Abstract: Endotoxin, also known as lipopolysaccharides (LPS), is the major mediator of septic shock due to Gram-negative bacterial infection. Chemically synthesized S3 peptide, derived from Sushi3 domain of Factor C, which is the endotoxin-sensitive serine protease of the limulus coagulation cascade, binds and neutralizes LPS activity. Fluorescent tagged-S3 is shown to detect LPS-containing bacteria. For large-scale production of S3 and to mimic other pathogen-recognizing molecules, tandem multimers of the S3 gene were constructed and expressed in E. coli. Tetramer of S3 for example is shown to display an enhanced inhibitory effect on LPS-induced activities. An affinity matrix based on tetramer of S3 is also shown to be particularly efficient at removing LPS.

Abstract: Endotoxin, also known as lipopolysaccharides (LPS), is the major mediator of septic shock due to Gram-negative bacterial infection. Chemically synthesized S3 peptide, derived from Sushi3 domain of Factor C, which is the endotoxin-sensitive serine protease of the limulus coagulation cascade, binds and neutralizes LPS activity. Fluorescent tagged-S3 is shown to detect LPS-containing bacteria. For large-scale production of S3 and to mimic other pathogen-recognizing molecules, tandem multimers of the S3 gene were constructed and expressed in E. coli. Tetramer of S3 for example is shown to display an enhanced inhibitory effect on LPS-induced activities. An affinity matrix based on tetramer of S3 is also shown to be particularly efficient at removing LPS.

Abstract: A universal secretory signal originally derived from a piscine vitellogenin (Vtg) gene is inserted into various expression vectors for driving the secretion of the recombinant protein into the culture medium. This enhances the detection, quantification and downstream scaled-up purification of a recombinant protein of interest. The secretory signal system is very versatile, being conveniently and widely applicable to an array of heterologous host cells such as bacteria, yeast, insect, piscine, and mammalian cell lines (e.g., COS, CHO, NIH/3T3). The said secretory system is also applicable as a reporter vector for secretion of reporter proteins/enzymes, thus, enabling the detection of the reporter proteins (e.g., CAT, GFP) in the culture medium.

Abstract: The horseshoe crab, Carcinoscorpius rotundicauda Factor C cDNA (CrFC21) has been cloned into a shuttle baculoviral vector and another vector suitable for expression in insect cells. The recombinant baculoviral DNA was then transfected into the insect cells for expression of recombinant Factor C. Recombinant Factor C was found to be immunoreactive and is capable of binding both free and bound/immobilized lipid A. It is enzymatically active when triggered by LPS. The rFC is probably of the two-chain form, being cleaved into the heavy and light chains after activation by Gram negative bacterial endotoxin. As low as 0.01 pg (0.001 ng/ml) of LPS was detectable by the rFC, thus, indicating its potentials as a novel generation of “limulus amoebocyte lysate.

Abstract: CrFC21 cDNA was cloned into two mammalian vectors: pCIneo and pCDNAI, both of which carry the strong CMV promoter for expression in mammalian cell lines. Various CrFC cDNA constructs transformed into P. pastoris and S. cerevisiae were expressed to yield full-length recombinant Factor C (rCrFC) protein of .about.130 kDa which is immunoreactive. The rCrFC is expressed in an intracellular, insoluble form. Intracellular localization of the nascent protein provides protection from premature digestion by proteases secreted by the host cell. Subsequent to its synthesis, rCrFC is solubilized and purified under pyrogen-free conditions. Using established protocols, the protein can be denatured and renatured to recover its biological functionality. By manipulation of the 5' end of CrFC26, truncated constructs containing this cDNA are expressed by S. cerevisiae to give immunoreactive rCrFC. The rCrFC produced from both CrFC21 and CrFC26 constructs, solubilized by Triton X-100 or SDS, is found to be immunoreactive.

Abstract: CrFC21 cDNA was cloned into two mammalian vectors: pCIneo and pCDNAI, both of which carry the strong CMV promoter for expression in mammalian cell lines. Various CrFC cDNA constructs transformed into P. pastoris and S. cerevisiae were expressed to yield full-length recombinant Factor C (rCrFC) protein of .about.130 kDa which is immunoreactive. The rCrFC is expressed in an intracellular, insoluble form. Intracellular localization of the nascent protein provides protection from premature digestion by proteases secreted by the host cell. Subsequent to its synthesis, rCrFC is solubilized and purified under pyrogen-free conditions. Using established protocols, the protein can be denatured and renatured to recover its biological functionality. By manipulation of the 5' end of CrFC26, truncated constructs containing this cDNA are expressed by S. cerevisiae to give immunoreactive rCrFC. The rCrFC produced from both CrFC21 and CrFC26 constructs, solubilized by Triton X-100 or SDS, is found to be immunoreactive.

Abstract: Full-length and deletion subclones of cDNAs for Factor C of Carcinoscorpius rotundicauda are provided. These cDNAs have been cloned into .lambda.gt 22 and pGEM 11Zf(+). Further manipulations of the 5' and 3' ends of these cDNAs have been carried out, and these cDNAs have been further subcloned into other expression vectors such as pGEMEX-1, pET 3b, and the yeast shuttle vectors YEpsec 1 and pEMBLyex 4, and pPIC 9 and pHIL D2. Also provided are host cells transformed with expression vectors containing DNA molecules encoding proteins having Factor C-like enzymatic activity, methods of producing such proteins, methods for purifying Factor C zymogens, and methods for protecting Factor C zymogens from autoactivation by Gram negative bacterial endotoxin while the proenzyme is being purified and/or processed from amoebocyte lysates or from recombinant clones, or during storage or subsequent handling. This protection is afforded by the addition of 5-30% Me.sub.2 SO, which reversibly inhibits the Factor C zymogen.

Abstract: Full-length and deletion subclones of cDNAs for Factor C of Carcinoscorpius rotundicauda are provided. These cDNAs have been cloned into .lambda.gt 22 and pGEM 11Zf(+). Further manipulations of the 5' and 3' ends of these cDNAs have been carried out, and these cDNAs have been further subcloned into other expression vectors such as pGEMEX-1, pET 3b, and the yeast shuttle vectors YEpsec 1 and pEMBLyex 4, and pPIC 9 and pHIL D2. Also provided are host cells transformed with expression vectors containing DNA molecules encoding proteins having Factor C-like enzymatic activity, methods of producing such proteins, methods for purifying Factor C zymogens, and methods for protecting Factor C zymogens from autoactivation by Gram negative bacterial endotoxin while the proenzyme is being purified and/or processed from amoebocyte lysates or from recombinant clones, or during storage or subsequent handling. This protection is afforded by the addition of 5-30% Me.sub.2 SO, which reversibly inhibits the Factor C zymogen.