Abstract
Carcinoscorpius rotundicauda Factor C cDNA has been cloned and expressed in Pichia pastoris to produce a recombinant full-length Factor C (rFC) which is both immunoreactive and functional. The presence of a functional endotoxin-binding domain on rFC was ascertained by LPS-binding assays. One involved the relative binding affinity of rFC to electroblotted lipid A moiety of LPS. The second assay showed that rFC competed against native Factor C contained in C. rotundicauda amebocyte lysate (CAL) to bind LPS. Purification of rFC enhanced its binding affinity to LPS. By agglutination, rFC caused bacteriostasis of Gram-negative bacteria within 2 h. In an in vivo system, rFC also decreased the mortality of actinomycin D-sensitized/LPS-challenged mice. The rFCEE, bearing the 5' terminal LPS binding domain displayed a lowered affinity for LPS. This is in contrast to the rFCSN subclone that is devoid of the 5' end of Factor C, and which does not bind LPS. The presence of a fully-functional endotoxin binding domain in rFC probably requires a full-length protein for co-operative interaction of its downstream sequences. Thus, rFC has potential in the detection and removal of contaminating LPS from biological specimens and fluids for injection, since it is capable of binding both free and bound lipid A.
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