in medical, pharmaceutical, and food sectors. The wide application of Limulus amebocyte lysate (LAL) has led to a sharp decline in the number of horseshoe crabs. Moreover, the LAL assay has limitations such as interbatch variations and difficulty in quantification. The recombinant factor C (rFC) assay is stable between batches, highly sensitive, and capable of quantitation, and thus it can be used as an alternative for the LAL assay. However, the high cost and complex procedures involved in producing recombinant factor C have limited the widespread application of this method. In order to simplify the preparation and reduce the production cost of recombinant factor C, this study focuses on the production of recombinant factor C based on the baculovirus expression system. Multiple measures such as a high-yield and anti-apoptotic vector qBac-IIIG, the optimal signal peptide, and the optimized codon were used to reach the goal of endotoxin detection with cell supernatant. This method simplifies the steps of protein purification. The sensitivity of the supernatant reached 0.05 EU/mL in a 1-L fermentation system, and 500 000 detecting reactions can be supported per liter of fermentation broth. This study increases the yield and activity of recombinant factor C, simplifies the procedures of protein purification, and reduces the cost, laying a foundation for the promotion and application of recombinant factor C in endotoxin detection.