Abstract
The expression of the different domains of a complex serine protease, recombinant Factor C of the horseshoe crab, Carcinoscorpius rotundicauda, was achieved using pCIneo and pEGFP-N1 vectors. In an in vitro coupled transcription-translation assay, the truncated CrFC cDNA insert coding for the initial 331 amino acids expressed the expected 36 kDa polypeptide lacking the antigenic epitopes. The highly disulfide-bonded Cys-rich and EGF-like domains at the N-terminus of CrFC which bind LPS were fused to GFP. When expressed in COS-1 cells, this fusion protein did not alter the localisation of the chromophore and remained soluble, indicating that other domains could be responsible for the membrane-bound nature of the recombinant Factor C, rFC. The expression of this LPS-binding domain of rFC as a soluble protein suggests possibilities of obtaining active rFC in mammalian cells.
Similar content being viewed by others
References
Coxon, A. and Bester, T. H. (1995) Chem. Biol. 2, 119-121.
Ding, J. L., Chai, C., Pui, A.W.M. and Ho, B. (1997) J. Endotoxin Research 4, 33-43.
Ding, J. L., Navas, M. A. A. and Ho, B. (1995) Mol. Mar. Biol. Biotech. 4, 90-103.
Eldering, E., Nuijens, J. H. and Hack, C. E. (1988) J. Biol. Chem. 262, 11776-11779.
Hampton, R. Y., Koning, A., Wright, R. and Rine, J. (1996) Proc. Natl. Acad. Sci. USA 93, 828-833.
Iwanaga, S. (1993) Curr. Opin. Immunol. 5, 74-82.
Kaether, C. and Gerdes, H. H. (1995) FEBS lett. 369, 267-271.
Miura, Y., Tokunaga, F., Miyata, T., Moriasu, M., Yoshikawa, K. and Iwanaga, S. (1992) J. Biol. Chem. 269, 542-547.
Muta, T., Miyata, T., Misumi, Y., Tokunaga, F., Nakamura, T., Toh, Y., Ikehara, Y. and Iwanaga, S. (1991) J. Biol. Chem. 265, 22426-22433.
Ormo, M., Cubitt, A. B., Kallio, K., Gross, L. A., Tsien, R. Y. and Remington, S. J. (1996) Science 273, 1392-1395.
Rizzuto, R., Brini, M., Pizzo, P., Murgia, M. and Pozzan, T. (1995) Current Biol. 5, 635-642.
Roopashree, S. D., Chai, C., Ho, B. and Ding, J. L. (1995) Biochem. Mol. Biol. Intl. 35, 841-849.
Roopashree, S. D., Ho, B. and Ding, J. L. (1996) Mol. Mar. Biol. Biotech. 5, 334-343.
Roopashree, S. D., Ho, B. and Ding, J. L. (1997) Biotechnol. Letters 19, 357-361.
Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) In: Molecular Cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, USA. 2nd Edition.
Toh, Y., Mizutani, A., Tokunaga, F., Muta, T. and Iwanaga, S. (1991) Cell. Tissue Res. 266, 137-147.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Dwarakanath, R., Ho, B. & Ding, J. The Cys-rich and EGF-like domains of Carcinoscorpius rotundicauda Factor C yields soluble fusion protein with GFP. Biotechnology Letters 19, 1147–1150 (1997). https://doi.org/10.1023/A:1018461131484
Issue Date:
DOI: https://doi.org/10.1023/A:1018461131484