Endotoxin detection--from limulus amebocyte lysate to recombinant factor C.
Gram negative bacterial endotoxin is a biological pyrogen that causes fever when introduced intravenously. The endotoxin, also known as lipopolysaccharide (LPS), is found in the outer membrane of Gram-negative bacteria. During Gram-negative sepsis, endotoxin stimulates host macrophages to release inflammatory cytokines. However, excessive inflammation causes multiple organ failure and death. Endotoxins, which are ubiquitous pathogenic molecules, are a bane to the pharmaceutical industry and healthcare community. Thus early and sensitive detection of endotoxin is crucial to prevent endotoxaemia. The limulus amebocyte lysate (LAL) has been widely used for ~30 years for the detection of endotoxin in the quality assurance of injectable drugs and medical devices. The LAL constitutes a cascade of serine proteases which are triggered by trace levels of endotoxin, culminating in a gel clot at the end of the reaction. The Factor C, which normally exists as a zymogen, is the primer of this coagulation cascade. In vivo, Factor C is the perfect biosensor, which alerts the horseshoe crab of the presence of a Gram-negative invader. The hemostatic end-point entraps the invader, killing it and limiting further infection. However, as an in vitro endotoxin detection tool, variations in the sensitivity and specificity of LAL to endotoxin, and the dwindling supply of horseshoe crabs are posing increasing challenges to the biotechnology industry. This has necessitated the innovation of an alternative test for endotoxin. Thus, Factor C became the obvious, albeit tricky target for the recombinant technology effort. This chapter documents the backwater of mining the natural blood lysate of the endangered species to the monumental effort of genetic engineering, to produce recombinant Factor C (rFC). The rFC is a 132 kDa molecule, which was produced as a proenzyme inducible by the presence of trace levels of endotoxin. The rFC forms the basis of the "PyroGene" kit, which is a novel micro-enzymatic endotoxin diagnostic assay for high-throughput screens of endotoxin. Using the rFC, Lonza Inc. has spawned the "PyroSense" which serves as checkpoints of the biotechnology production line. Thus, from cloning to commercial applications, the rFC has initiated a new era in endotoxin-testing for the quality assurance of biomedical products and for the healthcare industry, whilst sparing the endangered horseshoe crabs.
Ding
Jeak Ling
JL
Department of Biological Sciences, Faculty of Science, National University of Singapore, 14 Science Drive 4, Singapore, 117543, Singapore. dbsdjl@nus.edu.sg
Ho
Bow
B
D000818:Animals; D060829:Arthropod Proteins; D001681:Biological Assay / Q000295:instrumentation / Q000379:methods; D001798:Blood Proteins / Q000378:metabolism; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D004731:Endotoxins / Q000737:chemistry*; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism*; D006737:Horseshoe Crabs / Q000166:cytology*; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D012772:Shock, Septic / Q000276:immunology / Q000382:microbiology
D060829:Arthropod Proteins; D001798:Blood Proteins; D004731:Endotoxins; D004792:Enzyme Precursors; D008565:Membrane Proteins; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins; C025357:endotoxin binding proteins; D012697:Serine Endopeptidases; C057151:Limulus clotting factor C