The production of recombinant Carcinoscorpius rotundicauda Limulus clotting factor C in E. coli starts with the co-insertion of the gene of interest (691-1019aa of Carcinoscorpius rotundicauda Limulus clotting factor C) into an expression vector with an N-terminal 6xHis-tag gene, followed by transformation into E. coli cells. These cells are grown under conditions that induce protein expression. After sufficient growth, cells are lysed to release the recombinant protein. Purification is carried out using the affinity chromatography technique. The purity of the protein is confirmed with SDS-PAGE, reaching up to 85%.
Carcinoscorpius rotundicauda Limulus clotting factor C, identified as a serine protease zymogen, plays a central role in initiating a cascade of pathways that lead to the coagulation of Gram-negative bacteria [1]. Studies have shown that factor C of Carcinoscorpius rotundicauda is both immunoreactive and functional, indicating its significance in the immune response of the horseshoe crab [2].
Factor C from Carcinoscorpius rotundicauda has been extensively studied for its ability to bind endotoxins and induce bacteriostasis, showcasing its importance in the defense mechanisms of the horseshoe crab [3]. The interaction of factor C with lipopolysaccharides (LPS) triggers a series of events leading to the activation of clotting enzymes and the subsequent clotting process, essential for encapsulating and eliminating bacteria [4].
References:
[1] M. Khan, T. Muthamilselvan, H. Kang, & I. Hwang, Plant produced endotoxin binding recombinant proteins effectively remove endotoxins from protein samples, Scientific Reports, vol. 12, no. 1, 2022. https://doi.org/10.1038/s41598-022-20776-6
[2] A. Pui, B. Ho, & J. Ding, Yeast recombinant factor c from horseshoe crab binds endotoxin and causes bacteriostasis, Journal of Endotoxin Research, vol. 4, no. 6, p. 391-400, 1997. https://doi.org/10.1177/096805199700400602
[3] J. Ding, C. Chai, A. Pui, & B. Ho, Expression of full length and deletion homologues of carcinoscorpius rotundicauda factor c in saccharomyces cerevisiae: immunoreactivity and endotoxin binding, Journal of Endotoxin Research, vol. 4, no. 1, p. 33-43, 1997. https://doi.org/10.1177/096805199700400105
[4] S. Srimal, T. Miyata, S. Kawabata, T. Miyata, & S. Iwanaga, The complete amino acid sequence of coagulogen isolated from southeast asian horseshoe crab, carcinoscorpius rotundicauda1, The Journal of Biochemistry, vol. 98, no. 2, p. 305-318, 1985. https://doi.org/10.1093/oxfordjournals.jbchem.a135283
