National University of Singapore

Jeak Ling Ding

BoW Ho

Nguan Soon Tan

Toong Jin Lam

Bow Ho

Recombinant fragments of Factor C are disclosed. These proteins and peptides show great potency in recognizing, binding to, neutralizing and removing endotoxin. These molecules can thus be used for anti-microbial, anti-endotoxin, and anti-sepsis therapy. SSCrFCES is a 38 kDa protein representing the LPS-binding domain of Factor C. The ability of SSCrFCES to bind lipid A was analyzed using an ELISA-based assay as well as surface plasmon resonance. Surface plasmon resonance similarly carried out for SSCrFC-sushi-1,2,3-GFP, SSCrFC-sushi-1GFP, and SSCrFC-sushi-3GFP confirmed their superior affinity for endotoxin. The 50% endotoxin-neutralizing concentration of SSCrFCES against 200 EU of endotoxin is 0.069 &#x3bc;M, suggesting that SSCrFCES is an effective inhibitor of LAL coagulation cascade. Although partially attenuated by human serum, as low as 1 &#x3bc;M of SSCrFCES inhibits the LPS-induced secretion of hTNF-&#x3b1; and hIL-8 by THP-1 and human pheripheral blood mononuclear cells with a potency more superior than polymyxin B. SSCrFCES is non-cytotoxic, with a clearance rate of 4.7 ml/minute. The LDof SSCrFCES for LPS lethality in mice is achieved at 2 &#x3bc;M. These results demonstrate the endotoxin-neutralizing capability of SSCrFCES in vitro and in vivo, as well as its potential for use in the treatment of endotoxin-induced septic shock. Also embodied in this application is the use of the sushi peptides and their mutant derivatives as potent antimicrobials.

Factor C for treating gram-negative bacterial infection

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 thecoagulation 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. 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.

Sushi peptide multimer

Recombinant fragments of Factor C are disclosed. These proteins and peptides show great potency in recognizing, binding to, neutralizing and removing endotoxin. These molecules can thus be used for anti-microbial, anti-endotoxin, and anti-sepsis therapy. SSCrFCES is a 38 kDa protein representing the LPS-binding domain of Factor C. The ability of SSCrFCES to bind lipid A was analyzed using an ELISA-based assay as well as surface plasmon resonance. Surface plasmon resonance similarly carried out for SSCrFC-sushi-1,2,3-GFP, SSCrFC-sushi-1GFP, and SSCrFC-sushi-3GFP confirmed their superior affinity for endotoxin. The 50% endotoxin-neutralizing concentration of SSCrFCES against 200 EU of endotoxin is 0.069 &#x3bc;M, suggesting that SSCrFCES is an effective inhibitor of LAL coagulation cascade. Although partially attenuated by human serum, as low as 1 &#x3bc;M of SSCrFCES inhibits the LPS-induced secretion of hTNF-&#x3b1; and hIL-8 by THP-1 and human pheripheral blood mononuclear cells with a potency more superior than polymyxin B. SSCrFCES is non-cytotoxic, with a clearance rate of 4.7 ml/minute. The LDof SSCrFCES for LPS lethality in mice is achieved at 2 &#x3bc;M. These results demonstrate the endotoxin-neutralizing capability of SSCrFCES in vitro and in vivo, as well as its potential for use in the treatment of endotoxin-induced septic shock. Also embodied in this application is the use of the sushi peptides and their mutant derivatives as potent antimicrobials. Further embodied in this application is the use of sushi peptides or sushi recombinant proteins to remove endotoxin from liquids.

Recombinant proteins and peptides for endotoxin biosensors, endotoxin removal, and anti-microbial and anti-endotoxin therapeutics

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.

Isolated nucleic acids encoding a secretory signal for expression and secretion of heterologous recombinant proteins

Recombinant fragments of Factor C are disclosed. These proteins and peptides show great potency in recognizing, binding to, neutralizing and removing endotoxin. These molecules can thus be used for anti-microbial, anti-endotoxin, and anti-sepsis therapy. SSCrFCES is a 38 kDa protein representing the LPS-binding domain of Factor C. The ability of SSCrFCES to bind lipid A was analyzed using an ELISA-based assay as well as surface plasmon resonance. Surface plasmon resonance similarly carried out for SSCrFC-sushi-1,2,3-GFP, SSCrFC-sushi-1GFP, and SSCrFC-sushi-3GFP confirmed their superior affinity for endotoxin. The 50% endotoxin-neutralizing concentration of SSCrFCES against 200 EU of endotoxin is 0.069 &mgr;M, suggesting that SSCrFCES is an effective inhibitor of LAL coagulation cascade. Although partially attenuated by human serum, as low as 1 &mgr;M of SSCrFCES inhibits the LPS-induced secretion of hTNF-&agr; and hIL-8 by THP-1 and human pheripheral blood mononuclear cells with a potency more superior than polymyxin B. SSCrFCES is non-cytotoxic, with a clearance rate of 4.7 ml/minute. The LD of SSCrFCES for LPS lethality in mice is achieved at 2 &mgr;M. These results demonstrate the endotoxin-neutralizing capability of SSCrFCES in vitro and in vivo, as well as its potential for use in the treatment of endotoxin-induced septic shock. Also embodied in this application is the use of the sushi peptides and their mutant derivatives as potent antimicrobials. Further embodied in this application is the use of sushi peptides or sushi recombinant proteins to remove endotoxin from liquids.

The horseshoe crab, 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 &ldquo;limulus amoebocyte lysate.&rdquo;

Assays for endotoxin

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. Solubilized rCrFC was purified as a proenzyme and reversibly protected from activation by addition of Me.sub.2 SO.

Expression of Carcinoscorpius rotundicauda Factor C in eukaryotes

Expression of carcinoscorpius rotundicauda factor C in eukaryotes

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.

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Singapore, Singapore

Jeak Ling Ding