Abstract
Limulus Factor C is a potent antagonist of endotoxin from Gram-negative bacteria. A fusion construct containing full length Factor C has been cloned intoSpodoptera frugiperda Sf9 cell. Stable Sf9 cell transfectants were obtained using Zeocin selection for 2 weeks. The recombinant Factor C (rFC) was secreted into the culture medium at 9 mg l−1. Both the crude and partially purified rFC were able to detect lipid A at 10 pg ml−1 in an ELISA-based lipid A binding assay.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Ding JL, Chai C, Pui WM, Ho B (1997) Expression of full length and deletion homologues of Carcinoscorpius rotundicauda Factor C in Saccharomyces cerevisiae: immunoreactivity and endotoxin binding. J. Endotoxin Res. 4: 33–43.
Ding JL, Navas MAA III, Ho B (1995) Molecular cloning and sequence analysis of Factor C cDNA from the Singapore horseshoe crab, Carcinoscorpius rotundicauda. Mol. Mar. Biol. Biotech. 4: 90–103.
Ding JL, Tan NS, Ho B, Lam TJ (1996) Isolated nucleic acids encoding a secretory signal for expression and secretion of heterologous recombinant proteins. US Patent Application No. 09/426, 776; PCT/SG99/00108, WO 00/26366.
Hegedus DD, Pfeifer TA, Theilmann DA, Kennard ML, Gabathuler R, Jefferies WA, Grigliatti TA (1999) Differences in the expression and localization of human melanotransferrin in lepidopteran and dipteran insect cell lines. Protein Expres. Purif. 15: 296–307.
Ho B (1983) An improved Limulus gelation assay. Microbios. Lett. 24: 81–84.
Nakamura T, Morita T, Iwanaga S (1986) Lipopolysaccharidesensitive serine-protease zymogen (factor C) found in Limulus hemocytes. Isolation and characterization. Eur. J. Biochem. 154: 511–521.
Pui AWM, Ho B, Ding JL (1997) Yeast recombinant Factor C from horseshoe crab binds endotoxin and causes bacteriostasis. J. Endotoxin Res. 4: 391–400.
Pui AWM, Roopashree SD, Ho B, Ding JL (1998) Recombinant Factor C from Carcinoscorpius rotundicauda binds endotoxin. In: New Development in Marine Biotechnology. New York: Plenum Press, pp. 151–154.
Roopashree SD, Chai C, Ho B, Ding JL (1995) Expression of Carcinoscorpius rotundicauda Factor C cDNA. Biochem. Mol. Biol. Intl. 35: 841–849.
Roopashree SD, Ho B, Ding JL (1996) Expression of Carcinoscorpius rotundicauda Factor C in Pichia pastoris. Mol. Mar. Biol. Biotech. 5: 334–343.
Roopashree SD, Ho B., Ding JL (1997a) Recombinant COS-1 cells express Carcinoscorpius rotundicauda Factor C. Biotechnol. Lett. 19: 357–361.
Roopashree SD, Ho B, Ding JL (1997b) The cys-rich and EGF-like domains of Carcinoscorpius rotundicauda Factor C yields soluble fusion protein with GFP. Biotechnol. Lett. 19: 1147–1150.
Tan NS, Ho B, Ding JL (2000) High-affinity LPS binding domain(s) in recombinant Factor C of a horseshoe crab neutralizes LPSinduced lethality. FASEB J. 14: 859–870.
Theilmann DA, Steward S (1992) Molecular analysis of the transactivating IE-2 gene of Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus. Virology 187: 84–96.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Wang, J., Ho, B. & Ding, J.L. Functional expression of full length Limulus Factor C in stably transformed Sf9 cells. Biotechnology Letters 23, 71–76 (2001). https://doi.org/10.1023/A:1026764514737
Issue Date:
DOI: https://doi.org/10.1023/A:1026764514737